Tumor Microenvironments in Organs: From the Brain to the Skin – Part A (Advances in Experimental Medicine and Biology, 1226) 3030362132, 9783030362133

Table of contents :
Preface
Contents
Contributors
1: The Intestinal Tumour Microenvironment
1.1 Physiology and Function of the Intestines
1.2 Intestinal Cancers
1.2.1 Staging of Intestinal Tumours
1.2.2 Discrepancy Between Large and Small Intestine
1.2.3 Genetic Mechanisms of Tumour Development
1.2.4 Left- and Right-Sided Colorectal Tumours
1.2.5 Consensus Molecular Subtypes of CRC Defined in Part by the TME
1.3 Immune Contribution to TME
1.3.1 T Cells
1.3.2 Tumour-Associated Macrophages
1.3.3 Neutrophils
1.3.4 Natural Killer Cells
1.4 Fibroblast Contribution to TME
1.4.1 CAFs Promote Tumour Chemoresistance
1.4.2 CAF-Secreted Cytokines Create a Tumour-Permissive Environment
1.4.3 CAFs Promote Tumour Invasion
1.5 Microbial Contribution to TME
1.5.1 Bacterial Metabolites Promote or Protect Against CRC
1.5.2 Bacterial Proteins May Promote CRC Progression
1.5.3 Two-Way Relationship: TME Influences Microbiota
1.6 Conclusions
References
2: Gastric Tumor Microenvironment
2.1 Gastric Cancer
2.2 Stromal Cells
2.2.1 Bone Marrow-Derived Cells (BMDCs)
2.2.2 Tumor-Associated Mast Cells (TAMCs)
2.2.3 Cancer-Associated Fibroblasts (CAFs)
2.2.4 Tumor-Associated Macrophages (TAMs)
2.2.5 Tumor-Infiltrating Neutrophils (TINs)
2.3 Extracellular Matrix
2.4 MicroRNAs
2.5 Exosomes
2.6 Dysregulated Cellular Signaling
2.7 Concluding Remarks
References
3: Parathyroid Tumor Microenvironment
3.1 Introduction
3.2 Parathyroid Tumors
3.3 Microenvironment in Parathyroid Tumors
3.3.1 Parathyroid Tumor Histology
3.3.2 Tumor Angiogenesis
3.3.3 Lymphangiogenesis
3.3.4 Tumor Inflammatory Infiltration
3.3.5 Tumor-Associated Myofibroblasts
3.3.6 Tumor-Associated Mesenchymal Stem Cells
3.3.7 Potential Role of Parathyroid Tumors-Deregulated MicroRNAs in TME Modulation
3.4 Future Trends and Directions
References
4: Microenvironment in Cardiac Tumor Development: What Lies Beyond the Event Horizon?
4.1 Cardiac Tumors: A Snapshot of What We Know About Them
4.2 Applying the “Black Hole” Paradigm in Exploring Cardiac Tumorigenesis
4.3 Materials and Methods: The “Event Horizon Telescope”
4.4 Tumor Microenvironment (TME): Contextualizing Benchmark Knowledge from Noncardiac Tumors
4.5 What Makes Cardiac TME Different? What Lies Beyond the “Event Horizon”?
4.6 Future Directions
4.7 Epilogue
References
5: The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis
5.1 Introduction
5.2 Bone Marrow Tumor Microenvironment
5.2.1 Cells of Mesenchymal Origin
5.2.1.1 Osteoblasts
5.2.1.2 Osteocytes
5.2.1.3 Bone Marrow Adipocytes
5.2.2 Cells of Hematopoietic Origin
5.2.2.1 Osteoclasts
5.2.2.2 Immune Cells
5.2.3 Other Components of Bone Marrow Microenvironment
5.2.3.1 Endothelial Cells
5.2.3.2 Nerves
5.3 Conclusions and Future Directions
References
6: Adipose Tumor Microenvironment
6.1 Adipose Tissue as a Multifunctional Organ
6.2 A Role for Adipose Tissue in Cancer
6.3 IL6: The Cross Talk Mediator
6.3.1 Adipocytes
6.3.2 Preadipocytes
6.3.3 Macrophages
6.3.4 Fibroblasts
6.4 Extracellular Vesicles as a Possible Mediator of Cell-Cell Communication in the Adipose Tumor Microenvironment
6.5 The Future of Adipose Research
6.5.1 Realizing the Role of Adipose Tissue in Cancer Onset and Progression
6.5.2 Targeting the IL6:GP130 Axis
6.5.3 EVs: The Next-Generation Mediators of Cell-to-Cell Communication
References
7: Appendix Tumor Microenvironment
7.1 Introduction
7.2 Appendix Tumor Microenvironment (TME)
7.3 TME Immune Power
7.4 Tumor Immunology
7.5 Appendix Tumor Immunology
7.6 TME in Appendix Lymphomas
References
8: Spinal Cord Tumor Microenvironment
8.1 Introduction
8.2 The Spinal Cord
8.3 Neoplastic Diseases of the Spinal Cord
8.4 Impact of the Spinal Cord Microenvironment on Tumor Biology
8.4.1 Blood-Spinal Cord Barrier
8.4.2 Vascularization
8.4.3 Lymphatic Vessels
8.4.4 Astrocytes
8.4.5 Neurons
8.4.6 Pericytes
8.4.7 Macrophages and Microglia
8.4.8 Dendritic Cells
8.4.9 Neutrophil Granulocytes
8.4.10 Lymphoid Cells
8.4.11 Extracellular Matrix
8.5 Conclusion
References
9: Cancer Stem Cell Niche and Immune-Active Tumor Microenvironment in Testicular Germ Cell Tumors
9.1 Introduction
9.2 Microenvironment of Normal Testis
9.3 Microenvironment of Testicular Germ Cell Tumors
9.3.1 Pluripotent Stem Cell Niche in TGCTs
9.3.2 Hypoxia in the TGCT Microenvironment
9.4 Immunity and Testicle Tumor Microenvironment
9.4.1 Immune Cell Infiltration of TGCTs and Cytokines Modulating TGCT Microenvironment
9.4.2 Immune Function Gene Polymorphisms in Testicular Germ Cell Tumors
9.4.3 Immune-Related Biomarkers in TGCTs
9.5 Conclusion
References
10: Tumour Microenvironment in Skin Carcinogenesis
10.1 Introduction
10.2 Tumour Microenvironment: Key Player in Carcinogenesis
10.3 Basal Cell Carcinoma
10.3.1 Tumour-Associated Macrophages
10.3.2 Tumour-Infiltrating Lymphocytes
10.3.3 Confocal Assessment of BCC Inflammatory Infiltrate
10.3.4 Cancer-Associated Fibroblasts
10.3.5 Cytokines
10.4 Squamous Cell Carcinoma
10.4.1 Tumour-Associated Macrophages
10.4.2 Tumour-Infiltrating Lymphocytes
10.4.3 Confocal Assessment of SCC Inflammatory Infiltrate
10.4.4 Cancer-Associated Fibroblasts
10.4.5 Natural Killer Cells
10.4.6 Extracellular Matrix
10.4.7 Cytokines
10.5 Melanoma
10.5.1 Tumour-Associated Macrophages
10.5.2 Tumour-Infiltrating Lymphocytes
10.5.3 Cancer-Associated Fibroblasts
10.5.4 B Lymphocytes
10.5.5 Dendritic Cells
10.5.6 Confocal Assessment of Cutaneous Melanoma Inflammatory Infiltrate
10.5.7 The Acidification of the Tumour Microenvironment
10.5.8 Cytokines
10.6 Conclusion
References
Index

Citation preview

Advances in Experimental Medicine and Biology 1226

Alexander Birbrair  Editor

Tumor Microenvironments in Organs From the Brain to the Skin – Part A

Advances in Experimental Medicine and Biology Volume 1226 Series Editors: Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux UMR 5287, Pessac Cedex, France John D. Lambris, University of Pennsylvania, Philadelphia, PA, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This book series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2018 Impact Factor: 2.126. More information about this series at http://www.springer.com/series/5584

Alexander Birbrair Editor

Tumor Microenvironments in Organs From the Brain to the Skin – Part A

Editor Alexander Birbrair Department of Radiology Columbia University Medical Center New York, NY, USA Department of Pathology Federal University of Minas Gerais Belo Horizonte, Minas Gerais, Brazil

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-36213-3    ISBN 978-3-030-36214-0 (eBook) https://doi.org/10.1007/978-3-030-36214-0 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

This book’s initial title was Tumor Microenvironment. However, due to the ­current great interest in this topic, we were able to assemble more chapters than would fit in one book, covering tumor microenvironment biology from different perspectives. Therefore, the book was subdivided into several volumes. This book Tumor Microenvironments in Organs: From the Brain to the Skin – Part A presents contributions by expert researchers and clinicians in the multidisciplinary areas of medical and biological research. The chapters provide timely detailed ­overviews of recent advances in the field. This book describes the tumor microenvironment in different tissues during cancer development. Further insights into the differences between microenvironments of distinct organs will have important implications for our understanding of cancer initiation, development, and progression. The authors focus on the modern methodologies and the leading-edge concepts in the field of cancer biology. In recent years, remarkable progress has been made in the identification and characterization of different components of the tumor microenvironment in several tissues using state-of-the-art techniques. These advantages facilitated the identification of key targets and definition of the molecular basis of cancer progression within different organs. Thus, the present book is an attempt to describe the most recent developments in the area of tumor biology, which is one of the emergent hot topics in the field of molecular and cellular biology today. Here, we present a selected collection of detailed chapters on what we know so far about the variable tumor microenvironments in several tissues. Ten chapters written by experts in the field summarize the present knowledge about the tumor microenvironment in distinct organs during tumor development. Roslyn Kemp and colleagues from the University of Otago discuss the intestinal tumor microenvironment. Armando Rojas and colleagues from the Catholic University of Maule describe the gastric tumor microenvironment. Sabrina Corbetta and colleagues from the University of Milan compile our understanding of the parathyroid tumor microenvironment. Konstantinos Mylonas and ­colleagues from the National and Kapodistrian University of Athens summarize current knowledge on the tissue microenvironment in ­cardiac tumor development. Yusuke Shiozawa from Wake Forest School of Medicine updates us with what we know about the roles of bone marrow-­resident cells as a microenvironment for bone metastasis. Raphael Pollock and colleagues from the Ohio State University address the importance of the tumor microenvironment in the adipose tissue. Luca Roncati and colleagues from the University Hospital of Modena focus on the tumor microenvironment in the appendix. Laurèl Rauschenbach v

Preface

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from University Hospital Essen introduces our current knowledge about the ­spinal cord tumor microenvironment. Michal Chovanec and colleagues from Comenius University and National Cancer Institute talk about the cancer stem cell niche in testicular germ cell tumors. Finally, Monica Neagu and colleagues from the University of Bucharest give an overview of the tumor microenvironment in skin carcinogenesis. It is hoped that the articles published in this book will become a source of reference and inspiration for future research ideas. I would like to express my deep gratitude to my wife Veranika Ushakova and Mr. Murugesan Tamilselvan from Springer, who helped at every step of the execution of this project. Belo Horizonte, Minas Gerais, Brazil

Alexander Birbrair

Contents

1 The Intestinal Tumour Microenvironment��������������������������������    1 J. K. H. Leman, L. Munoz-Erazo, and R. A. Kemp 2 Gastric Tumor Microenvironment ��������������������������������������������   23 Armando Rojas, Paulina Araya, Ileana Gonzalez, and Erik Morales 3 Parathyroid Tumor Microenvironment ������������������������������������   37 Chiara Verdelli, Valentina Vaira, and Sabrina Corbetta 4 Microenvironment in Cardiac Tumor Development: What Lies Beyond the Event Horizon?��������������������������������������   51 Konstantinos S. Mylonas, Ioannis A. Ziogas, and Dimitrios V. Avgerinos 5 The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis ����������������������������   57 Yusuke Shiozawa 6 Adipose Tumor Microenvironment��������������������������������������������   73 Abbie Zewdu, Lucia Casadei, Raphael E. Pollock, and Danielle Braggio 7 Appendix Tumor Microenvironment������������������������������������������   87 Luca Roncati, Paolo Gasparri, Graziana Gallo, Giuditta Bernardelli, Giuliana Zanelli, and Antonio Manenti 8 Spinal Cord Tumor Microenvironment ������������������������������������   97 Laurèl Rauschenbach 9 Cancer Stem Cell Niche and Immune-Active Tumor Microenvironment in Testicular Germ Cell Tumors����������������  111 Katarina Kalavska, Lucia Kucerova, Silvia Schmidtova, Michal Chovanec, and Michal Mego 10 Tumour Microenvironment in Skin Carcinogenesis ����������������  123 Simona Roxana Georgescu, Mircea Tampa, Cristina Iulia Mitran, Madalina Irina Mitran, Constantin Caruntu, Ana Caruntu, Mihai Lupu, Clara Matei, Carolina Constantin, and Monica Neagu Index������������������������������������������������������������������������������������������������������  143 vii

Contributors

Paulina  Araya Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile Dimitrios  V.  Avgerinos  Department of Cardiothoracic Surgery, New York Presbyterian Hospital, Weill Cornell Medicine, New York City, NY, USA Giuditta  Bernardelli Department of Medical and Surgical Sciences, Institute of Pathology, University Hospital of Modena, Modena (MO), Italy Danielle  Braggio Program in Translational Therapeutics, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Department of Surgery, The Ohio State University, Columbus, OH, USA Ana Caruntu  Department of Oral and Maxillofacial Surgery, “Carol Davila” Central Military Emergency Hospital, Bucharest, Romania Faculty of Medicine, Department of Preclinical Sciences, “Titu Maiorescu” University, Bucharest, Romania Constantin Caruntu  “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Department of Dermatology, “Prof. N.  Paulescu” National Institute of Diabetes, Nutrition and Metabolic Diseases, Bucharest, Romania Lucia  Casadei  Program in Translational Therapeutics, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Department of Surgery, The Ohio State University, Columbus, OH, USA Michal  Chovanec Faculty of Medicine, 2nd Department of Oncology, Comenius University and National Cancer Institute, Bratislava, Slovakia Carolina  Constantin Immunology Department, “Victor Babes” National Institute of Pathology, Bucharest, Romania Colentina Clinical Hospital, Bucharest, Romania Sabrina  Corbetta Department of Biomedical, Surgical and Odontoiatric Sciences, University of Milan, Milan, Italy Endocrinology and Diabetology Service, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy ix

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Graziana Gallo  Department of Medical and Surgical Sciences, Institute of Pathology, University Hospital of Modena, Modena (MO), Italy Paolo Gasparri  Department of Medical and Surgical Sciences, Institute of Pathology, University Hospital of Modena, Modena (MO), Italy Simona  Roxana  Georgescu “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania “Victor Babes” Clinical Hospital for Infectious Diseases, Bucharest, Romania Ileana  Gonzalez Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile Katarina  Kalavska Translational Research Unit, Faculty of Medicine, Comenius University and National Cancer Institute, Bratislava, Slovakia Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia R. A. Kemp  Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Lucia  Kucerova Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia J. K. H. Leman  Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Mihai  Lupu Department of Dermatology, MEDAS Medical Center, Bucharest, Romania Antonio Manenti  Department of Medical and Surgical Sciences, Section of Surgery, University Hospital of Modena, Modena (MO), Italy Clara  Matei “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Michal Mego  Translational Research Unit, Faculty of Medicine, Comenius University and National Cancer Institute, Bratislava, Slovakia Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia Faculty of Medicine, 2nd Department of Oncology, Comenius University and National Cancer Institute, Bratislava, Slovakia Cristina Iulia Mitran  “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania “Cantacuzino” National Medico-Military Institute for Research and Development, Bucharest, Romania

Contributors

Contributors

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Madalina  Irina  Mitran “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania “Cantacuzino” National Medico-Military Institute for Research and Development, Bucharest, Romania Erik  Morales Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile L. Munoz-Erazo  Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Maurice Wilkins Centre for Biodiscovery, Auckland, New Zealand Konstantinos  S.  Mylonas First Department of Surgery, Laikon General Hospital, National and Kapodistrian University of Athens, Athens, Greece Monica Neagu  Immunology Department, “Victor Babes” National Institute of Pathology, Bucharest, Romania Colentina Clinical Hospital, Bucharest, Romania Faculty of Biology, University of Bucharest, Bucharest, Romania Raphael  E.  Pollock Program in Translational Therapeutics, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Department of Surgery, The Ohio State University, Columbus, OH, USA Laurèl  Rauschenbach Department of Neurosurgery, University Hospital Essen, Essen, Germany DKFZ Division of Translational Neuro-Oncology at the West German Cancer Center (WTZ), German Cancer Consortium (DKTK) Partner Site, University Hospital Essen, Essen, Germany Armando  Rojas Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile Luca  Roncati  Department of Medical and Surgical Sciences, Institute of Pathology, University Hospital of Modena, Modena (MO), Italy Silvia  Schmidtova  Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia Yusuke Shiozawa  Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University Health Sciences, Winston-Salem, NC, USA Mircea  Tampa “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania “Victor Babes” Clinical Hospital for Infectious Diseases, Bucharest, Romania Valentina  Vaira Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

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Chiara Verdelli  Laboratory of Experimental Endocrinology, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy Giuliana Zanelli  Department of Medical and Surgical Sciences, Institute of Pathology, University Hospital of Modena, Modena (MO), Italy Abbie  Zewdu  Program in Translational Therapeutics, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Department of Surgery, The Ohio State University, Columbus, OH, USA Ioannis  A.  Ziogas  Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA

Contributors

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The Intestinal Tumour Microenvironment J. K. H. Leman, L. Munoz-Erazo, and R. A. Kemp

Abstract

Keywords

The tumour microenvironment (TME) of intestinal tumours is highly complex and comprises a network of stromal cells, tumour cells, immune cells and fibroblasts, as well as microorganisms. The tumour location, environmental factors and the tumour cells themselves influence the cells within the TME.  Immune cells can destroy tumour cells and are associated with better patient prognosis and response to therapy; however, immune cells are highly plastic and easily influenced to instead promote tumour growth. The interaction between local immune cells and the microbiome can lead to progression or regression of intestinal tumours. In this chapter, we will discuss how tumour development and progression can influence, and be influenced by, the microenvironment surrounding it, focusing on immune and fibroblastic cells, and the intestinal microbiota, particularly in the context of colorectal cancer.

Angiogenesis · Colorectal cancer · Immune cell · Fibroblast · Intestines · Macrophage · Microbiota · Microsatellite instability · Natural killer cell · Neutrophil · T cell · Tumour

J. K. H. Leman · R. A. Kemp (*) Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand e-mail: [email protected] L. Munoz-Erazo Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand Maurice Wilkins Centre for Biodiscovery, Auckland, New Zealand

Abbreviations AJCC

American Joint Committee on Cancer APC Adenomatous polyposis coli BLIMP-1 B lymphocyte-induced maturation protein 1 B-RAF Rapidly accelerated fibrosarcoma BTF Bacteroides fragilis toxin CAF Cancer-associated fibroblast CCL C-C motif chemokine ligand CCR C-C motif chemokine receptor CD Cluster of differentiation CIN Chromosomal instability CMS Consensus molecular subtypes CRC Colorectal cancer CTLA-4 Cytotoxic T lymphocyte-associated antigen 4 CXCR2 C-X-C chemokine receptor 2 DC Dendritic cell dMMR Mismatch repair deficient DFS Disease-free survival

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_1

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J. K. H. Leman et al.

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ECM EGF EGFR

Extracellular matrix Epidermal growth factor Epidermal growth factor receptor FADa Adhesin A FAP Fibroblast activation protein FOXP3 Forkhead box P3 GEF Guanine nucleotide exchange factor GLUT1 Glucose transporter 1 IFNγ Interferon gamma IL- Interleukin iNOS Inducible nitric oxide synthase KRAS Kirsten rat sarcoma viral oncogene homolog LCRC Left-sided colorectal cancer MHC Major histocompatibility complex MMP Matrix metalloprotease MMR Mismatch repair MSI Microsatellite instability NK Natural killer OS Overall survival p53 Tumour protein 53 PBMC Peripheral blood mononuclear cell PD-1 Programmed cell death protein 1 PD-L1 Programmed death-ligand 1 PIK3CA Phosphatidylinositol-4,5-­ bisphosphate 3-kinase catalytic subunit alpha RCRC Right-sided colorectal cancer ROS Reactive oxygen species SCFA Short-chain fatty acids TAM Tumour-associated macrophages TAN Tumour-associated neutrophil TCR T-cell receptor TGFα Transforming growth factor alpha TGFβ Transforming growth factor beta TIAM1 T-lymphoma invasion and metastasis-­inducing protein 1 TIGIT T-cell immunoreceptor with Ig and ITIM domains TME Tumour microenvironment TNM Tumour node metastasis Treg Regulatory T cell VCAM Vascular cell adhesion molecule VEGF Vascular endothelial growth factor αSMA Alpha smooth muscle actin

The intestine is exposed to a variety of environmental factors and exogenous antigens from food and commensal microbes. Genetic and environmental factors, such as diet, contribute to a high degree of heterogeneity in intestinal tumours. The enteral immune system is specialised to neutralise pathogens while maintaining tolerance to food and commensal microbial antigens [1]. Maintenance of homeostasis requires many layers of regulation from immune cells and from the epithelial colonocytes. Intestinal tumours usually develop sporadically, and interactions between the tumour and other cells and molecules of the intestine can influence tumour progression. The cells and molecules surrounding tumour cells are known as the tumour microenvironment (TME) and influence the growth of tumour cells. Understanding how interactions of the cells and molecules in the TME interact could improve prognostic accuracy and treatment choice for individuals with intestinal cancers such as colorectal cancer (CRC). Many of the mechanisms of interaction between the tumour cells and the TME still need to be resolved in order to better predict response to treatment and target tumour cells with the most effective therapies. Because cancers of the small intestine are relatively rare compared to colorectal cancer, fewer molecular characterisations have been performed, and so we have focused on tumours which develop in the colon and rectum. In this chapter, the role of the TME in the progression of intestinal cancers will be discussed, with particular focus on CRC.

1.1

Physiology and Function of the Intestines

The small intestine is the part of the gastrointestinal tract between the stomach and the colon and comprises three sections: the duodenum, the jejunum and the ileum (Fig.  1.1). The small intestine absorbs the majority of the nutrients consumed (reviewed in Schofield et  al. [2]). In

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Fig. 1.1  The anatomy of the small intestine and the colon

order to absorb these nutrients, the surface area of the mucosa in the small intestine is increased by intestinal villi, which project into the lumen. The colon is the structure which continues from the jejunum down to the rectum (Fig. 1.1). The colon reabsorbs water from undigested food, passes waste from the body and is home to a large bacterial ecosystem (reviewed in Jandhyala et  al. [3]). Bacteria digest products passing through the intestines and produce essential vitamins such as vitamin K (reviewed in Jandhyala et  al. [3]). The colon has four sections: the ascending colon on the right-hand side of the body, the transverse colon, the descending colon and the sigmoid colon, which then becomes the rectum (Fig. 1.1). In the ileal epithelium, aggregations of lymphoid tissue known as Peyer’s patches enable immune surveillance of the intestine. Because each section of the intestine is exposed to different types of antigen at different concentrations, the immune system is specialised to tolerate different types of antigen in the different intestinal regions [1, 4]. The antigens, immune cells, microbial community and intestinal cells themselves vary between each region of the intestine, and so tumours originating in various sections of the intestine have different characteristics [5–7].

1.2

Intestinal Cancers

1.2.1 Staging of Intestinal Tumours Small intestine and colorectal cancers are staged using the clinical American Joint Committee on Cancer (AJCC) tumour node metastasis (TNM) system. This system classifies tumours based on the extent of growth. Tumours restricted to the epithelium, which have not penetrated deeper tissues, are classified as AJCC stage 0. Tumour invasion into the lamina propria, submucosa and serosa is used to determine stages I and II. Invasion of tumour cells to the lymph nodes is classified as stage III tumours, and if the tumour has metastasised to other organs, it is classified as a stage IV tumour. Tumours of a higher stage are associated with worse prognosis for the patient (reviewed in Compton [8]). The AJCC TNM staging is used to make treatment decisions and can give an indication of patient outcome (reviewed in Compton [8]). Staging does not always accurately predict tumour growth and response to treatment. For most individuals with stage II CRC, surgical removal of the tumour is curative, but between 20 and 40% of these patients suffer relapse within five years of surgical resection and may have benefited from treatment with adjuvant chemo-

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therapy [9, 10]. Differentiating between highand low-risk stage II patients remains challenging. The cells and molecules of the TME can directly promote or impede the growth and spread of tumour cells and the response to treatment. Methods to incorporate these cells into existing staging methods are currently being developed. For example, a worldwide validation of the Immunoscore, which is used to predict outcome based on the frequency of T cells infiltrating colorectal tumours, showed that it can be additive to current TNM staging in predicting outcomes for patients with CRC [11].

1.2.2 Discrepancy Between Large and Small Intestine Tumours are 40–50 times more likely to develop in the colon than in the small intestine [12]. Small intestine cancer is rare, accounting for only two percent of gastrointestinal cancer cases in the United States [13]. The reason for the disparity in occurrence of small bowel cancers compared to colon cancers is not known, but several hypotheses have been developed which implicate exposure time to food and different microbial communities in the development of tumours [6]. The mucosa of the small intestine is exposed to food and potential carcinogens for less time than the colon. The material is passed through more quickly than in the colon due to the action of the muscles encircling the mucosa (reviewed in Neugut et  al. [14]). There is also a much lower bacterial presence in the small intestine, which reduces the risk of exposure to carcinogenic organisms (reviewed in Pan and Morrison [15]).

1.2.3 Genetic Mechanisms of Tumour Development Cancers can arise in any section of the intestinal tract, usually beginning as a benign polyp, which may develop into a malignant tumour over time (reviewed in Manne et  al. [7]). The majority of colorectal tumours are sporadic in origin rather than caused by heritable genetic factors (reviewed

J. K. H. Leman et al.

in Mundade et  al. [16]). Inflammation has also been implicated in CRC development as inflammatory bowel diseases are a risk factor for CRC (reviewed in Axelrad et al. [17]). Overall mutation burden is a marker of good prognosis in CRC and has been linked with immunogenicity [18, 19]. Six key mutations in genes involved in regulation of cell cycle have been identified as driver mutations in CRC [20]. These are adenomatous polyposis coli (APC), rapidly accelerated fibrosarcoma (BRAF), phosphatidylinositol-­4,5-­ bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Kirsten rat sarcoma viral oncogene homolog (KRAS), SMAD4 and tumour protein 53 (p53). Mutations in p53, KRAS and SMAD4 have also been associated with metastasis in CRC [20]. Along with acquisition of driver mutations, two pathways for tumour development in CRC have been identified (reviewed in Armaghany et al. [21]). These are the chromosomal instability (CIN) pathway and the microsatellite instability (MSI) pathway (Fig. 1.2). An accumulation of mutations in genes, which suppress tumour development, and epigenetic changes are associated with CRC tumour development in both pathways (reviewed in Armaghany et  al. [21]) (Fig. 1.2). The CIN pathway can be caused by mutations in several of the driver genes mentioned above, with numerous studies reporting that mutations in APC promote CIN in CRC, as APC contributes to cytoskeletal regulation (reviewed in Pino and Chung [22]) (Fig. 1.2). Transforming growth factor beta (TGFβ), a multifunctional cytokine with a suppressive effect on multiple immune cell lineages, has also been suggested to contribute to chromosomal instability as introduction of TGFβ to culture media of cervical epithelial cell lines resulted in chromosomal structural aberrations and end-to-end fusing of chromosomes, which may indicate a role for TGFβ in CIN tumour development [23] (Fig. 1.2). Microsatellite instability occurs when DNA mismatch repair (MMR) mechanisms are impaired (Fig. 1.2). Tumours which are mismatch repair deficient (dMMR), leading to microsatellite instability, acquire mutations at a higher rate than tumours which are microsatellite stable

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Fig. 1.2  Genetic pathways of tumorigenesis in colorectal cancer. Mutations in APC often result in CIN tumours, and TGFβ has been implicated in this process. Mutations in DNA mismatch repair mechanisms (MMR) can pro-

mote the development of MSI tumours in the colon. An accumulation of mutations and epigenetic changes is associated with tumour development

(reviewed in Yuza et al. [24]). Microsatellites are repetitive regions of DNA which are prone to mutation, and deficiencies in MMR cause these mutations to become fixed. MSIhigh tumours are thought to be more immunogenic due to the accumulation of frameshift mutations, which generate peptides to which the immune system can respond [19]. Tumours which have many mutations in the microsatellite regions of the DNA (MSIhigh) are often infiltrated by immune cells to a high level, and this is associated with improved disease-free survival [25, 26].

ences could be due to differences between the intestinal cells of the left and right bowel or to differences in the antigens, such as bacteria and bile acids, to which the cells are exposed [27]. There is evidence that the immune system and the microbial communities vary along the length of the colon [1, 6]. A study by Dejea et  al. [6] showed that bacterial biofilms associated with RCRCs are at a much higher frequency than with LCRCs [6].

1.2.4 Left- and Right-Sided Colorectal Tumours The relationship between the side of the bowel in which the tumour develops and the characteristics of the TME is not well understood. Generally, tumours originating on the right-hand side of the bowel (RCRC; ascending) are more likely to be MSIhigh, more heavily infiltrated by immune cells, and thus more responsive to immunotherapies than tumours originating in the left-sided colon (LCRC; descending) [5, 15]. Gene expression  studies of right-side and left-side colon biopsies revealed distinct expression profiles, and the researchers noted higher transcriptional activity in the descending colon [27]. These differ-

1.2.5 Consensus Molecular Subtypes of CRC Defined in Part by the TME Conventional TNM staging does not capture the molecular heterogeneity of CRC tumours and therefore does not always predict the tumour growth or response to treatment [9]. For this reason, the CRC consensus molecular subtypes (CMS) were recently defined, using gene expression data from multiple cohorts of CRC patients [28]. Guinney et al. [28] defined four CMS subtypes (CMS1–4) using mutation status and enrichment of signalling pathways both within the tumour and in  the surrounding cells. These subtypes were associated with distinct outcomes for patients. Many of the genes which were used to define tumour subtypes were expressed by

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cells in the TME, which shows the relevance of the factors surrounding the tumour cells in the progression and outcome of the disease. CMS1 tumours, known as ‘MSI immune’, are characterised as MSIhigh and are enriched for genes involved in immune activation and infiltration, which indicates that immune cells are an important part of the TME of CMS1 tumours. Immune infiltration and activity coincide with microsatellite instability in CRC tumours, and MSIhigh patients are more responsive to immunotherapies than patients with microsatellite stable tumours [25, 26, 29]. However, patients with metastatic CMS1 tumours were less responsive to other types of therapy, such as anti-epidermal growth factor receptor (EGFR) therapy, than individuals with tumours of other subtypes. Patients with CMS2 tumours had significantly longer overall survival (OS) than patients with CMS1 tumours treated with  anti-EGFR [30]. Therapies that block EGFR and its downstream signalling are used for some CRC patients with metastatic disease, but it is not yet clear what factors predict response to treatment. EGFR is expressed by most CRC tumours, and ligands such as EGF and TGFα bind to activate EGFR signalling. EGFR signalling activates proteins involved in promoting cell survival and proliferation, which may enhance tumour cell growth. While it appears that immune cells in the TME contribute to the response to immunotherapy, it is not clear whether factors in the TME make patients with CMS1 tumours less responsive to anti-EGFR treatment [31]. CMS2 tumours (‘canonical’) are characterised by activation of MYC and WNT pathways and usually develop in the descending colon and rectum (LCRC). WNT promotes proliferation and a stemlike phenotype in the epithelial stem cells at the base of each crypt (reviewed in Schatoff et al. [32]). The Cancer Genome Atlas Network found mutations in the WNT signalling pathway in 93% of colorectal cancers [33]. A study of murine colorectal cancer showed that tumour-derived WNT proteins inhibited the antitumour T-cell response [34]. WNT inhibited cluster of differentiation (CD)4  T-cell production of IFNγ but enhanced the production of

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interleukin (IL)-17, which may promote tumour progression in CRC [34]. Interferon gamma (IFNγ) is a potent antitumour cytokine (reviewed in Kosmidis et  al. [35]). MYC signalling is largely regulated by c-Myc, a phosphoprotein and can act as a promoter or repressor of genes involved in cell cycle, survival, adhesion and protein synthesis (reviewed in Dang et al. [36]). MYC activation promotes expression of the immunosuppressive molecules CD47 and programmed death-ligand 1 (PD-L1). CD47 acts as an anti-phagocytosis signal to macrophages and has been shown to impair antitumour T-cell activity in head and neck squamous cell carcinoma [37]. PD-L1 is a ligand for programmed cell death protein 1 (PD-1), a receptor expressed by T cells that dampens T-cell activation and function [38]. The level of MYC expression correlated to PD-L1 and CD47 expression and MYC  bound to the promoters of the genes for PD-L1 and CD47 in both mouse and human cell lines [39]. Knockdown of MYC resulted in enhanced CD4+ T cell recruitment to the tumour and overall survival [39]. This recruitment was dependent on PD-L1 and CD47 expression, as constitutive expression of CD47 or PD-L1 by the tumour cells blocked tumour regression [39]. In CRC, the association between c-Myc expression and patient survival in CRC remains controversial, as discussed in a meta-analysis by He et al. [40]. CMS3 (‘metabolic’) tumours are characterised by metabolic dysregulation, as they are enriched in several pathways involved in metabolism of various sugars, amino acids and lipids [28]. Mutations in the KRAS gene were also overrepresented in CMS3 tumours. Mutations in KRAS promote metabolic resilience in CRC. KRAS-mutated CRC cell lines displayed enhanced glucose uptake and survival in low-­ glucose conditions due to upregulation of glucose transporter 1 (GLUT1) [41]. KRAS mutations also promoted metabolic flexibility with regard to amino acid use and enabled adaptation to low-glutamine conditions through upregulation of asparagine biosynthesis [42]. CMS4 (‘mesenchymal’) tumours are defined by several TME-related characteristics. CMS4

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tumours are enriched for pathways involved in stromal infiltration, TGFβ activation, matrix remodelling pathways, angiogenesis and the complement inflammatory system [28]. Individuals with CMS4 tumours were more likely to have worse relapse-free and overall survival and were associated with a more advanced TNM stage than individuals with CMS1–3 tumours [28]. TGFβ activation and angiogenesis are likely to contribute to the worse prognosis of individuals with CMS4 tumours (discussed in detail below).

1.3

Immune Contribution to TME

The immune system is an important part of the TME.  Immune cells such as T cells, macrophages, natural killer (NK) cells and neutrophils infiltrate tumours and destroy tumour cells as part of immunosurveillance. Tumour cells and the cells of the TME can ‘programme’ immune cells into tumour-permissive or tumour-promoting phenotypes. Immune cells are highly plastic, and changes in the cytokine milieu or metabolic environment affect immune cell phenotype and function (reviewed in Leman et  al. [43]). Infiltration of immune cells is associated with both good and poor prognosis in some cancers, but the relationships between immune cells, tumour cells and TME factors are highly complex and situation-­dependent. Thus, it is important to consider the frequency and phenotypes of immune cells present in a tumour when assessing the TME [44].

1.3.1 T Cells T cells recognise tumour antigen, presented by antigen-presenting cells on major histocompatibility complex (MHC), through the T-cell receptor (TCR) (Fig.  1.3). Peptide-MHC interactions activate T cells. Activated T cells can kill the tumour directly, through the production of cytotoxic molecules such as perforin and granzyme, or recruit other immune cells to the site by production of inflammatory cytokines. A high infil-

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tration of CD3+ and CD8+ T cells into colorectal tumours at the invasive margin and centre of the tumour has been  associated with improved disease-­free survival (DFS) [11, 44] (Fig.  1.3). The Immunoscore was developed to quantify the T-cell infiltrate and improved prognostic accuracy compared to conventional staging [11]. As T cells are heterogeneous and plastic, and some subsets are not associated with improved DFS, quantification of specific T-cell subsets could improve the accuracy of Immunoscore ([45]; reviewed in Leman et al. [43]). Regulatory T cells (Tregs) are generally defined by the expression of the transcription factor forkhead box P3 (FOXP3) and suppress immune responses mainly through the inhibition of antigen-presenting cell activity, sequestration of IL-2 and the production of IL-10 (reviewed in Schmidt et al. [46]). There are multiple studies of the role of Tregs in CRC. A high infiltration of FOXP3+ cells into colorectal tumours was associated with poor DFS in some studies [47], but in other studies, it was associated with favourable outcome for CRC patients [48]. However, inclusion of the transcription factor B lymphocyte-­induced maturation protein 1 (BLIMP-1) into analysis of the relationship between Tregs and prognosis revealed that BLIMP-1+ FOXP3+ Tregs were associated with longer DFS in patients with stage II CRC [45] (Fig. 1.3). T-cell responses can be inhibited by negative regulatory pathways that are exploited by the tumour. Tumour cells can express ligands to receptors that inhibit TCR signalling and reduce T-cell cytokine production and proliferation (Fig.  1.3). A high ratio of cells expressing the inhibitory receptor PD-1 to CD8 cells, correlated with poor relapse-free and overall survival rates in CRC patients [49]. Checkpoint blockade therapies target inhibitory interactions between receptors expressed by T cells, such as PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and their ligands expressed by cancer cells. These therapies have shown success in treating patients with MSIhigh CRC, but not those with microsatellite stable tumours, which indicates that factors other than MSI status may be

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Fig. 1.3  Interactions between tumour cells, immune cells and microbiota in the TME of colorectal tumours. Macrophages can modify the ECM of colorectal tumours to enhance tumour cell invasion. CD68+ macrophages are associated with apoptosis at the tumour margin. High infiltrates of BLIMP-1+ FOXP3+ Tregs and CD3+CD8+ T cells into colorectal tumours are associated with increased DFS.  Tumour cells can inhibit T-cell responses by expressing inhibitory ligands. The antitumour T-cell response requires activation with tumour peptide, presented by an antigen-presenting cell, such as a DC, in the context of MHC.  NK cells can prime DCs by secreting cytokines and can also directly kill tumour cells. However,

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NK cells and T cells can be inhibited by FAP2-expressing F. nucleatum. The bacteria which make up the microbiota ferment dietary fibre to produce SCFA such as butyrate. Butyrate induces Treg development and downregulates IL-6 and IL-12 production by macrophages. Primary bile acids are secreted by the host to enhance fat metabolism, and a high-fat diet is associated with increased bile acids. Bacteria deconjugate bile acids, and secondary bile acids have been shown to promote invasion and proliferation in epithelial cells, which could contribute to CRC progression. The role of neutrophils in CRC is currently being investigated and is likely dependent on phenotype and location

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associated with a functional T-cell response. The expression of multiple inhibitory receptors could limit the efficacy of single checkpoint blockade inhibitors. The efficacy of anti-CTLA-4 and ­anti-­PD-­1 blockade for stage II/III CRC patients is currently being tested (reviewed in Arora and Mahalingam [50]).

1.3.2 Tumour-Associated Macrophages The relationship between macrophages and CRC progression is controversial, but it has been reported that the presence of macrophages in colorectal tumours was associated with improved patient survival [51]. A positive association between CD68+ macrophages in the invasive margin of colorectal tumours and tumour cell apoptosis indicates a direct role for macrophages in tumour killing [52] (Fig. 1.3). However, tumour-infiltrating macrophages (TAMs) are a heterogeneous and highly plastic population, and different populations of macrophages interact with tumour cells and other TME components in different ways (reviewed in Mantovani [53]). Classical categorisation of macrophages into conventionally activated M1 and alternatively activated M2 phenotypes indicated that M1-like macrophages were involved in tumour suppression and immunostimulation, whereas M2-like macrophages promoted metastasis and a more invasive phenotype (reviewed in Mantovani [53]). Further characterisation of colorectal TAMs has muddied this distinction. A study which defined colorectal M1 TAMs as inducible nitric oxide synthase (iNOS)+, and M2 macrophages as CD163+, showed that a high infiltration of M2-like CD163+ macrophages was associated with fewer lymph node metastases and a better clinical outcome, whereas M1-like iNOS+ macrophages were not. This difference  shows that there is heterogeneity in M1 and M2 macrophage subsets and that full functional characterisation is required to predict the clinical impact of macrophages in CRC [54]. Flow cytometric analysis has shown that in comparison to macrophages in the non-tumour bowel, TAMs from CRC tumours

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had a more activated phenotype, which was dependent on factors of the TME, including IL-6 [55]. There is evidence that macrophages have a role in remodelling the extracellular matrix (ECM) to promote tumour growth through the production of proteases and matricellular proteins (Fig.  1.3). Cross-linking of  the ECM enhanced tumour invasion [56]. Expression of proteases and other remodelling enzymes make the TME a more permissive environment for tumour growth. Populations of macrophages (defined in the study  as Ly6Chigh or F4/80high) infiltrated orthotopically implanted murine colorectal tumours in a C-C motif chemokine receptor type 2 (CCR2)-dependent manner. Tumours that were CCR2−/− had impaired macrophage infiltration, and tumour progression in these mice was significantly reduced [57]. TAMs from CCR2-sufficient mice expressed significantly higher levels of many proteins involved with the ECM, including several types of collagen, collagen synthesis proteins and modulators and matrix metalloproteases (MMPs), which degrade ECM components [57]. Many of the proteins identified in murine models were also enriched in human colorectal tumours compared to healthy colon. MMP-9 was expressed by a subpopulation of CD68+ macrophages in human colorectal tumours and was suggested to play a role in tumour invasion [58]. This work shows that macrophages are an influential part of the TME and can promote tumour invasion in both murine and human CRC through the expression of ECM remodelling proteins.

1.3.3 Neutrophils Neutrophils respond to invading pathogens using cytotoxic mechanisms, such as phagocytosis, and the release of reactive oxygen species (ROS) and lysozymes [59]. In cancer, tumour-associated neutrophils (TANs) have been classified in the literature into two phenotypes, N1 and N2. The N1 TANs are considered to have antitumour functions, while the N2 TANs are pro-tumour [60]. Like the M1/M2 classification for macro-

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phages, the N1/N2 has been criticised as an oversimplification of a functionally dynamic immune subset [61]. Prognostic outcome in several cancers has been predicted by quantification of the neutrophil to lymphocyte ratio, with a high ratio corresponding to worse outcome [62–64]. However, these studies investigate absolute numbers of neutrophils rather than identifying any subtypes. In CRC, a high neutrophil to lymphocyte ratio corresponds to worse overall, disease-free, recurrence-­free and diseasespecific survival [65]. In contrast, quantification of neutrophils revealed a correlation between a high number of TANs and improved patient survival in a stage II cohort of CRC patients [66]. The majority of studies of human neutrophil and CRC cell interactions have been performed in  vitro [59]. However, several studies using murine models have investigated potential effects of TANs on CRC. C-C motif chemokine ligand 15 (CCL15) expression was  associated with shorter relapse-free survival in both primary and metastatic CRC [67, 68]. One potential mechanism explaining this association is the recruitment of myeloid cells expressing CCR1, a receptor for CCL15 [69]. In mouse models, CCR1+ myeloid cells facilitated primary CRC invasion and metastasis, while knockout of CCR1  in myeloid cells resulted in reduction of both primary and metastatic tumour growth [59].

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tory NK cells are CD56bright [74, 75]. NK cells secrete cytokines to recruit and prime conventional type 1 dendritic cells (DC) towards a general antitumour response [70] (Fig.  1.3). In a cohort of post-chemotherapy rectal cancer patients, an increase in the frequency of CD56+ cells was associated  with an increase in  overall survival [76]. This finding is also supported by Li et al., who determined that, in vitro, the colorectal cancer cell line HRT-18 was susceptible to NK cytotoxic activity [77]. NK cells do not always enhance the antitumour response. TGFβ signalling can significantly reduce  proliferation, cytotoxicity and cytokine production of NK cells [78]. TGFβ levels were increased in patients with metastatic CRC compared to healthy volunteers [79]. This increase  could reduce the efficacy of NK cell antitumour responses. In addition, expression of the NK cell activation marker NKp44 was reduced on circulating CD56dim NK cells in CRC patients compared with healthy controls [75, 80]. A pro-tumour role for circulating CD56bright NK cells has been observed in CRC [81]. CD56bright NK cells from a cohort of 59 CRC patients showed elevated levels of proangiogenic vascular endothelial growth factor (VEGF) and angiogenin compared with CD56bright NK cells from healthy controls [81]. Angiogenesis is the formation of new blood vessels and is an important part of tumour growth. Actively dividing tumour cells have a high demand for nutrients and oxygen, which are supplied by the vasculature (reviewed 1.3.4 Natural Killer Cells in Rajabi and Mousa [82]). This indicates a role Natural killer cells detect and neutralise trans- for NK cells in promoting angiogenesis. NK cells are a potential target for immunoformed cells without prior neoantigen recognition [70] (Fig.  1.3). NK cells were positively therapy. In vitro, activated peripheral blood correlated with reduced prostate cancer incidence mononuclear cell (PBMC)-derived NK cells had [71]. In CRC patients, high numbers of intratu- effective tumour-killing capacity when cultured moural NK cells were associated with good prog- with colorectal cell line 3D spheroid cultures [83]. Currently, a clinical trial is underway invesnosis [72, 73]. The definition of NK cells is not consistent tigating the activity of an antibody which blocks across the literature. Broadly speaking, NK cells NK cell inhibition and enhances NK cell activity are divided into two types based on expression of against target cells in patients with squamous cell CD56, which is also expressed by NK T cells carcinoma of the head and neck [84]. Interim [74]. However, the level of CD56 expression can results report a 31% response rate in combination indicate NK cell function  –  cytotoxic effector-­ with anti-EGFR treatment, with some patients type NK cells are CD56dim and immunoregula- experiencing partial remission [84].

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1.4

Fibroblast Contribution to TME

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Fibroblasts synthesise ECM and maintain the integrity of tissue and communication between immune cells and epithelial cells (reviewed in

Tommelein et  al. [85]). Fibroblasts secrete a range of cytokines and chemokines, which attract immune cells to the TME and contribute to inflammation (reviewed in Tommelein et  al. [85]). Inflammation, defined as the accumulation of inflammatory cells, chemokines, cytokines

Fig. 1.4  Interactions of CAFs in the TME of colorectal tumours. Tumours-derived TGFβ activates fibroblasts and promotes expression of FAP and αSMA.  FAP degrades constituents of the ECM to enhance tumour cell invasion. CAFs promote chemoresistance in tumour cells by secretion of H19-containing endosomes and by promoting upregulation of TIAM1. CAF-derived IL-6 induces VEGF

secretion and enhances angiogenesis and also induces tumour cell expression of VCAM, which recruits macrophages. CAF-derived IL-8 and CCL2 induce an M2-like macrophage phenotype, and CAF-activated macrophages inhibit NK cell cytotoxicity. CAF-derived TGFβ downregulates MHC expression by DCs and is also thought to inhibit T-cell function in the TME of colorectal tumours

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and inflammatory molecules, has long been implicated in CRC development [17, 86]. Factors in the tumour can activate fibroblasts and alter their function. Tumour-derived TGFβ activates fibroblasts in the context of CRC [87] (Fig.  1.4). Other cytokines, including IL-4, IL-6, prostaglandin and insulin-like growth factor II, also promote cancer-associated fibroblast (CAF) differentiation in CRC (reviewed in Tommelein et  al. [85]). CAFs (also referred to as myofibroblasts or activated fibroblasts) were increased in colorectal tumours compared to non-tumour bowel and were enriched for genes involved in migration and invasion [88]. CAFs also expressed higher levels of activation proteins such as alpha smooth muscle actin (αSMA) and fibroblast activation protein (FAP), which have both been implicated in colorectal tumour invasion [89, 90] (Fig.  1.4). TGFβ is produced by immune cells, other CAFs and the tumour cells themselves. The role of CAFs in CRC has been reviewed recently [85, 91], so this section will discuss the role of CAFs in promoting chemoresistance, inflammation and invasion in CRC.

1.4.1 CAFs Promote Tumour Chemoresistance CAFs decrease the susceptibility of tumour cells to chemotherapeutic drugs, a process called chemoresistance (reviewed in Ziani et al. [92]). CAFs induced chemoresistance by enhancing stemness, activating WNT signalling and promoting expression of T-lymphoma invasion and metastasisinducing protein-1 (TIAM1) [93] (Fig.  1.4). TIAM1 was expressed at a higher level in the tumours of CRC patients who did not respond to chemotherapy and in cell lines which had been made resistant to chemotherapy through continuous treatment. CAF-conditioned media promoted TIAM1 expression, which enhanced chemoresistance in CRC cell lines [93]. TIAM1 is a guanine nucleotide exchange factor (GEF) and was initially shown to promote invasion of T lymphoma cells [94]. GEFs are important modulators of cell cycle and cytoskeletal activity and are often dysregulated in cancer (reviewed in Kazanietz and Caloca [95]). TIAM1 is recruited to WNT-

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responsive promoters to enhance WNT signalling, which could promote a proliferative, stemlike phenotype in colorectal tumour cells [32, 96]. CAFs can also induce chemoresistance by delivering RNA to tumour cells (Fig.  1.4). Exosomes are an important means of intercellular communication and, in the context of CRC, can deliver RNA from the cells of the TME to tumour cells. CAFs isolated from CRC patients could promote cancer cell stemness and resistance to chemotherapeutic drugs in CRC cell lines through secretion of exosomes containing the non-coding RNA H19 [97]. H19 derepressed WNT/βcatenin signalling by sequestering free strands of micro-RNA 141. In doing so, H19 promoted β-catenin production and WNT expression in CRC cell lines, which enhanced resistance to chemotherapeutic drugs both in vitro and in vivo [97].

1.4.2 CAF-Secreted Cytokines Create a Tumour-Permissive Environment CAFs interact with macrophages, NK cells, dendritic cells and T cells to enhance tumour progression (Fig.  1.4). CAF-derived CCL-2 recruited macrophages into the TME and promoted a pro-­ tumour, M2-like phenotype [98, 99]. CAFs have also been shown to recruit monocytes via IL-8 production, which binds the C-X-C chemokine receptor 2 (CXCR2) expressed by monocytes [100]. The IL-8-CXCR2 pathway also promoted an M2-like macrophage phenotype as evidenced by the upregulation of CD206 and CD163 and of IL-10 mRNA [100]. CAF-induced macrophages promoted the migration of CRC cells in vitro and also suppressed NK cell cytotoxicity (Fig.  1.4). Co-culture of NK cells with CAF-induced macrophages resulted in less cytotoxicity against a CRC cell line compared to NK cells cultured with M1 macrophages [100]. This result showed that CAFrecruited and activated macrophages could inhibit the antitumour response of other immune cells in the TME.  CAF-derived TGFβ could also inhibit the antitumour T-cell response (Fig.  1.4). CAFs secrete TGFβ, which promoted tumour cell migration in vitro [101]. TGFβ signalling downregulates

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MHCII expression by dendritic cells, along with co-stimulatory molecules CD80 and 86 (reviewed in Ziani et al. [92]) (Fig. 1.4). CAF-derived TGFβ may also directly impair T-cell function as TGFβ has been shown to inhibit cytotoxicity and promote cell death in CD8+ T cells [102, 103]. CAFs can also promote angiogenesis and tumour cell survival through the production of IL-6 [104]. CAF-derived IL-6 stimulated stromal fibroblasts to produce VEGF-A, which resulted in increased blood vessel formation [104] (Fig. 1.4). VEGF stimulates blood vessel formation and has been associated with more advanced stages of CRC [105]. CAF-derived IL-6 also acts upon tumour cells to promote macrophage recruitment. IL-6 upregulated vascular cell adhesion molecule (VCAM) on CRC tumour cells, which promoted infiltration of macrophages [100] (Fig. 1.4).

1.4.3 CAFs Promote Tumour Invasion

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system. Some species of bacteria, such as Fusobacterium nucleatum, have been implicated in tumour progression due to their prevalence in individuals with CRC and the expression of proteins which can modulate the immune response or tumour progression directly. Increasing evidence suggests that functions of the microbial community as a whole, and the metabolic products they produce, may also have an important impact on CRC (reviewed in Louis et al. [109]). Bacteria ferment the undigested fraction of the waste passing through the intestine and produce a wide range of metabolites which can modulate tumour growth and immune cell function, as well as providing essential nutrients to colonic epithelial cells (reviewed in Louis et al. [109]).

1.5.1 Bacterial Metabolites Promote or Protect Against CRC

The composition of the microbiome, and the genes expressed by the components of the microCAFs express several proteins that have been biome, respond rapidly to changes in diet. With linked to enhanced tumour invasion. TGFβ-­ change from a plant-based diet to an animal-­ activated CAFs upregulate FAP and αSMA to based diet, characterised by increased fat and promote tumour invasion in CRC [106, 107]. protein and reduced fibre, changes in the proporFAP is an endopeptidase that cleaves type 1 col- tions of genes involved in short-chain fatty acid lagen, a major constituent of the ECM, which (SCFA) and bile acid metabolism were observed enables invasion of tumour cells [108]. FAP [110]. SCFAs such as acetate and butyrate are the expression by fibroblasts enhanced migration of fermentation products of undigestible carbohyHCT116 CRC tumour cells [107]. αSMA expres- drates such as fibre and were reduced in the sion by desmin-negative myofibroblasts was switch from a plant to animal diet. Bile acids are associated with high venous invasion by tumour released to aid in fat absorption, and bile acid cells in a cohort of CRC patient tissues [90]. Like hydrolases were increased in animals on an macrophages, CAFs can modify the ECM to per- animal-­based diet [110]. mit tumour cell invasion, thus contributing to Bile acids are increased in individuals with tumour growth and spread (Fig. 1.4). high-fat diets and are altered by the microbial community of the intestines (Fig.  1.3). Altered bile acids have been implicated in CRC tumour progression. Most bile acids are reabsorbed in the 1.5 Microbial Contribution ileum, but about 10% reach the colon where they to TME are deconjugated from their taurine or glycine Bacteria contribute to the normal development groups (making them more hydrophobic) by bacand functioning of the gut, produce and alter met- terial bile salt hydrolases. Bile salt hydrolases are abolic products and are important for the devel- found in over 100 genera of bacteria in the human opment and maintenance of a functional immune gut [111]. Unconjugated, or secondary bile acids,

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can contribute to CRC progression in several ways. The increased hydrophobicity of secondary bile acids facilitated membrane disruption cell, which caused ROS production and DNA damage (reviewed in Louis et  al. [109]). Bile acids can also act as signalling molecules. While bile acids can induce apoptosis in hepatocytes and colonic epithelial cells, several studies found that low doses of bile acids could activate β-catenin and promote proliferation and invasiveness (reviewed in Barrasa et al. [112]) (Fig. 1.3). Secondary bile acids can also promote cell proliferation and stemness by binding EGFR and signalling through c-Myc [113]. The relationship between diet, bile acids and colorectal carcinogenesis is compelling but complex, and the role of the microbial community provides a key mechanistic link as to how diet can affect CRC progression. Microbial metabolites can also support the antitumour response in the colon. SCFAs produced by microbes promote homeostasis and inhibit tumour progression in the gut. One of the three main SCFAs, butyrate, promoted immune homeostasis by inducing a Treg phenotype in colonic Tregs in mice [114, 115] (Fig. 1.3). Butyrate also regulated macrophage-mediated inflammation by decreasing the inflammatory cytokines IL-6 and IL-12 [116] (Fig. 1.3). Butyrate caused apoptosis and inhibited proliferation and invasion in CRC cell lines, and butyrate-­supplemented diets were protective against CRC in rats [117–119]. This inhibition may tie the relationship between diet and risk of CRC to the bacterial metabolic processes occurring in the gut and highlights the fact that these processes can be protective against colorectal cancer development and progression.

1.5.2 B  acterial Proteins May Promote CRC Progression CRC patients with bacterial biofilms had reduced expression of E-cadherin in the crypts [6]. E-cadherin is a tumour-suppressor protein and a crucial mediator of cell-cell adhesion, a property which is greatly reduced or lost in malignant cells (reviewed in Pećina-Slaus [120]). Downregulation of E-cadherin is associated with tumour progression

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and with epithelial-mesenchymal transition; thus, E-cadherin has been annotated as a tumour-­ suppressor protein (reviewed in Christou et  al. [121]). Bacteria can directly cleave E-cadherin from epithelial cells (reviewed in [122]) (Fig.  1.3). For example, F. nucleatum, a commensal bacterium of the oral cavity, expresses Adhesion A (FADa), which binds to E-cadherin on epithelial cells. FADa binding to E-cadherin on tumour tissues promoted tumour growth and inflammatory gene expression in a mouse model of CRC [123]. Another colonic commensal, enterotoxigenic Bacteroides fragilis, is associated with early colorectal lesions and produces a toxin that interacts with E-cadherin [124]. Bacteroides fragilis toxin (BTF) promoted tumour development by cleaving E-cadherin from intestinal epithelial cells [125] (Fig. 1.3). F. nucleatum could also promote tumour progression through modulation of the antitumour immune response. F. nucleatum expresses an adhesion protein, Fap2, which has a pro-tumour immunomodulatory role in CRC (Fig. 1.3). Fap2-­ expressing F. nucleatum inhibited NK and T-cell cytotoxicity by signalling through the inhibitory receptor T-cell immunoreceptor with Ig and ITIM domains (TIGIT) [126]. Co-culture of F. nucleatum with NK cells resulted in reduced cytotoxicity against colorectal tumour cell lines. F. nucleatum also inhibited TIGIT-expressing T cells via Fap2 [126]. In numerous studies, F. nucleatum was enriched in both tumour biopsy and stool samples of CRC patients (reviewed in Jahani-Sherafat et al. [127]).

1.5.3 Two-Way Relationship: TME Influences Microbiota As with the other elements of the TME, the microbiota are not only influencers of, but are also influenced by  the TME.  Defining causal relationships in such a heterogeneous environment always presents challenges, but some associations between tumour characteristics and microbiota composition are evident. A recent study investigating the relationship between MMR and microbiome showed that MMR deficiency was the greatest contributor to change in

1  The Intestinal Tumour Microenvironment

microbiota amongst CRC patients [128]. Tumour location (proximal or distal) was the next most important factor. B. fragilis and F. nucleatum were enriched in dMMR samples and also in tumours compared to matched non-tumour bowel [128]. Fusobacteria were associated with dMMR tumours, which develop more frequently in the proximal colon. The role of Fusobacteria in tumour progression and prognosis may be affected by the location of the tumour [128].

1.6

Conclusions

The intestinal tract is a vastly complex environment, and a myriad of factors contribute to the microenvironment of tumours which develop there. Immune cells, fibroblasts and bacteria interact with each other and the tumour cells and also influence the extracellular milieu by secreting signalling molecules, remodelling the extracellular matrix and promoting blood vessel development. Immune cells and fibroblasts can be reprogrammed by the tumour cells to create a more permissive environment for tumour growth. However, immune cells can also mediate a potent antitumour effect, and thus the modulation of immune responses to enhance tumour clearance and improve prognosis for patients is becoming increasingly well studied (reviewed in Kakimi et al. [129]). Heterogeneity between patients, as a result of genetic mutation, diet and environmental factors, also affects the cells of the TME, which highlights the need for more personalised approaches to colorectal cancer treatment. Understanding how each component of the TME interacts with the tumour and the rest of the TME would help target treatments to those patients who will benefit, thus reducing the current disease burden of intestinal cancers.

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2

Gastric Tumor Microenvironment Armando Rojas, Paulina Araya, Ileana Gonzalez, and Erik Morales

Abstract

Keywords

A compelling body of evidence has demonstrated that gastric cancer has a very particular tumor microenvironment, a signature very suitable to promote tumor progression and metastasis. Recent investigations have provided new insights into the multiple molecular mechanisms, defined by genetic and epigenetic mechanisms, supporting a very active cross talk between the components of the tumor microenvironment and thus defining the fate of tumor progression. In  this review, we intend to highlight the role  of very active contributors at gastric cancer TME, particularly cancer-associated ­ fibroblasts, bone marrow-derived cells, tumor-associated macrophages, and tumorinfiltrating neutrophils, all of them surrounded by an overtime changing extracellular matrix. In addition, the very active cross talk between the components of the tumor microenvironment, defined by genetic and epigenetic mechanisms, thus defining the fate of tumor progression, is also reviewed.

Gastric cancer · Chronic inflammation · Tumor microenvironment · Helicobacter pylori · Tumor-associated macrophages · Cancer-associated fibroblasts · MicroRNAs · Exosomes · Extracellular matrix · Tumor-­ associated mast cells · Tumor-infiltrating neutrophils

A. Rojas (*) · P. Araya · I. Gonzalez · E. Morales Biomedical Research Laboratories, Medicine Faculty, Catholic University of Maule, Talca, Chile e-mail: [email protected]

2.1

Gastric Cancer

Gastric cancer (GC) is the fourth most common malignancy and the second leading cause of cancer-­related death worldwide [1]. For many years, various actions as part of public health policies were undertaken throughout the world in order to minimize the incidence and mortality rates from gastric cancer. Although both parameters have declined in several developed countries, the GC burden has remained high in several countries of Asia, Latin America, and Central and Eastern Europe [2]. Helicobacter pylori (H. pylori) infection is considered the most important risk factor of GC. In 1994, the International Agency for Research on Cancer (IARC) of the World Health Organization declared H. pylori as a group I human carcinogen [3]. It is estimated that about half of the world’s population is infected with this bacterium [4]. However, GC development cannot be only

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_2

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explained by infection with H. pylori because GC develops in only a small proportion of H. pylori-­infected people, ranging between 2% and 5% [5]. Therefore, other factors such as host-related (genetic), environmental, and bacterial virulence factors, such as CagA, VacA, and adhesins, are considered to be responsible for the disparities observed not only between populations but also between geographical regions. In the nineteenth century, Rudolph Virchow first launched the idea about a putative connection between inflammation and cancer. At present, it is estimated that as many as 20% of all cancer is somehow associated with chronic infection and inflammation [6]. In addition, several pieces of evidence derived from both epidemiological studies and basic research have even demonstrated that organ-specific carcinogenesis is linked to the chronic and local inflammatory milieu. This association is widely documented not only for the H. pylori-induced gastric inflammation and the occurrence of gastric cancer but also for prostate cancer [7], colon cancer [8], and gallbladder carcinoma [9]. After many years of intensive research, our understanding on how a tumor develops has dramatically changed, starting from a very simple view of a malignant cell itself to a very complex niche, termed tumor microenvironment (TME). Tumor cells are part of a dynamic network composed of extracellular matrix (ECM), stromal cells, as well as immune and inflammatory cells that drive cancer cells fate and where all these components cross talk through a huge reciprocal exchange of molecular information [10]. Of note, this very particular niche is efficiently manipulated by tumor cells, not only to favor the recruitment of cells but also and more importantly for reeducating various cell types, thus rendering a tumor-supporting microenvironment. In the particular case of gastric cancer, we intended in this review to summarize the particular contributions of stromal cells, particularly immune cells and carcinoma-associated fibroblasts; changes in the extracellular matrix; as well as different novel actors in the complex network

of signals, such as exosomes, microRNAs, and a deregulated cellular signaling. Altogether, these actors markedly support the dampening of the immune response as well as tumor growth and metastasis (see Fig. 2.1).

2.2

Stromal Cells

2.2.1 B  one Marrow-Derived Cells (BMDCs) For many years, epithelial cancers were thought to be originated from the transformation of tissue stem cells. Stem cells, due to their fundamental properties of longevity and self-renewal, appear to be the ideal cellular targets for the accumulation of genetic alterations. In fact, gastric epithelial homeostasis is known to be maintained by long-lived stem cells surrounded by a supportive stem cell niche. Therefore, gastric cancer may arise from stem cells that have accumulated gene mutations and their subsequent expansion [11]. Of note, gastric cancer is strongly linked to chronic inflammation caused by H. pylori infection. At present, BMDCs, which are the most primitive uncommitted adult stem cell, seem to be the ideal candidate for transformation if they are recruited into a chronically inflamed tissue, based on the pioneering studies employing mouse models of H. pylori-induced gastric cancer [12, 13]. The underlying mechanisms involved in the recruitment of BMDCs from the circulation seem to be mediated by the H. pylori-associated chronic inflammation, which results in the upregulation of proinflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor-α, and chemokines such as CXCL12 (also known as SDF-1α), which is known to contribute to the recruitment of progenitors [14–16]. Furthermore, the contribution of BMDCs is now recognized not only in the generation of gastric cancer stem cells but also in the formation of bone marrow-derived endothelial progenitor cells, thus contributing to angiogenesis in tumor formation [17, 18] as well as to the generation of cancer-associated fibroblasts [19].

2  Gastric Tumor Microenvironment

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Fig. 2.1  Compelling evidence supports the contribution of gastric cancer microenvironment to tumor growth and metastasis development. The active participation of many tumor stroma cells, the remodeling of extracellular matrix

in both the tumor niche and the surrounding space, and the establishment of very complex and aberrant signaling networks delineate the well-known aggressive tumor biology of this malignancy

2.2.2 T  umor-Associated Mast Cells (TAMCs)

[27]. This finding deserves particular attention considering that the role of the Th17 population in gastric cancer has remained controversial [28, 29]. It is known that IL-17 is a proinflammatory factor, which promotes angiogenesis [30, 31]. However, mast cells are able to release angiogenic mediators, other than IL-17, in the tumor microenvironment, such as the vascular endothelial growth factor (VEGF) and the fibroblast growth factor-2 (FGF-2). Furthermore, other mediators released from the mast cell secretory granules such as the serine proteases, tryptases, and chymases [32, 33] have potent proangiogenic activity. The capacity of tryptases to induce angiogenesis in tumors is particularly interesting, considering not only that they are the most abundant enzyme in MCs but also the wide range of other biological activities relevant to local angiogenesis and tumor progression [34].

Tumor-associated mast cells (TAMCs) have been reported as part of the cellular stroma in many human solid and hematological tumors, including gastric cancer [20–22]. These infiltrating cells produce profound effects at tumor microenvironment, supporting not only tumor progression [23], immunosuppression [24], and angiogenesis [25], but also they actively participate in remodeling the tumor microenvironment [26]. This cell population seems to be recruited into the tumor microenvironment through the CXCL12-CXCR4 chemotaxis axis [24]. Noteworthy, a recent report showed that most IL-17-producing cells at the tumor microenvironment in gastric cancer patients were mast cells

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2.2.3 Cancer-Associated Fibroblasts (CAFs)

2.2.4 Tumor-Associated Macrophages (TAMs)

Most tumors are associated with a biologically active type of fibroblasts known as cancer-­ associated fibroblasts (CAFs), which are the most prominent components of the tumor microenvironment and play important roles in gastric cancer growth and progression [35–38]. CAF also represents an active source of a myriad of biological mediators supporting, by several mechanisms, tumor growth, angiogenesis, invasion, and metastasis. These mediators include multiple metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and MMP-14), growth factors such as TGF-β, VEGF, as well as chemokines and cytokines such as CXCL12 (SDF-1), CXCL14, CXCL16, CCL2, CCL5, IL-4, and IL-6, among many others [39, 40]. Very recently, IL-6 secreted by CAFs was reported to play an important role in the progression of gastric cancer, by promoting migration and epithelial to mesenchymal transition (EMT) of gastric cancer cells, by a mechanism dependent on the activation of JAK2-STAT3 signaling pathway [41]. Noteworthy, CAFs also act as guides for stromal dissemination, by generating ECM tracks that pave the way for the collective invasion of the cancer cells that have not undergone a full EMT [42]. CAFs are also key actors in sculpturing an immunosuppressive tumor microenvironment. The IL-6 produced by CAFs restricts the maturation of dendritic cells and redirects monocytes toward macrophage differentiation [43]. Additionally, they actively participate not only in the recruitment of macrophages into the TME but also in the phenotypic polarization process toward the M2 phenotype [44]. Through secretion of TGF-β and other cytokines, the immunosuppressive role of CAFs has been shown to affect immune response in TME, either by the recruitment of both regulatory T cells and myeloid-derived suppressor cells or by inhibiting the activity of both natural killer (NK) and T cells in the TME [45].

It is known that infiltrating macrophages may represent up to 50% of the total tumor mass; therefore, they must be considered as a very relevant actor in the biology of the tumor microenvironment [46, 47]. TAMs are a very heterogeneous population, and once infiltrated, they differentiate into a continuum of specialized phenotypes whose extremes are described as proinflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes [48]. It is widely accepted that TAMs bearing an M2 phenotype have mostly protumoral functions, promoting tumor cell survival, proliferation, and dissemination [49]. Interestingly, transcriptional reprogramming is an important mechanism for signal integration and cell function of polarized macrophages, demonstrating that marked changes are produced in both cellular proteome and transcriptome during macrophage polarization [50, 51]. Furthermore, marked changes are also produced in “canonical” signaling pathways where skewed signaling pathways are triggering to reinforce the protumoral activities of M2 macrophages based on their abilities to enhance tumor growth and invasion and promoting angiogenesis [52–54]. TAMs in the peritoneal cavity of gastric cancer patients with peritoneal dissemination are reported to be M2 phenotype, and they contributed to the development of peritoneal dissemination via activation of the EGFR signaling pathways [55]. Although the predictive value of TAMs density remained controversial for many years, recent reports have reinforced the notion that the amount of TAMs in tumor stroma predicts the size, the stage, and the metastasis of a gastric tumor, suggesting that TAMs may be useful for risk-patient screening, early diagnosis, and prognosis [56–58].

2  Gastric Tumor Microenvironment

2.2.5 Tumor-Infiltrating Neutrophils (TINs) Neutrophils are very abundant at gastric cancer microenvironment, and their role in tumor progression has remained controversial. However, an emerging body of evidence supports that this cell population has important functions in the tumor microenvironment. Due to the myriad of factors produced and released by neutrophils, they promote carcinogenesis, tumor growth, and metastasis as well as angiogenesis and immunosuppression by several mechanisms [59–64]. Once infiltrated, and in analogy to macrophage polarization, neutrophils are reported to be polarized into N1 and N2 phenotypes. This condition may be an explanation of the controversial roles of TINs in TME when considered as a whole population without their polarized phenotypes [65]. In the particular case of gastric cancer, recent findings have revealed that TINs promote gastric cancer cell migration and invasion through the activation of the ERK pathway, as well as the induction of EMT, indicating that neutrophils may play an important role in gastric cancer metastasis [66]. It is well known that neutrophils are an important source of IL-17a in the setting of inflammation. This cytokine is widely found in the inflammatory microenvironment of various tumors, including GC, where it supports tumor progression and metastasis [67]. Noteworthy, a very recent report showed that TIN-derived IL-17a also promotes EMT of GC cells through JAK2/STAT3 signaling [68]. Additionally, TINs also play an active role in the immunosuppression at TME by suppressing T-cell proliferation, as well as IFN-γ production through a PD-L1–dependent mechanism. The expression of PD-L1 is markedly induced by gastric cancer cells-derived GM-CSF [69]. Finally, TIN density seems to correlate with gastric cancer outcomes, and it has been suggested as a useful independent prognostic factor to be considered in any predictive model to stratify patients with different prognosis [70].

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2.3

Extracellular Matrix

Far beyond the initial perception of ECM, as a provider of the mechanical and structural support needed for tissue functions, it is now considered as a physiologically active component of living tissue, regulating cell-cell communication, cell adhesion, and cell proliferation, and its dysregulation contributes to neoplastic progression [71]. ECM is a very dynamic niche, constantly undergoing a remodeling process, by which components are degraded and modified, and thus providing a fine-tuning control of elasticity and compressive/tensile strength in tissues [72]. Degradation of the extracellular matrix (ECM) and basement membrane (BM) barriers is an essential step in the pathology of gastric cancer. This process is mediated by the actions of metalloproteinases (MMPs), which are produced by various cells, including leukocytes, macrophages, endothelial cells, and fibroblasts, as well as by tumor cells. The balance between the expression of MMPs and their tissue inhibitor (TIMPs) plays a critical role in maintaining the degradation and synthesis of ECM.  Loss of this balance may facilitate the growth and progression of tumor cells [73–75]. An intensive tumor-associated ECM remodeling leading to the stiffening of the tumor microenvironment is a hallmark in cancer progression. This remodeling is mainly mediated by ECM deposition, fiber alignment, and cross-linking, which in turn is able to promote tumor progression and malignancy, as well as to induce highly invasive cell phenotypes, and to promote transforming growth factor (TGF)-β-induced epithelial-­mesenchymal transition (EMT) [76]. Actually, a growing body of evidence indicates that selected MMPs and TIMPs play a crucial role in tumor growth, angiogenesis, and metastasis of gastric cancer [77]. Collagen I is a very abundant component of the extracellular matrix in gastric cancer, which is additionally cross-linked via enzyme- or nonenzyme-­mediated processes, thus enhancing matrix stiffness. As a consequence, the disruption of E-cadherin/β-catenin complex disruption is produced by a FAK signaling-dependent mechanism [78].

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Noteworthy, β-catenin accumulation within the nucleus or cytoplasm has been implicated in increased tumorigenesis, poor clinical results, and even with chemoresistance in gastric cancer [79]. The biomechanical properties of ECM are also tightly controlled by cross-linking, which in turn support tumor growth and development [80, 81]. Lysyl oxidases (LOX) are a family of copper-­ dependent amine oxidases, which modify collagens and elastin in the ECM, thereby catalyzing the covalent cross-linking of collagen fibers and thus increasing stiffness and tensile strength [82]. Noteworthy, LOX expression is associated with the high invasion activity of gastric cancer under hypoxic conditions [83]. Considering all data derived from clinical and experimental approaches, it is suggested that LOX expression may be a helpful predictive factor for patients with GC [84].

Many chemotherapeutic drugs can induce autophagy, as tumor response to the toxic insult, thus contributing to the development of acquired resistance to the pharmacological agent. Noteworthy, some miRNAs are capable of regulating autophagy by controlling the levels of masterpieces in the autophagy process, such as mTOR signaling [91, 92]. Finally, and in spite of multiple research efforts, the complex network of miRNAs in gastric tumor microenvironment deserves new initiatives for the full understanding of their impact on gastric cancer tumor biology.

2.5

Exosomes

Far beyond of the initial consideration of exosomes as garbage bins, where these small ­ cell-­derived vesicles were just involved in the removing of unnecessary proteins and other mol2.4 MicroRNAs ecules from the releasing cells [93], exosomes actively participate in the transfer of information The miRNAs have emerged as one of the most within the tumor microenvironment. important posttranscriptional regulators of gene At present, exosomes are known to transfer expression, being master regulators of up to 30% information locally within the TME, via autoprotein-coding genes in the human genome [85]. crine signals provided by exosomes, as well as Based on this particular feature, miRNAs are systemically to distant tissue sites. able to regulate multiple signaling pathways The content of exosomes varies from proteins within the tumor microenvironment [86]. to messenger RNA (mRNA) and microRNAs. All A growing body of evidence has demonstrated these components can be transferred from donor that several miRNAs showed a differential cells into either neighbor or distant cells to proexpression profiles in gastric cancer, suggesting duce a myriad of biological effects in the target their active participation on gastric carcinogene- cells [94]. In the particular case of gastric cancer, sis. For instances, miR-17-5p acts as an oncogene a compelling body of evidence showed that they (oncomiR), thus promoting tumor growth and are critically involved in GC progression, metasinvasion. Conversely, miR-203 is a tumor sup- tasis, angiogenesis, immune evasion, and drug pressor, which is able to inhibit tumor cell prolif- resistance [95–97]. eration, migration, and invasion [87]. Changes in Peritoneal dissemination is an important cause miRNAs profiles observed in gastric cancer of morbidity and mortality among patients with appeared very early in the onset of the disease, gastric cancer, with a five-year survival rate of even in the preneoplastic lesions in H. pylori-­ only 2% [98]. Recently, exosome-dependent infected subjects [88]. molecular transfer or signaling pathway activaA growing body of evidence supports the tion is reported as a crucial process in the four active participation of miRNAs in gastric carci- stages of peritoneal dissemination of gastric nogenesis by targeting the canonical NF-κB sig- ­cancer [99]. naling pathway for directing other complex Exosomes are reported to mediate drug processes, such as the epithelial-mesenchymal ­resistance by upregulating the expression of multransition [89, 90]. tidrug resistance components, particularly that of

2  Gastric Tumor Microenvironment

ABC transporters family members [100]. Furthermore, exosomes seem to be used by cancer cells to expel drugs outside the cell [101]. Another important role of exosomes in gastric cancer microenvironment is the capacity to modulate immune response either by favoring the ­differentiation of tumor-infiltrating lymphocytes into a Th-17 phenotype [102] or by strengthening the regulatory functions of mesenchymal stem cells, through the activation of an NF-kB-­ dependent mechanism, thus supporting tumor growth [103].

2.6

Dysregulated Cellular Signaling

Although the etiology of gastric cancer is now considered as multifactorial, the H. pylori infection is a major contributor in this respect, being responsible for more than 80% of all cases [104]. Due to the inflammatory nature of host response to H. pylori infection, many signaling pathways become deregulated, particularly those of oncogenic nature. In this sense, dysregulation of cell cycle regulators, such as cyclins D1 and E2, is frequently overexpressed in early stages of gastric carcinoma [105]. These cyclins bind to CDK4/6 and CDK2, respectively, and subsequently phosphorylate retinoblastoma protein, which is required for the transition from G1 to S phase. NF-κB signaling cascade accounts as one of the important triggers in inflammation-induced carcinogenesis. Since the earliest 2000, NF-kB is known to be constitutively activated in gastric cancer tissues [106]. Interestingly, some H. pylori virulence factors such as the cytotoxin-associated gene pathogenicity island (cagPAI) are required for NF-kappaB activation. The interaction of some H. pylori outer membrane proteins with host cell surface receptors is able to trigger the activation of NF-kB signaling. For instance, the outer inflammatory protein (OipA) can activate, in a cagPAI-­ independently way, NF-κB in gastric epithelial cells [107]. The H. pylori-induced IL-1β secretion is mediated by the activation of Toll-like receptor 2

29

(TLR-2) and the subsequent inflammasome activation [108]. Other TLRs, such as TLR-4 and TLR-5, are involved in the recognition of H. pylori, and subsequent activation of the NF-kB signaling pathway, leading to the release of proinflammatory mediators [109]. The receptor of advanced glycation end-­ products (RAGE) is now classified as a pattern recognition receptor and is depicting a signaling cascade through the activation of NF-kB and mitogen-activated protein kinase (MAPK) pathways. This receptor actively participates in the adhesion of H. pylori to the gastric epithelium and mediates the release of IL-8 [110]. H. pylori infection also induces Notch signaling, which in turn induces the expression of the cyclooxygenase-2 (COX-2) through the binding of the Notch1 receptor intracellular domain to the Cox-2 promoter gene [111]. In the normal stomach, Sonic Hedgehog (Shh) regulates gastric epithelial cell differentiation and function [112]. This signaling pathway is also dysregulated in gastric cancer [113]. Increased and constitutive expression is reported in gastric carcinogenesis and where the degree of promoter methylation seems to be involved in this dysregulated expression pattern in gastric cancer [114]. Noteworthy, H. pylori induces Shh via an NF-κB-dependent mechanism in the stomach [115]. Wnt/β-catenin pathway is crucial to embryo development and adult tissue homeostasis, and dysregulation of this signaling pathway can cause uncontrolled cell growth and cell malignant transformation [116]. Of note, Wnt/β-catenin signaling also plays an active role in the self-renewal of gastric cancer stem cells (GCSC) [117]. Furthermore, Wnt/β-catenin signaling contributes to tumor progression and metastasis not only by enhancing the proliferation and invasiveness of gastric cancer cell but also by supporting epithelial-­mesenchymal transition [79, 118, 119]. The Hippo signaling pathway is a key element in the homeostasis of the gastrointestinal tissues. Dysregulation of the Hippo pathway is associated with initiation, development, and distant metastasis of GC [120]. Noteworthy, the deregulated Hippo pathway seems to actively cross talk with Wnt/β-catenin, Notch, and TGF-β signaling

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pathways, thus promoting tumorigenesis coordinately [121–123]. TGF-β signaling pathway is involved in cell growth, cell differentiation, and apoptosis. Its paradoxical roles as a tumor suppressor and tumor promoter have extensively studied and where the critical factor is the timing of its action [124, 125]. During tumor initiation, TGF-β signaling promotes cell cycle arrest and apoptosis, thereby acting as a tumor suppressor. In contrast, TGF-β has been shown to promote tumor cell proliferation, EMT, and stemlike behavior as well as fibrosis, inflammation, and angiogenesis during tumor progression [125]. In addition, an imbalance in Smad4/7 expressions, which are members of the canonical TGF signaling pathway, is associated with gastric cancer cell differentiation, metastasis, and apoptosis, suggesting that the dysregulation of TGF signaling pathways play an important role not only in GC development [126] but also in lymph node metastasis [127, 128]. Finally, another dysfunctional signaling in gastric cancer microenvironment is that associated with the epidermal growth factor family of receptors (EGFRs), which has both redundant and restricted functions, and loss of regulation of activation-mediated receptor signaling underlies many human diseases, including cancer [129]. EGFR signaling is frequently deregulated in solid tumors, including gastric cancer, leading to abnormal activation of pro-proliferative and antiapoptotic pathways [130], as well as supporting angiogenesis, tumor cell motility, and metastasis [131]. Interestingly, EGFR signaling is activated in tumor-associated endothelial cells but not in endothelial cells within a no tumor-associated niche, suggesting that both receptor activation and expression are conditioned by the tissue-­ surrounding environment [132]. Noteworthy, it has been reported that CD24 signaling contributing to malignant progression of gastric cancer has been demonstrated recently [133, 134]. CD24 is a cell adhesion glycoprotein implicated in tumor cell proliferation, and its expression seems to be a novel prognostic factor in diffuse-type gastric adenocarcinoma [135].

This molecule actively regulates EGFR signaling by preventing EGFR internalization and degradation through RhoA, as recently reported [136].

2.7

Concluding Remarks

After many years of intense research, we are now aware that the tumor is not a simple mass of tumor cells; it is located in a very complex niche named tumor microenvironment. This niche, far beyond to be considered as a bystander of how tumor develops, is undoubtedly a key element determining not only the fate of tumor development but also the therapy success. This determining capacity goes through an intensive and reciprocal communication between cellular and noncellular components at the tumor microenvironment leading to high-proliferation and metastatic capability. Therefore, this complex communication network could not be underestimated for any attempt to develop novel therapies, preferably those based on a multi-target approach to give more effective long-term outcome, considering the poor response of gastric neoplasia to various existing treatment modalities, and the low five-year survival rate for patients diagnosed with this malignancy. We should be able to develop new strategies with the aim of turning the TME into an aggressive niche for the tumor cells instead of one that supports the growth of the tumors. However, and considering that not only TME differs between cancer types but also it changes over the time, particularly in gastric cancer, many questions are still unanswered, and much more research is needed to get a more comprehensive understanding of this complex niche.

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3

Parathyroid Tumor Microenvironment Chiara Verdelli, Valentina Vaira, and Sabrina Corbetta

Abstract

Parathyroid tumors are the second most common endocrine neoplasia, and it is almost always associated with hypersecretion of the parathormone (PTH), involved in calcium homeostasis, causing primary hyperparathyroidism (PHPT). Parathyroid neoplasia has a stromal component particularly represented in atypical adenomatous and carcinomatous lesions. Recently, data about the features and the function of the parathyroid tumor microenvironment (TME) have been accumulated. Parathyroid TME includes heterogeneous cells: endothelial cells, myofibroblasts, lymphocytes and macrophages, and mesenchy-

C. Verdelli Laboratory of Experimental Endocrinology, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy V. Vaira Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy S. Corbetta (*) Department of Biomedical, Surgical and Odontoiatric Sciences, University of Milan, Milan, Italy Endocrinology and Diabetology Service, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy e-mail: [email protected]

mal stem cells have been identified, each of them presenting a phenotype consistent with tumor-­ associated cells. Parathyroid tumors overexpress proangiogenic molecules including vascular endothelial growth factor (VEGF-A), fibroblast growth factor-2 (FGF2), and angiopoietins that promote both recruitment and proliferation of endothelial cell precursors, thus resulting in a microvessel density higher than that detected in normal parathyroid glands. Moreover, parathyroid tumor endocrine cells operate multifaceted interactions with stromal cells, partly mediated by the CXCL12/CXCR4 pathway, while, at present, the immune landscape of parathyroid tumors has just begun to be investigated. Studies about TME in parathyroid adenomas provide an example of the role of TME in benign tumors, whose molecular mechanisms and functions comprehension are limited. Keywords

Parathyroid tumors · Primary hyperparathyroidism · PTH · Parathyroid adenomas · Parathyroid carcinomas · Tumor-associated myofibroblasts · Tumor-associated endothelial cells · Tumor-infiltrating lymphocytes · Tumor-associated macrophages · Collagen matrix · CXCR4 · SDF-1/CXCL12 · VEGF-A · CD34 · CD31 · α-SMA

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_3

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C. Verdelli et al.

38

3.1

Introduction

Tumor microenvironment (TME) is a dynamic heterocellular place that modulates tumor progression [1]. It comprises cells such as infiltrated immune cells, cancer-associated endothelial cells (CAEs), and cancer-associated fibroblasts (CAFs), and all the components participate in the complicated cross talk with tumor cells to affect tumor initiation and progression. Besides, TME factors such as hypoxia and inflammation have major impact on cellular heterogeneity within tumors. Experimental evidences about TME in most common solid cancers are accumulating, providing new potential therapeutic approaches for the treatment of cancers, while data about the TME features and functions in benign tumors are limited. Primary hyperparathyroidism (PHPT) is the third most common endocrine disorder, frequently complicated by bone and renal involvement, with an increased risk of fragility bone fractures and kidney stones development and increased cardiovascular mortality. PHPT is most often identified in postmenopausal women [2]. PHPT is caused by parathormone (PTH) inappropriate secretion from parathyroid neoplasia.

Fig. 3.1 Components of tumor microenvironment (TME) in parathyroid tumors. TME is very heterogeneous and comprised of various cell types, such as tumor-associated fibroblasts (TAFs), endothelial cells (TAEs), immune cells, and local and bone marrow-­ derived mesenchymal stem and progenitor cells, and surrounding extracellular matrix. Parathyroid endocrine tumor cells interact with stromal cells

Parathyroid tumors display different features spanning throughout benign adenomas, hyperplasia, atypical adenomas, and the rare carcinomas. As far as the pathogenic mechanisms of parathyroid tumorigenesis are concerned, attention has been focused so far on the proliferation of epithelial cells, though parathyroid neoplasia has a stromal component that is particularly represented in atypical adenomatous and carcinomatous lesions, similarly to what observed in most solid common cancers, such as breast and colon carcinomas. In the present chapter, experimental data about TME in parathyroid tumors will be presented. Parathyroid TME included heterogeneous cells (Fig.  3.1 and Table  3.1), and they overexpress proangiogenic molecules including vascular endothelial growth factor (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietins that promote both recruitment and proliferation of endothelial cell precursors, thus resulting in a microvessel density higher than that detected in normal parathyroid glands. Also, parathyroid tumor endocrine cells operate multifaceted interactions with stromal cells, while, at present, the immune landscape of parathyroid tumors has just begun to be investigated (Table 3.1).

3  Parathyroid Tumor Microenvironment

39

Table 3.1  Cells detected in the parathyroid tumors microenvironment, related surface markers and specific features detected in parathyroid tumors Surface cell markers Angiogenic cells CD31

Cells

Features in PTs

References

Endothelial cells

CD34

Resident/BM endothelial progenitors

Increased in PAds Two subpopulations Increased in PAds Two subpopulations

CD45 Lymphangiogenic cells LYVE-1 Inflammatory cells CD4

BM cells

More abundant in PAds

Grabmaier [19] Corbetta [20] Garcia de la Torre [14], Viacava [15], Grabmaier [19] Corbetta [20] Grabmaier [19]

Lymphatic cells

Not varied among the histotypes

De la Torre [14]

Lymphocytes

–

CD8

Lymphocytes

Increased in PAds and hyperplasia

CD8+/CD3+

TILs

Variably reduced in PCas and atypical PAds

CD68

Macrophages

PD-L1

Tumor endocrine cells

More abundant in PCas than atypical PAds Reduced in most PCas and PAds

Silva-Figueroa [25] Shi [21], Haglund [22] Shi [21], Silva-Figueroa [25] Silva-Figueroa [25] Silva-Figueroa [25], Pan [27]

Myofibroblasts a-SMA Mesenchymal stem cells CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, MHC class I

Resident/BM activated fibroblasts

Support neoangiogenesis in PAds

Verdelli [30]

Resident cells

Identified in PAds Detectable levels of telomerase activity Expression of Sall4 gene Osteogenic, chondrogenic and adipogenic differentiation potential

Shih [34]

PTs, parathyroid tumors; PAds, parathyroid adenomas; PCas, parathyroid carcinomas; BM, bone marrow; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; TILs, tumor-infiltrating lymphocytes; PD-L1, programmed death-­ ligand 1; a-SMA, a-smooth muscle actin

3.2

Parathyroid Tumors

Parathyroid tumors are the second most common endocrine neoplasia after thyroid tumors. They are mainly benign as parathyroid malignancies represent less than 1.0% of all cases of PHPT [3]. The primary cause of PHPT is a benign overgrowth of parathyroid tissue causing excessive secretion of parathyroid hormone. The molecular etiology of PHPT is incompletely defined, though oncosuppressor genes

and epigenetic deregulation have been identified. With the exception of rare double adenomas, parathyroid adenomas and carcinomas are almost always uniglandular (solitary) lesions, whereas hyperplasia represents multiglandular proliferation, which can be asymmetrical and asynchronous. The presence of an atrophic rim of hypocellular non-tumor parathyroid tissue adjacent to a cellular proliferation often allows an accurate diagnosis of “parathyroid adenoma” in

C. Verdelli et al.

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the appropriate clinical and biochemical setting. However, in the absence of an atrophic rim, hyperplasia cannot be excluded at the morphologic level. Parathyroid carcinoma is commonly sporadic, though it can be part of a familial syndrome [i.e., primary hyperparathyroidism-jaw tumor syndrome (PHPT-JT; OMIM#145001), type 1 multiple endocrine neoplasia (MEN1; OMIM#131100), type 2A multiple endocrine neoplasia (MEN2A; OMIM#171400), and familial isolated hyperparathyroidism (FIHP; OMIM#617343) [3, 4]. It is often a large mass adherent to and/or infiltrating the surrounding tissues, characterized by a variable growth pattern, from sheetlike diffuse to nest or trabecular. The criteria to diagnose unequivocally a parathyroid carcinoma are based on histology features and include: • The invasion of the capsule to the surrounding structures with an extension tonguelike of the tumor tissue through the collagenous fibers of the capsule (the entrapment of tumor cell in the capsule is defined pseudoinvasion and can be observed in parathyroid adenomas) • A true vascular invasion with the tumor cells within a vessel and at least in part attached to the wall cells • Invasion of perineural spaces • Extension to the surroundings tissues • Metastasis Parathyroid tumors lacking the unequivocal histological signs of parathyroid carcinoma, namely, capsular, vascular, and/or perineural tumor invasion, but showing some features of malignancy (i.e., fibrous bands, questionable capsular invasion, increased mitotic figures, and adherence to surrounding tissues) are defined as “atypical adenomas” [3, 5]. Atypical parathyroid adenomas rarely recur with a fiveyear recurrence-­free survival rate of 91%, while parathyroid cancers have high recurrence rate within five years with a free survival rate of 60% [6].

3.3

Microenvironment in Parathyroid Tumors

3.3.1 Parathyroid Tumor Histology Histologically, the normal parathyroid glands are bounded by a thin fibrous capsule that overlies a network of adipose tissue, blood vessels, and glandular parenchyma. The amount of stromal fibroadipose tissue increases with aging, eventually comprising approximately 50% of the gland volume in elderly [7]. Chief cells are the major cell type of the parathyroid glands in healthy subjects. Chief cells synthesize and secrete PTH to correct or maintain normal blood calcium levels, by sensing changes in extracellular calcium. Chief cells undergo ultrastructural changes corresponding to different stages of the secretory cycle. Oxyphil cells derived from chief cells as aging or some metabolic derangement have the potential to produce PTH, PTH-related peptide (PTHrP), and calcitriol (i.e., 1,25-­dihydroxyvitamin D3) [7]. TME is particularly represented in the rare most aggressive lesions of parathyroid atypical adenomas and carcinomas. The diagnosis of carcinomas considers specific features of the TME in the involved parathyroid gland, and it should be limited to tumors with clear and unequivocal signs of invasive growth, such as neoplastic infiltration of adjacent tissues (as the soft tissues or the thyroid gland), vascular invasion, perineural space invasion, and/or documented metastases (WHO 4th edition) [8]. Both parathyroid atypical adenomas and carcinomas are often characterized by the presence of broad bands of fibrous connective tissue. The banding fibrosis appears as collagenous bands originating from the fibrous capsule and subdividing the tumor in variable-­ sized nodules. In some cases, the fibrous bands may create the entrapment of neoplastic cells in the context of the capsule, simulating capsular invasion (known as pseudocapsular) [5]. A parathyroid adenoma is composed of varying proportions of chief, clear, transitional oncocytic and oncocytic cells. Various architectural patterns have been reported, including arrange-

3  Parathyroid Tumor Microenvironment

ments in cords, nests, sheets, papillary, pseudopapillary, and follicles with palisaded ­ appearance around blood vessels [9]. The proliferation pattern is often surrounded by thin stroma. Therefore, benign parathyroid adenomas show a great variability in their cell proliferation pattern, which can differ also within the same tumor. It’s worth of note that spatial heterogeneity is considered as a fundamental biological feature of the tumor microenvironment [10].

3.3.2 Tumor Angiogenesis Angiogenesis is the physiological process of neovascular formation from preexisting blood vessels, which can occur during embryogenesis, adult tissue homeostasis, and tumorigenesis [11]. There are many endogenous stimulators of angiogenesis, such as vascular endothelial growth factor A (VEGF-A). It has been shown that tumor blood vessels are deeply different from the normal ones, both physiologically and morphologically. During tumor angiogenesis, formed vessels are disorganized and leaky. Moreover, there are strong differences at the molecular level between pathological and physiological angiogenesis. Parathyroid tissue can trigger spontaneous induction of angiogenesis in in vitro and in vivo models in a VEGF-dependent manner [12]. Autotransplantated parathyroid tissue after thyroidectomy is able to form new vasculature and produces PTH, maintaining calcium homeostasis [13]. It has been observed that the neoangiogenesis is more pronounced in adenomas and hyperplastic transplants compared with normal parathyroid glands transplants [13]. A great amount of factors contributes to the process of new vessel formation in PHPT, such as VEGF, transforming growth factor-β, and angiopoietins. As far as parathyroid tumors were concerned, microvascular density, assessed as count of CD34-expressing cells by immunohistochemistry, has been investigated in 77 parathyroid adenomas, 17 PHPT-related hyperplastic parathyroid glands, and 13 normal parathyroid glands [14, 15]. Parathyroid benign proliferative lesions have higher microvascular density than normal tissue

41

and higher VEGF-A and FGF2 mRNA expression levels. Microvascular density did not correlate with the secretory status and tumor size of the parathyroid lesions. Angiogenesis is also increased in malignant parathyroid lesions compared with parathyroid adenomas. Markers of angiogenesis, including VEGF, VEGFR2, CD105, and FGF2, have been found increased in parathyroid cancer lesions compared with adenomatous lesions, though overlap between benign and malignant parathyroid lesions prevents their usefulness as markers for the differential diagnosis [16]. Neoangiogenesis can be promoted by migrating cells from bone marrow [17], and PTH has been shown to influence homing and migration of bone marrow-derived cells [18]. The markers CD34 and CD31 are simultaneously expressed in endothelial cells, while the surface marker CD45 stains all hematopoietic cells except erythrocytes; CD45 indicates the bone marrow as origin of the cells. Grabmaier et  al. [19] detected increased transcript levels of the CD31, CD34, and CD45 genes in parathyroid adenomas compared with normal parathyroid glands. In agreement with the previous reports, the authors reported that parathyroid adenomas showed a significantly higher-­ vessel density than normal parathyroid tissue. Moreover, they identified two subpopulations of CD34+ cells (Fig.  3.1): one subpopulation consisted of endothelial cells lining vessel walls; the second subpopulation included single cells, disseminated in the parenchyma. A similar pattern of expression was detected by investigating the surface marker CD31 (Fig.  3.2), which also marked endothelial cells of vessel walls on one hand and scattered single cells in the parenchyma on the other hand. In the study, scattered single cells expressing CD31, CD34, or CD45 were significantly augmented compared to normal parathyroid glands and directly correlated with vessel density. Therefore, the authors concluded that parathyroid tumors may be enriched of bone marrow-­derived cells. Moreover, transcript levels of SDF-1/CXCL12 was increased, whereas its major inhibitor dipeptidylpeptidase IV/DPP IV was decreased in parathyroid adenomas, suggesting that the SDF-1/CXCL12 axis plays a role in

42 Fig. 3.2 (a–d) Immunostaining with anti-CD34 antibodies of formalin-­fixed paraffin-­embedded (FFPE) sections from four different parathyroid adenomas. The two subpopulations of CD34+ cells are evident: CD34+ cells lining the wall of vessels in panels a and b and CD34+ cells scattered in the parenchyma in panels b and c; in panel d, CD34+ cells surrounded parathyroid acinar structures; bars, 200 μn (personnel unpublished data)

C. Verdelli et al.

3  Parathyroid Tumor Microenvironment

the migration of bone marrow-derived cells into parathyroid adenomas. The two subpopulations of CD34+ cells identified in parathyroid tumors by Grabmaier [19] were similar to the finding previously reported by Corbetta et  al. [20]. The subpopulation of cells lining small vessels (Fig. 3.1) displayed endothelial antigens, namely, factor VIII, isolectin, laminin, and CD146, while the subpopulation constituted of single cells scattered throughout the parenchyma did not express endothelial markers. In the present study, the parathyroid-­ derived CD34+ cells were negative for the hematopoietic and mesenchymal markers CD45, Thy-1/CD90, CD105, and CD117/c-kit, excluding their migration from the bone marrow. Some CD34+ cells co-expressed the parathyroid specific genes glial cell missing B (GCM2), parathormone (PTH), and calcium-sensing receptor (CASR). When cultured, these cells released significant amount of PTH. Parathyroid-derived CD34+ cells, but not CD34- cells, proliferated slowly and differentiated into mature endothelial cells.

3.3.3 Lymphangiogenesis Data about lymphangiogenesis in parathyroid tumors are limited. Investigating 13 normal parathyroid glands, 77 parathyroid adenomas, and 17 PHPT-related parathyroid hyperplasia, lymphatic vascular density (LVD) did not differ among the histotypes, and VEGF-C expression was unrelated to LVD.  LYVE-1 staining demonstrated lymphatics localized at the periphery of the lesions or in the vascular hilum and much less frequent or undetectable in the central portion [14].

3.3.4 Tumor Inflammatory Infiltration Immuno-oncology and the immune-targeted therapies have revolutionized the approach to cancer treatment. Immune cells express immune checkpoint receptors that, when bound to their

43

ligands, induce an inhibitory signal that downregulates immune response. Shi et al. applied flow cytometry to characterize cell types in 20 parathyroid adenomas and 5 corresponding normal glands and observed higher but varying levels of tumor CD8+ lymphocytes as compared to the inflammatory cells in the normal glands [21]. The parathyroid adenoma-­derived T lymphocytes expressed the leukocyte common antigen CD45 and the T-cell receptor unit CD3. CD3 staining on parathyroid adenoma tissue sections showed three predominant patterns: widely separated single cells, clustered cells extravasated into the parenchyma of the gland but adjacent to vascular structures, and focal, densely packed infiltrates. This observation, in association with the cell surface markers pattern CD45+/CD3+/CD19−/CD24−/CD44+, suggests that the intratumoral parathyroid lymphocytes are tumor-infiltrating lymphocytes (TILs). A second study investigated the inflammatory infiltrates in parathyroid tumors, finding concordant results. Parathyroid adenomas and hyperplasia were investigated by immunohistochemistry for the surface cell markers CD4, CD8, CD20, and CD45 [22]. Parathyroid tumors had prominent germinal center-like nodular lymphocytic infiltrates consisting of T and B lymphocytes and/or diffuse infiltrates of predominantly CD8+ T lymphocytes. In the majority of cases with adjacent normal parathyroid tissue, the normal rim was unaffected by the inflammatory infiltrates. Presence of inflammatory infiltrates was associated with higher levels of serum PTH and oxyphilic differentiation. The number of CD8+ lymphocytes in the TME is a cardinal prognostic factor for cancer, playing a role in antitumor immunity. CD8+ lymphocytes act through promoting cancer cell apoptosis, though it should be considered that other TME cells, such as cancer-­ associated fibroblasts, act as suppressors of CD8+ lymphocytic infiltration to the core of tumor [23]. Programmed death-ligand 1 (PD-L1) is one of the critical immune checkpoint proteins acting through binding to its receptor PD-1. Stratification based on T lymphocytes status and PD-L1 expression is related to overall survival in cancer patients [24]. Four distinct tumor microenviron-

44

ments based on the expression of PD-L1 and the presence of tumor-infiltrating lymphocytes (TILs; defined by the expression of the cell surface markers CD3, CD8 identifying the ­lymphocytes and CD68 identifying the macrophages) have been proposed: immunotype I (adaptive resistance, TILs+ and PD-L1+), immunotype II (immunologic ignorance, TILs− and PD-L1−), immunotype III (intrinsic induction, TILs− and PD-L1+), and immunotype IV (tolerance, TILs+ and PD-L1−). Parathyroid carcinomas and atypical parathyroid adenomas exhibit a similar spectrum of immune microenvironments. The predominant tumor microenvironments in parathyroid carcinomas were those with absence of PD-L1 and/or without the presence of TILs (immunotypes II and IV), suggesting that for these cancers, non-PD-L1-based therapies that induce TILs responses or other immunosuppressive pathways should be considered [25]. However, about 25% of parathyroid carcinomas showed a significant expression of PD-L1 and may be candidates for anti-PD-L1 therapy. Both parathyroid carcinomas and atypical parathyroid adenomas harbor intratumoral CD68+ macrophages. This finding suggests the potential to modulate the immune response by TILs in parathyroid tumors. Parathyroid carcinomas had a lesser CD3+ lymphocyte density than did atypical adenomas, while CD68+ macrophages were more abundant. Macrophage accumulation is related to promotion of angiogenesis and local neoplasm invasion [26]. In agreement with the first report, in a further series of 26 parathyroid carcinoma and 37 adenoma samples, PD-L1 expression was deficient in the majority of parathyroid tumors [27].

3.3.5 Tumor-Associated Myofibroblasts In the tumor microenvironment (TME), heterogeneous populations of cells with various or overlapping functions contribute to tumorigenesis [28], and fibroblasts play a crucial role. Cancer-associated fibroblasts (CAFs) are likely derived from resident fibroblasts, adipocytes,

C. Verdelli et al.

bone marrow-derived mesenchymal precursor cells, and endothelial and epithelial cells, some of which are stimulated by cytokines, such as transforming growth factor-β (TGFβ). Myofibroblasts are known as activated fibroblasts with high expression levels of α-smooth muscle actin (α-SMA) and play a critical role in reinforcing contractility in connective tissues. In addition to α-SMA, other markers are fibroblast activation protein (FAP), which is expressed on the surface of fibroblasts, comprises p95 and p105 subunits, and serves as a serine protease, and fibroblast-specific protein 1 (FSP1), which is an intermediate filament-associated protein considered as a marker of quiescent fibroblasts. CAFs can contribute to cancer progression through multiple mechanisms, including the promotion of proliferation, the enhancement of invasion, metastasis, and vascularization, and antitumor effects. CAFs secrete numerous stromal cell growth factors and regulate angiogenesis, immune cell recruitment and polarization in a pro-tumorigenic manner by secreting growth factors and ligands such as CXCL12. Myofibroblasts, or tumor-associated fibroblasts (TAF), are one of the major populations of the tumor stromal cells. It was shown that about 40% of tumor stromal cells are bone marrow-­ derived. Most of the fibroblast-specific protein (FSP)-positive and fibroblast activation protein (FAP)-positive TAFs originate from bone marrow-­ mesenchymal stem cells, whereas α-SMA+ TAFs and perivascular stromal cell (pericytes) are mainly derived from the adipose tissue adjacent to the tumor [29]. In parathyroid adenomas, intraparenchymal α-SMA+ cells were confined to zones with an acinar pattern of cell proliferation similar to those observed in the normal gland (Fig.  3.4). The zones with a trabecular pattern of cell proliferation were poor of myofibroblasts [30]. In parathyroid adenomas, α-SMA+ cells surrounded new microvessels, consistent with a possible role in neoangiogenesis (Fig.  3.4, panels b, d). Interestingly, in human fetal parathyroids (19 and 25 weeks of gestational age), α-SMA+ cells were exclusively found lining blood vessels in formation. In parathyroid atypical adenomas and carci-

3  Parathyroid Tumor Microenvironment

45

Fig. 3.3 (a–d) Immunostaining with anti-CD31 antibodies of FFPE sections from four different parathyroid adenomas. Images are from contiguous sections of the series presented in Fig. 3.2. The CD31 staining resembled the pattern of CD34 expression; bars, 200 μn (personnel unpublished data)

nomas, the parathyroid epithelial cells proliferating in sheets were not sustained by α-SMA+ cells. Conversely, α-SMA+ cells were abundantly represented in the stroma of fibrous bands, in the septa departing from the capsula,

and in the peripheral capsula [30]. TAFs-related genes mRNA expression, namely, α-SMA, SDF-1/CXCL12, FAP, and vimentin, was detected by RT-PCR and confirmed by TaqMan real-time PCR in parathyroid samples. While α-SMA,

46

C. Verdelli et al.

Fig. 3.4 (a–d) Immunostaining with anti-α-SMA antibodies of FFPE sections from four different parathyroid adenomas. Images are from contiguous sections of the series presented in Fig. 3.2. α-SMA+ cells surrounded the walls of vessels in panels a, b, and d and also delimited small neo-vessels as shown in panels a and b (white arrows); moreover, α-SMA+ were well represented in presence of acinar structures of parathyroid endocrine cells (panels c and d); bars, 200 μn (personnel unpublished data)

SDF-1/CXCL12, and vimentin mRNAs were homogeneously expressed almost in all normal and neoplastic tissue samples, FAP was undetectable in normal parathyroid glands, while it was expressed at variable levels in both parathyroid

adenomas and carcinomas. FAP expression is known to characterize TAFs in epithelial-derived solid tumors, and it is positively correlated with tumor cell proliferation, myofibroblasts content, and blood vessels density. A subset of α-SMA+

3  Parathyroid Tumor Microenvironment

47

cells co-expressed the hematopoietic surface CD105, and MHC class I and negative for CD34, marker CD34, suggesting the origin from bone CD133, CD117, CD114, CD31, CD62P, EGF-R, marrow stromal cell progenitors. Moreover, a ICAM-­3, CD26, CXCR4, CD106, CD90, and subset of α-SMA+ cells co-expressed GCM2 at MHC class II, similar to mesenchymal stem nuclear level, a specific parathyroid marker, and cells. The parathyroid-derived stem cells TBX1, an embryonic nuclear transcription factor expressed detectable levels of telomerase activinvolved in development of endodermic pharynx, ity along with the pluripotency Sall4 gene and suggesting the occurrence of epithelial-to-­ possessed osteogenic, chondrogenic, and adipomesenchymal transition as a source of TAFs in genic differentiation potentials [34]. Expression parathyroid adenomas. of calcium-sensing receptor gene along with Human bone marrow-mesenchymal stem cells α-SMA was induced, and cellular uptake of were induced to express TAF-specific features by extracellular calcium ions was observed. parathyroid adenomas-derived cells [30]. Mesenchymal stem cells are multipotent proMoreover, parathyroid adenomas-derived cells genitor cells with the potential to differentiate induced the expression of VEGF-A in human into diverse types of tissue cells, including bone marrow-mesenchymal stem cells, suggest- osteoblasts, adipocytes, chondrocytes, and myoing the role of parathyroid adenomas-induced fibroblasts [47]. These cells closely interact TAFs in neoangiogenesis. Interestingly, the with cells of the immune system in the tissue VEGF-A-increased expression was prevented by microenvironment during repair from tissue the treatment with the CXCR4 inhibitor damage and in the tumor microenvironment, AMD3100. Treatment with AMD3100 also contributing to tumor growth, resistance to therinhibited the PTH mRNA expression levels in apy, and metastases. Mesenchymal stem cells parathyroid endocrine cells. This finding might were isolated from almost every tissue in the be attractive as AMD3100/Plerixafor has been body in addition to bone marrow. They were developed in clinical setting [31–33]. also isolated from various tumor tissues as In conclusions, cells from benign tumors such tumor could stimulate resident mesenchymal as parathyroid adenomas show the ability to acti- stem cell proliferation, located at perivascular vate cells of mesenchymal origin. Benign TAFs sites, or recruit circulating mesenchymal stem are involved in early tumor neoangiogenesis. cells. Tumor-associated mesenchymal stem CXCL12/CXCR4 pathway is expressed and cells contribute to distinct steps of tumorigeneactive in parathyroid adenomatous cells, and sis. They facilitate tumor-associated inflammaCXCL12/CXCR4 might play a role in parathy- tion, reprogram innate immune cells, and roid tumor angiogenesis and PTH synthesis suppress adaptative immunity. The proliferation modulation, and therefore, it might be a target phase is characterized by exogenous tissue prefor new therapeutic approaches to patients with cursor cell recruitment and resident cell differPHPT. entiation and proliferation. Mesenchymal stem cells have a preferential homing to the tumor. Bone marrow-derived mesenchymal stem cells 3.3.6 Tumor-Associated also utilize the CXCL12/CXCR4 autocrine loop Mesenchymal Stem Cells to impel their own migration toward tumors. The tumor-associated mesenchymal stem cells Tissue resident mesenchymal stem cells may be undergo proliferation and differentiation into a source of TAMs. Stem cells have been isolated myofibroblasts to build a collagen network. from human tumor parathyroids, and they Moreover, the tumor-associated mesenchymal showed a surface phenotype consistent with stem cells support the survival and growth of mesenchymal stem cells. Namely, the surface cancer cells as well as cancer stem cells and phenotype of the cells was positive for CD73, supply angiogenic factors for CD166, CD29, CD49a, CD49b, CD49d, CD44, neovascularization.

48

3.3.7 P  otential Role of Parathyroid Tumors-Deregulated MicroRNAs in TME Modulation

C. Verdelli et al.

the differentiation, maintenance, and function of Treg lymphocytes [42]. Additionally, miR-26b and miR-30b, which are significantly downregulated in cancer-­ MicroRNAs (miRs) are abundant noncoding associated fibroblasts in breast cancer [43], were RNA of about 20 bps, exerting an inhibitory epi- found downregulated also in parathyroid cancers genetic effect on the gene expression, which have compared with parathyroid adenomas [38, 39]. been demonstrated to be involved in a number of MiR-26b downregulation in cancer-associated cell function such as cell cycle regulation, prolif- fibroblasts promoted the migration of fibroblasts eration, apoptosis, and neurogenesis. Altered and further promoted the invasion of breast canmiRNAs expression in both the stromal and cer cells [43]. Alteration of miR-126, miR-30b/d, tumor cells could drive tumorigenesis. and miR-199a-5p/3p in cancer cells elicits distal Angiogenesis is under the control of proan- impacts on tumor microenvironment to promote giogenic and antiangiogenic factors. Tumor cells tumor progression through various non-cell-­ could produce and secrete such factors into sur- autonomous mechanisms [44]. MiR-199b-5p rounding environment to promote vessel growth. was significantly downregulated and negatively The endogenous activators and inhibitors are fre- associated with PTH levels in the sporadic paraquently targeted in tumor cells by miRNAs, thyroid tumors but was upregulated in the herediwhich thereby regulate angiogenesis in a non-­ tary parathyroid tumors [45]. cell-­autonomous manner. Indeed, it’s mandatory to demonstrate that The importance of miRNAs in endothelial these TME-related miRNAs are expressed in the cells was demonstrated by the silencing of Dicer, TME cells in parathyroid tumors; such data are an enzyme responsible for miRNAs maturation. lacking up to now. Knockdown of Dicer in endothelial cells inhibited cell proliferation, migration, and cord formaFuture Trends and Directions tion [35], indicating that miRNAs are important 3.4 in the function of endothelial cells. Many studies have shown that miRNAs in endothelial cells Considering the TME features in parathyroid regulate the cellular response to angiogenic fac- tumors, neoangiogenesis and desmoplasia (fibrotors by targeting surface receptors and signaling plasia), which characterize the proliferation phase of tumor progression [46], are well repremolecules [36]. Epigenetic deregulation has been investigated sented and, likely, play a crucial role in determinin parathyroid tumors. Among the deregulated ing the pattern of proliferation and in modulating miRNAs detected in parathyroid tumors com- the parathyroid tumor’s sensitivity to extracellupared with normal parathyroid glands [37–41], lar calcium and their secretory activity. Besides there were some miRNAs involved in angiogen- neoangiogenesis and desmoplasia, most of paraesis. The endothelial-associated miRNAs miR-­ thyroid tumors share similar PD-L1 and tumor-­ 222, miR-296-5p, miR-126-3p, and miR-126-5p associated lymphocytes deficiency as the most were found to be downregulated in parathyroid common cancers. Though the TME characterisadenomas and cancers compared with normal tics, functions, and cross talk with the tumor parathyroid glands [37–39]. MiR-126-3p and endocrine cells need to be further elucidated, the miR-126-5p have been experimentally shown to available data suggest the possibility to treat directly target the 3’UTR region of VEGF-A parathyroid tumors with TME-targeted therapy, mRNA in different types of cancer and subse- such as inhibitors of the CXCL12/CXCR4 pathquently impairing the pro-angiogenesis signaling way and non-PD-L1-mediated inhibitors of the of VEGF/VEGFR-2  in endothelial cells. These immune checkpoint. Moreover, parathyroid miRNAs are generally downregulated in cancer. tumors highlight that TME may play a critical Moreover, miR-126 has been reported to regulate role also in the development of benign tumors.

3  Parathyroid Tumor Microenvironment

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C. Verdelli et al. State of the art. J Cell Physiol 234:1099–1110. https:// doi.org/10.1002/jcp.27051 37. Corbetta S, Vaira V, Guarnieri V, Scillitani A, Eller-­ Vainicher C, Ferrero S et al (2010) Differential expression of microRNAs in human parathyroid carcinomas compared with normal parathyroid tissue. Endocr Relat Cancer 17:135–146. https://doi.org/10.1677/ ERC-09-0134 38. Hu Y, Zhang X, Cui M, Su Z, Wang M, Liao Q, Zhao Y (2018) Verification of candidate microRNA markers for parathyroid carcinoma. Endocrine 60:246– 254. https://doi.org/10.1007/s12020-018-1551-2 39. Rahbari R, Holloway AK, He M, Khanafshar E, Clark OH, Kebebew E (2011) Identification of differentially expressed microRNA in parathyroid tumors. Ann Surg Oncol 18:1158–1165. https://doi.org/10.1245/ s10434-010-1359-7 40. Vaira V, Elli F, Forno I, Guarnieri V, Verdelli C, Ferrero S, Scillitani A, Vicentini L, Cetani F, Mantovani G, Spada A, Bosari S, Corbetta S (2012) The microRNA cluster C19MC is deregulated in parathyroid tumours. J Mol Endocrinol 49:115–124. https://doi.org/10.1530/JME-11-0189 41. Verdelli C, Forno I, Morotti A, Creo P, Guarnieri V, Scillitani A, Cetani F, Vicentini L, Balza G, Beretta E, Ferrero S, Vaira V, Corbetta S (2018) The aberrantly expressed miR-372 partly impairs sensitivity to apoptosis in parathyroid tumor cells. Endocr Relat Cancer 25:761–771. https://doi.org/10.1530/ERC-17-0204 42. Qin A, Wen Z, Zhou Y, Li Y, Li Y, Luo J et al (2013) MicroRNA-126 regulates the induction and function of CD4(+) Foxp3(+) regulatory T cells through PI3K/ AKT pathway. J Cell Mol Med 17:252–264. https:// doi.org/10.1111/jcmm.12003 43. Verghese ET, Drury R, Green CA, Holliday DL, Lu X, Nash C et  al (2013) MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion. J Pathol 231:388–399. https://doi. org/10.1002/path.4248 44. Suzuki HI, Katsura A, Matsuyama H, Miyazono K (2015) MicroRNA regulons in tumor microenvironment. Oncogene 34:3085–3094. https://doi. org/10.1038/onc.2014.254 45. Hwang S, Jeong JJ, Kim SH, Chung YJ, Song SY, Lee YJ, Rhee Y (2018) Differential expression of miRNA199b-5p as a novel biomarker for sporadic and hereditary parathyroid tumors. Sci Rep 8:12016. https://doi.org/10.1038/s41598-018-30484-9 46. Kalluri R (2016) The biology and function of fibroblasts in cancer. Nat Rev Cancer 16:582–598. https:// doi.org/10.1038/nrc.2016.73 47. Li P, Gong Z, Shultz LD, Ren G (2019) Mesenchymal stem cells: From regeneration to cancer. Pharmacol Ther 200:42–54. https://doi.org/10.1016/j. pharmthera.2019.04.005

4

Microenvironment in Cardiac Tumor Development: What Lies Beyond the Event Horizon? Konstantinos S. Mylonas, Ioannis A. Ziogas, and Dimitrios V. Avgerinos

Abstract

Cardiac tumors are found in less than 1% of adult and pediatric autopsies. More than threefourths of primary cardiac neoplasms are benign, with myxomas and rhabdomyomas being the most common cardiac tumors seen in adults and children, respectively. Primary malignant cardiac tumors are extremely rare, whereas metastatic lesions can be seen in approximately 8% of patients dying from cancer. Attempting to understand why the heart is so resistant to carcinogenesis and which failsafe mechanisms malfunction when cardiac tumors do develop is particularly challenging considering the rarity of these tumors and the fact that when relevant clinical studies are published, they rarely focus on molecular pathogenesis. Apart from cancer cells, solid Dr. Konstantinos S.  Mylonas and Dr. Ioannis A.  Ziogas contributed equally and share first-authorship. K. S. Mylonas (*) First Department of Surgery, Laikon General Hospital, National and Kapodistrian University of Athens, Athens, Greece I. A. Ziogas Department of Surgery, Vanderbilt University Medical Center, Nashville, TN, USA D. V. Avgerinos Department of Cardiothoracic Surgery, New York Presbyterian Hospital, Weill Cornell Medicine, New York City, NY, USA

tumors are comprised of a concoction of noncancerous cells, and extracellular matrix constituents, which along with pH and oxygen levels jointly constitute the so-called tumor microenvironment (TME). In the present chapter, we explore mechanisms through which TME may influence cardiac carcinogenesis. Keywords

Heart tumors · Heart neoplasms · Cardiac tumors · Cardiac neoplasms · Myxoma · Rhabdomyoma · Cardiac fibroma · Rhabdomyosarcoma · Cardiac hemangioma · Cardiac lymphangioma · Cardiac metastasis · Tumor microenvironment · Resistance · Tumor development · Carcinogenesis

4.1

Cardiac Tumors: A Snapshot of What We Know About Them

Heart tumors are benign or malignant neoplasms that can either arise primarily or metastasize to the heart, with the latter being the most common scenario [1]. The prevalence of primary cardiac tumors is less than 0.05% both in adult and pediatric autopsy series. More than three-fourths of the primary neoplasms are benign, with myxomas being the most common cardiac tumors seen

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_4

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in adults. Rhabdomyomas are the most prevalent pediatric cardiac tumors, even though the majority does not evoke any symptoms and have a tendency to spontaneously regress [2–7]. Malignant tumors represent a very rare entity, with sarcoma being the most commonly described biologically aggressive lesion [2, 8]. On the other hand, metastatic cardiac tumors are not uncommon based on large autopsy series, where 8% of the patients dying from cancer were found to have metastatic disease in the heart [9]. The three proposed routes of cardiac metastasis include hematogenous spread, an invasive disease directly from the mediastinum, and extension of the tumor into the right atrium after the invasion of the vena cava [10]. Malignant melanoma is a tumor with great metastatic potential to the heart [11, 12], while other tumors that have been accused of causing metastatic heart disease include, but are not limited to, breast, lung, esophageal, or thyroid cancer, renal or hepatocellular carcinoma, and soft tissue sarcomas [13].

4.2

Applying the “Black Hole” Paradigm in Exploring Cardiac Tumorigenesis

Attempting to understand why the heart is so resistant to carcinogenesis and which fail-safe mechanisms malfunction when cardiac tumors do develop is particularly challenging considering the rarity of these tumors and the fact that when relevant clinical studies are published, they rarely (if ever) focus on molecular pathogenesis. As of yet, all we really know about cardiac neoplasms is their clinical presentation, histology, and how to best manage them using available evidence. Due to the paucity of relevant data, no study has attempted to summarize the unique biology of cardiac tumors. Nevertheless, as studying Black Holes can shed light into the origin of the universe, studying the unique properties of cardiac microenvironment can help us better understand how to battle cancer as a whole. To fully understand the anecdotal approach of our chapter, a few lines of astrophysics are warranted. A Black Hole’s mass is concentrated at a

single point deep in its core and cannot be observed. This is called a “gravitational singularity,” and there the space-time curvature becomes infinite [14]. The region of space around the singularity is known as the “event horizon” and marks the limit after within which nothing can be seen, and nothing can escape, because the necessary escape velocity would equal or exceed the speed of light (a physical impossibility). In the present chapter, we attempt to study what lies beyond the event horizon of cardiac tumor development to better understand why the heart is biologically programmed to withstand the forces of carcinogenesis.

4.3

Materials and Methods: The “Event Horizon Telescope”

The present review was conducted in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines and in line with the protocol agreed by all authors [15]. Eligible studies were identified through search of the PubMed bibliographical database (end-of-search date: May 12, 2019) by two independent reviewers (KSM, IAZ). The following MeSH terms were utilized in combination with Boolean operators (AND, OR, NOT): “heart,” “cardiac,” “teratoma,” “fibroma,” “rhabdomyosarcoma,” “myxoma,” “hemangioma,” “lymphangioma,” “non-Hodgkin,” and “microenvironment.” Any discrepancies were identified and resolved through quality control discussions with the senior author (DA) whenever necessary. Reference lists were hand-searched for potentially relevant, missed studies utilizing systematic “snowball” procedure guidelines. Eligible studies were (a) published in English, (b) designed as original experimental studies in either humans or animals, (c) randomized controlled trials (RCTs), or nonrandomized either prospective or retrospective clinical studies, (d) discussing the effect of cardiac tumor microenvironment (TME) in cardiac tumorigenesis. Excluded studies met at least one of the following criteria: (a) not published in English, (b)

4  Microenvironment in Cardiac Tumor Development: What Lies Beyond the Event Horizon?

reviews and meta-analyses, (c) editorials, perspectives and letters to the editor, and (e) articles irrelevant to cardiac TME.

4.4

Tumor Microenvironment (TME): Contextualizing Benchmark Knowledge from Noncardiac Tumors

Apart from cancer cells, solid tumors are comprised of a concoction of noncancerous cells, and extracellular matrix constituents, which along with pH and oxygen levels jointly constitute the so-called tumor microenvironment (TME) [16]. The group of noncancerous cells (also termed as tumor stroma) includes T cells, dendritic cells, neutrophils, macrophages, as well as fibroblasts, and vascular endothelial cells [17]. Our understanding of TME mainly stems from studying solid organs such as the liver, the kidney, and the gonads. Many of the same principles apply when the occasional heart neoplasm develops. Indeed, we now know that the initial steps in tumorigenesis include a sequence of biological events that take place in a normal cell and lead to hyperplasia, irrepressible growth, and cellular immortality [18, 19]. TME remodeling plays a vital role in tumor proliferation and enlargement, via alterations in tumor cell metabolism, which mediate the processes of acidosis, hypoxia, and oxidative stress, ultimately resulting in dysplasia [20, 21]. Later stages of tumorigenesis are also facilitated by TME via autocrine and paracrine communications between the TME cells, which lead to further progression and maturation of the tumor. This process results in greater extracellular matrix stiffness, blood and lymphatic vessel formation, regional necrosis, and metastasis [18]. Therefore, TME may be related to cancer prognosis and its susceptibility to chemotherapy and surgical modalities [22]. The impact of TME on tumorigenesis manifests on a local and on a systemic level. Locally, tumor vessels are poorly aligned and absurdly organized with disturbed blood flow [23]. This chaotic vascular system leads to tumor hypoperfusion which usually poses a barrier in the deliv-

53

ery of chemotherapeutic agents, as well as in the recruitment of neutrophils and other immune cells, which fight against cancer development. On the other hand, metastasis is the leading cause of tumor-related mortality and is commonly accomplished via the dissemination of tumor cells in a distant organ. Recent data suggest that the primary tumor can mediate the process of metastasis by preparing distant target organs via secretion of factors (i.e., vascular endothelial growth factor A [VEGF-A], tumor necrosis factor-alpha [TNF-a], etc.) even before the arrival of the metastatic tumor cells, a concept known as the “premetastatic niche” [24]. Notably, different types of signals arrive at distant organs that do not represent future metastatic sites. For example, there is evidence that mice with cancer exhibit poor vascular function in the kidney and the heart, when in this model those organs are not potential targets for metastasis. This impaired function of the vasculature was mediated by neutrophil extracellular traps (NETs), which led to the vascular occlusion [25].

4.5

 hat Makes Cardiac TME W Different? What Lies Beyond the “Event Horizon”?

Compared to most organs, the heart has displayed great resistance against malignancy (Fig.  4.1). The terminal differentiation of cardiomyocytes and their low turnover rates have been reported as the main mechanisms contributing to this phenomenon [26]. However, these theories remain debatable as more than 70% of the adult heart is actually comprised of non-cardiomyocyte cells, such as fibroblasts, vascular smooth muscle cells, and endothelial cells [27]. It is noteworthy that cardiac TME has a significant role in the resistance of the heart in carcinogenesis. The hepatic TME displays a strong preference not only for primary carcinogenesis but also for the establishment of metastatic tumors in the liver, and hence the hypothesis that liver stromal cells assist in the maintenance of cancer cells has gained great appreciation [28].

K. S. Mylonas et al.

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Fig. 4.1  Mechanisms through which cardiac microenvironment protects again tumor development

On the contrary, data from work comparing human stromal cells from bone marrow, adipose, liver, and cardiac tissues showed that cardiac stromal cells do not favor either the induction of tumorigenesis primarily or the “seeding” of cancer cells in a metastatic fashion, thus creating a deterring TME against carcinogenesis [29]. The mechanism of action is yet to be elucidated; however, direct interaction of tumor cells to stroma or secretion of cytokines or other factors into the microenvironment milieu may induce the inhibition of proliferation and loss of tumor cell viability. The different functions between bone marrow and heart stromal cells were also reported by Rossini et  al. [30], who demonstrated that the infusion of bone marrow mesenchymal stromal cells in heart tissue led to the inhibition of the tumor-suppressive TME. Histogenesis is an additional factor in the equation of carcinogenesis, as it affects the alignment of stromal cells, and hence may determine the anatomical sites of the heart with a higher predisposition for tumorigenesis, and probably the type of tumor that may arise in each specific site [31]. Even though tumor size is thought of as an important predictor of the extent of the disease, data show that TME might play a more crucial role in metastasis [32]. To elaborate, Qian et al. highlighted that mice deficient in PKD1 (an activator of the PI3K-PDK-Akt signaling pathway) exhibited slower tumor growth, impaired

smooth muscle cell development, and a disorganized vascular network [32], which can ultimately lead to the escape of tumor cells in the systemic circulation and metastasis [32, 33]. Undoubtedly, further research is required before deducing meaningful conclusions in order to achieve better understanding of the similarities and differences of TME in the heart that renders it less susceptible to carcinogenesis when compared to other tissues, as well as of the potential modifications that can be applied that will pave the way for safer cellular treatment modalities.

4.6

Future Directions

The present chapter concludes with plans for the future. Modifying tumor microenvironment through stem cell biology and pharmaceutical interventions is the next frontier that needs to be crossed. The safety and efficacy of stem cell therapy, either in the form of direct transplantation to the heart or cell-based tissue engineering, has been conveyed both in patients with congenital and acquired heart disease [34–37]. Preliminary studies are now suggesting that stem cell therapy may be helpful against cardiac tumor development as well. Notably, cardiosphere-derived cells (CDCs) exert their effects in a paracrine fashion via nanoscale extracellular vesicles (EVs), such as exosomes [38]. A study by Cedars-Sinai Heart

4  Microenvironment in Cardiac Tumor Development: What Lies Beyond the Event Horizon?

Institute showed that the injection of CDCderived EVs in a mouse fibrosarcoma model led to inhibition of tumor growth and to an increase in apoptosis along with decreased tumor angiogenesis [26]. Common cardiac medications may also affect cardiac microenvironment in a way that increases tolerance against tumorigenesis [39, 40]. Indeed, there is evidence that cardiac glycosides, such as digoxin, can block the differentiation of stromal fibroblasts to cancer-associated fibroblasts via suppressing tumor growth factorbeta [TGF-β] [41].

4.7

Epilogue

Although we are still a long way from fully understanding the unique biological properties of the heart in terms of tumor resistance, studying cardiac microenvironment may be the answer to more effectively battling cancer. Moving forward, we should take a closer look at the tumor vascular network, the cancer-related inflammation, as well as the cross talk between TME and cancer cells in cardiac tumors [42]. As more and more studies shed light on the “event horizon” of cardiac tumorigenesis, more therapeutic targets will become available in the future, and the armamentarium against heart and other solid organ cancer will continue to grow.

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56 22. Netea-Maier RT, Smit JWA, Netea MG (2018) Metabolic changes in tumor cells and tumor-associated macrophages: a mutual relationship. Cancer Lett 413:102–109 23. Baluk P, Hashizume H, McDonald DM (2005) Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev 15(1):102–111 24. Cedervall J, Dimberg A, Olsson A-K (2015) Tumorinduced local and systemic impact on blood vessel function. Mediat Inflamm 2015:418290 25. Cedervall J, Zhang Y, Huang H, Zhang L, Femel J, Dimberg A et al (2015) Neutrophil extracellular traps accumulate in peripheral blood vessels and compromise organ function in tumor-bearing animals. Cancer Res 75(13):2653–2662 26. Grigorian-Shamagian L, Fereydooni S, Liu W, Echavez A, Marban E (2017) Harnessing the heart’s resistance to malignant tumors: cardiac-derived extracellular vesicles decrease fibrosarcoma growth and leukemia-related mortality in rodents. Oncotarget 8(59):99624–99636 27. Tirziu D, Giordano FJ, Simons M (2010) Cell communications in the heart. Circulation 122(9):928–937 28. Chambers AF, Groom AC, MacDonald IC (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2(8):563–572. https://doi. org/10.1038/nrc865 29. Kellner J, Sivajothi S, McNiece I (2015) Differential properties of human stromal cells from bone marrow, adipose, liver and cardiac tissues. Cytotherapy 17(11):1514–1523 30. Rossini A, Frati C, Lagrasta C, Graiani G, Scopece A, Cavalli S et al (2010) Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin. Cardiovasc Res 89(3):650–660. https:// doi.org/10.1093/cvr/cvq290 31. Gaur K, Majumdar K, Kisku N, Gondal R, Sakhuja P, Satsangi DK (2017) Primary intracardiac leiomyoma arising from cardiomyocyte progenitors at the right ventriculoseptal interface: case report with literature review. Cardiovasc Pathol 28:46–50 32. Qian X-J, Li X-L, Xu X, Wang X, Feng Q-T, Yang C-J (2015) Alpha-SMA-Cre-mediated excision of PDK1 reveals an essential role of PDK1  in regulating morphology of cardiomyocyte and tumor progression in tissue microenvironment. Pathol Biol (Paris) 63(2):91–100

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5

The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis Yusuke Shiozawa

Abstract

It has been appreciated that the cross talk between bone metastatic cancer cells and bone marrow microenvironment influence one another to worsen bone metastatic disease progression. Bone marrow contains various cell types, including (1) cells of mesenchymal origin (e.g., osteoblasts, osteocytes, and adipocytes), (2) cells of hematopoietic origin (e.g., osteoclast and immune cells), and (3) others (e.g., endothelial cells and nerves). The recent studies have enabled us to discover many important cancer-derived factors responsible for the development of bone metastasis. However, many critical questions regarding the roles of bone microenvironment in bone metastatic progression remain elusive. To answer these questions, a deeper understanding of the cross talk between bone metastatic cancer and bone marrow microenvironment is clearly warranted. Keywords

Bone marrow microenvironment · Tumor microenvironment · Bone metastasis · Vicious cycle · Cells of mesenchymal origin · Cells of hematopoietic origin · Blood

Y. Shiozawa (*) Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University Health Sciences, Winston-Salem, NC, USA e-mail: [email protected]

formation · Bone formation · Osteoblasts · Osteocytes · Adipocytes · Osteoclasts · Immune cells · Endothelial cells · Nerves

5.1

Introduction

Bone metastasis is one of the major causes of cancer death. Full understanding of the underlying mechanisms of bone metastasis, however, remains unrevealed. The presence of micrometastatic cells in the marrow at early stage of cancer has been associated with a poor prognosis [1] since micrometastatic cells can result in full-­ blown bone metastases at later stage [2] and metastatic progression of these cells may be environmentally directed by influences from the bone marrow-resident cells (Fig. 5.1). Indeed, it has been suggested that the cross talk between bone metastatic cancer cells and bone marrow microenvironment plays a crucial role in ­regulating the dissemination cascade of cancer cells to the bone and the progression of micrometastatic cells within the bone [3–5]. Bone marrow contains many cell types, including cells of hematopoietic and mesenchymal origin. Interactions among these cells are crucial for regulating blood and bone formation. Osteoblasts [6–8], adipocytes [9, 10], endothelial cells [11, 12], and nerves [13, 14] are known to serve as a specific microenvironment for hematopoietic stem cells. Osteoblasts, osteoclasts, osteocytes, and endothelial cells collectively

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_5

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Fig. 5.1  The model of the bone marrow tumor microenvironment Bone marrow contains many cell types that directly influence blood formation (e.g., osteoblasts, adipocytes, endothelial cells, nerves) and bone formation (e.g., osteoblasts, osteocytes, osteoclasts, endothelial cells). Recent studies have revealed that bone metastatic cancer cells adopt mechanisms whereby these bone-resident cells maintain hematopoiesis and bone remodeling in

order to gain access to and grow within the bone. Bone marrow-­resident immune cells are also involved in the process of bone metastasis in both a direct and an indirect manner. By interacting one another, all these cells play crucial roles in controlling bone metastatic progression as a bone marrow tumor microenvironment. Graphics adapted from Smart Servier Medical Art (https://smart. servier.com/)

organize bone remodeling [15]. We have demonstrated that bone metastatic cancer cells target and commandeer the microenvironment for hematopoietic stem cells, using similar mechanisms whereby hematopoietic stem cells home to the marrow to gain access to the bone [16]. Furthermore, bone metastatic cancer cells cause “vicious cycle” of bone metastasis by disrupting the balance between normal osteoclastogenesis and osteoclastogenesis [17, 18]. These evidences suggest that a microenvironment in the marrow also conversely affects the development and progression of bone metastasis.

Stephen Paget famously hypothesized in his “seed and soil” theory that metastatic cancer cells (seed) seek a specific metastatic site (soil) in order to survive outside of the primary tumor site [19]. Although the molecular mechanisms whereby bone metastatic cancer cells (seed) disseminate to bone have been extensively studied, tumor-supportive roles of bone marrow microenvironment (soil) are still largely unknown. In this review, we will explore what is currently known about bone marrow microenvironment-mediated bone metastatic progression and also suggest future directions for the microenvironment-­ targeted therapies for bone metastatic disease.

5  The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis

5.2

Bone Marrow Tumor Microenvironment

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and rapamycin delayed the early colonization of MCF-7 cells to osteoblasts [22]. Similarly, human prostate cancer cell line C4-2B4 cells [23] 5.2.1 Cells of Mesenchymal Origin and human breast cancer cell line MDA-MB-231 cells [24] bound to murine pre-osteoblast cell 5.2.1.1 Osteoblasts line MC3T3-E1 cells through homophilic cadOsteoblasts are the cells of mesenchymal origin, herin-­11–cadherin-11 interaction. responsible for new bone formation, and known The interaction between bone metastatic canto be involved in regulating the homing and cer cells and osteoblasts might be involved in the maintenance of hematopoietic stem cells [20]. bone metastatic progression within the marrow. Several lines of evidence suggest that osteoblasts More PC-3 cells bound to murine primary calalso play significant roles in the development of varial osteoblasts obtained from Annexin II bone metastasis. When human prostate cancer (Anxa2) wild-type mice than those from Anxa2 cell line PC-3 cells (intracardiac injection) and knockout mice, and PC-3 cells grew greater on hematopoietic stem cells (intravenous injection) Anxa2 wild-type osteoblasts [25]. When PC-3 were simultaneously injected into irradiated cells were implanted into athymic nude mice severe combined immunodeficient (SCID) mice, with vertebral bodies obtained from either Anxa2 both PC-3 cells and hematopoietic stem cells wild-type or Anxa2 knockout mice, more growth homed to osteoblasts and localized very closely of PC-3 within Anxa2 wild-type vertebral bodies each other [16]. Moreover, PC-3 cells and human was observed compared to that within Anxa2 prostate cancer cell line C4-2B cells prevented knockout vertebral bodies [25]. When receptor the binding of hematopoietic stem cells to murine for Anxa2 were downregulated in PC-3 cells, primary calvarial osteoblasts [16]. Likewise, metastatic burdens within athymic nude mice more numbers of intracardially inoculated human were inhibited [25]. Anxa2 treatments induced prostate cancer cell line PC-3NW1 cells homed AXL expression, a receptor for growth to lateral endocortical surfaces of tibia of athymic ­ arrest-­ specific 6 (GAS6), in PC-3 cells [26]. nude mice where more osteoblasts reside com- Similarly, when PC-3 cells were cocultured with pared to medial endocortical surfaces [21]. MC3T3-E1 cells, PC-3 cells become dormant by However, there were no differences in between upregulating AXL expression [27]. Additionally, the minimum distance from disseminated when primary human osteoblasts were coculPC-3NW1 cells to lateral endocortical surfaces tured with PC-3 cells, the secretion of GAS6 and medial endocortical surfaces [21]. When ani- from osteoblasts was increased [26]. The GAS6 mals were treated with a C-X-C motif chemokine (osteoblasts)/AXL (PC-3 cells) interaction receptor 4 (CXCR4) antagonist AMD3100, dif- induced the expression of transforming growth ferential dissemination patterns of PC-3NW1 factor (TGF)-β1, TGF-β2, TGF-β receptor 2 cells to lateral and medial endocortical surfaces (TGFBR2), and TGFBR3  in PC-3 cells [27]. were no longer detected, and the minimum dis- TGF-β2 was responsible for osteoblast-mediated tance from disseminated PC-3NW1 cells to both PC-3 dormancy [27]. Moreover, PC-3 cells endocortical surfaces was extended [21]. When became stemlike phenotype (CD133 positive/ human breast cancer cell line MCF-7 cells were CD44 positive) on wild-type murine primary calinoculated into athymic nude mice by an intra-­ varial osteoblasts, while the conversion of PC-3 iliac artery injection, they initially colonized to cells to stemlike phenotype was prevented when alkaline phosphatase (ALP)-positive/collagen they were cocultured with osteoblasts obtained I-positive osteoblasts through E-cadherin from GAS6 knockout mice [28]. The GAS6 (MCF-7 cells) and N-cadherin (osteoblasts) derived from osteoblasts induced the conversion interaction [22]. This cancer-to-osteoblast inter- of PC-3 cells into stemlike phenotype by activataction activated mammalian target of rapamycin ing mTOR pathway through another GAS6 (mTOR) pathway, and mTOR inhibitors torin 1 receptor, Mer [28]. When PC-3 cells were

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implanted into SCID mice with vertebral bodies obtained from either GAS6 wild-type or GAS6 knockout mice, more stemlike PC-3 cells were observed near osteoblasts within GAS6 wildtype vertebral bodies [28]. When PC-3 cells pretreated with rapamycin were intracardially inoculated into SCID mice, the stemlike conversion of PC-3 cells within the bone marrow were diminished [28]. When Jagged1-overexpressing bone-tropic MDA-MB-231 subline SCP28 cells were cocultured with MC3T3-E1 cells, SCP28-derived Jagged1 induced interleukin 6 (IL-6) secretion from MC3T3-E1 by upregulating Hey1 (a downstream of notch pathway), resulting in the increased proliferation of SCP28 cells on MC3T3-E1 cells [29]. The γ-secretase inhibitor MRK-003, which abolishes notch activity [29], and human monoclonal anti-Jagged1 antibody 15D11 [30] attenuated (1) the increased proliferation of Jagged1-overexpressing SCP28 cells mediated by MC3T3-E1 cells and (2) bone metastatic burden of intracardially inoculated Jagged1-overexpressing SCP28 cells in athymic nude mice. Moreover, the combination of 15D11 and paclitaxel synergistically prevented bone metastatic progression of Jagged1-overexpressing SCP28 cells compared to 15D11 or paclitaxel alone [30]. Interestingly, 15D11 and paclitaxel combination also inhibited bone metastatic progression of low Jagged1-expressing parental SCP28 cells since paclitaxel induced Jagged1 expression in osteoblasts, leading to chemoresistance [30]. When osteoblasts were induced into senescence using a conditional transgenic mouse model [fibroblasts accelerate stromal-supported tumorigenesis (FASST) mouse: Col-Cre-ERT2 mouse x ROSAlox-stop-lox-p27Kip1 mouse], greater numbers of intracardially injected murine breast cancer cell line NT2.5 cells disseminated to bone [31]. In FASST mice, senescent osteoblasts indirectly enhanced the growth of disseminated NT2.5 cells by stimulating osteoclastogenesis via IL-6 production [31]. In addition, bone disseminated murine multiple myeloma cell line 5TGM1 cells (intravenous injection into C57BL/KaLwRijHsd mice) became dormant and consequently resistant to

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melphalan when they localized near osteoblasts [32]. However, when animals were treated with receptor activator of nuclear factor κ-Β ligand (RANKL) to stimulate osteoclast activities, the dormancy of 5TGM1 cells was reversed [32]. These data suggest that osteoblasts are important for the early dissemination and colonization of bone metastatic cancer cells as well as the regulation of dormancy, stem cell-like conversion, and later disease outgrowth of bone metastatic cancer cells.

5.2.1.2 Osteocytes Bone is a dynamic organ continuously remodeled throughout life. Bone remodeling is controlled by proper balance between osteoblastogenesis and osteoclastogenesis [15]. Osteocytes are also known to be involved in regulating bone remodeling. Osteocytes negatively regulate osteoblastic activities by releasing dickkopf-1 (DKK1) and sclerostin (inhibitors of Wnt pathway which are important for osteoblastic differentiation) and enhance osteoclastic resorption by releasing RANKL when they undergo programmed cell death [15, 33–35]. Osteocytes are the terminally differentiated cells of osteoblastic lineage and reside within the bone matrix [33–35]. Approximately 90–95% of adult skeletal tissues consist of osteocytes [33–35]. However, it has been only recently appreciated the roles of osteocytes as a tumor microenvironment. Osteocyte death and enhanced osteoclast activity were observed in osteolytic bone lesions of multiple myeloma patients [36]. Higher levels of pro-­osteoclastogenesis cytokine IL-11 were found in bones of multiple myeloma patients with osteolytic lesions than those without osteolytic lesions [36]. Conditioned medium (CM) obtained from the coculture between human multiple myeloma cell line JJN3 cells and human pre-osteocyte cell line HOB-01 cells stimulated osteoclastic formation of human primary peripheral blood mononuclear cells by enhancing the secretion of IL-11 and C-C motif chemokine ligand 3 (CCL3) [36]. These data suggest that osteocytes create favorable environment for bone metastatic cancer cells by promoting osteolytic bone damage.

5  The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis

Osteocytes not only provide the environment for cancer but also directly influence cancer progression. CM obtained from murine osteocyte cell line MLO-Y4 cells stimulated in  vitro cell proliferation of human prostate cancer cell lines (PC-3 and DU145) and human breast cancer cell lines (MDA-MB-231 and MCF-7) [37]. CM obtained from murine osteocyte cell line MLO-­ A5 cells also enhanced the proliferation of murine breast cancer cell line 4  T1.2 cells [38, 39]. However, when 4T1.2 cells were three-­ dimensional (3D) cocultured with MLO-A5 cells, the size of MLO-A5/4T1.2 spheroid was shrank [38, 39]. Additionally, in vitro 3D coculture system between human osteocytes and prostate cancer organoids revealed that prostate cancer organoids significantly impair bone structure through the induction of osteocyte apoptosis while promoting bone mineralization [40]. In this model, prostate cancer organoids significantly enhanced the protein expression of fibroblast growth factor 23 (FGF23) and DKK1  in osteocytes but reduced sclerostin [40]. However, these changes failed to be observed in 2D coculture conditions [40], suggesting that the 3D culture system provides a unique opportunity to study the interaction between osteocytes and cancer cells. MLO-Y4 CM also enhanced the anchorage-­ independent growth, migration, and invasion of MDA-MB-231 cells and murine breast cancer cell line Py8119 cells [41]. These phenomenon were inhibited with bisphosphonate [alendronate and zoledronic acid (ZOL)] treatments and fluid flow-induced shear stress by opening connexin 43 hemichannels in osteocytes [41]. The growth of Py8119 cells in the bone mediated by intratibial injections was enhanced when cells were injected into osteocyte-specific connexin 43 knockout mice or mice with osteocyte-specific connexin 43 junction impairment [41]. Alendronate upregulated ATP release from MLO-Y4 cells, and this ATP inhibited the MLO-­ Y4 CM-mediated anchorage-independent growth, migration, and invasion and in vivo primary tumor growth of MDA-MB-231 cells and Py8119 cells through P2X7 receptors [42]. In addition, these were reversed by adenosine treat-

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ment [42]. ATP treatments prevented the growth of intratibially injected Py8119 cells [42]. Likewise, CM from MLO-Y4 cells primed with CM of human prostate cancer cell lines (PC-3 and C4-2B) induced the growth, migration, and invasion of PC-3 and C4-2B cells by upregulating growth-derived factor 15 (GDF15) in MLO-­ Y4 cells and enhancing early growth response 1 (EGR1) expression in prostate cancer cells [43]. When C4-2B cells were co-inoculated with murine primary osteocytes into the flank of mice, these osteocyte/C4-2B mixtures grow greater than C4-2B cells alone, due in part to the upregulation of EGR1 in C4-2B cells [43]. The growth of osteocyte/C4-2B mixture was diminished when GDF15 was downregulated in osteocytes [43]. When PC-3 or C4-2B cells were injected into the tibia of mice in which GDF15 expression in tibia was downregulated using siRNA, prostate cancer cell growth in bones and EGR1 expression in prostate cancer cells were decreased compared to those in bones infected with control siRNA [43]. However, MLO-A5-CM conversely reduced the migratory ability of 4T1.2 cells through type I collagen by downregulating SNAIL [an epithelial to mesenchymal transition (EMT) inducer] and upregulating phosphorylation of Akt [38, 39]. Further study is warranted to conclude the effects of osteocytes on migratory ability of bone metastatic cancer cells. Osteocytes are mechanosensitive cells [33– 35], and the impact of mechanical stress on tumor microenvironmental abilities of osteocytes has been elucidated. Fluid flow-induced shear stress stimulated the differentiation of MLO-Y4 cells regardless of the existence of CM derived from MDA-MB-231  in the culture [44]. Fluid flow-­ induced shear stress on the 3D coculture between MLO-Y4 cells and human umbilical vein endothelial cells (HUVECs) inhibited the extravasation of MDA-MB-231 cells [45–47]. This was in part due to the reduction of IL-6  in MLO-Y4 cells (chemoattractant for cancer cells; inhibition of osteoclast apoptosis), the reduction of intercellular adhesion molecule 1 (ICAM-1) in HUVECs (cancer cell adhesion to endothelial cells; inhibition of osteoclast apoptosis), and the reduction of matrix metallopeptidase 9 (MMP9) in

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MDA-MB-231 cells (cancer cell migration; reduction of endothelial junctional integrity) [45–47]. When hydrostatic pressure was applied to MLO-Y4 cells, MLO-Y4-CM promoted the growth, migration, and invasion of human prostate cancer cell lines (PC-3, DU145, and LNCaP) in part due to the upregulation of CCL5 and MMP2 and MMP9 [48]. When human multiple myeloma cell line RPMI 8226 cells were cocultured with HOB-01 cells or MLO-Y4 cells, RPMI 8226 cells induced death of HOB-01 and MLO-Y4 cells by activating autophagy [49]. This multiple myeloma-­ induced osteocyte death and autophagy were inhibited by proteasome inhibitors, bortezomib, and MG 262 [49]. Consistently, multiple myeloma patients treated with regimens including bortezomib showed significantly higher osteocyte viability in the bone lesions compared to those treated with regimens without bortezomib (p  =  0.017) [49]. Moreover, a combination agent of nanoparticle loaded with a natural compound, plumbagin and ZOL (PUCZP), prevented the MLO-Y4-mediated osteoclastogenesis of murine bone marrow monocytes mediated by downregulating of RANKL and sclerostin expression in MLO-Y4 cells [50]. PUCZP also attenuated bone metastatic progression and cancer-­induced bone resorption in athymic nude mice intracardially inoculated with MDA-MB-231 cells by downregulating of RANKL and sclerostin expression in osteocytes, reducing osteoclastogenesis, and inducing apoptosis of tumor cells [50]. These findings suggest that targeting osteocytes is a potential new therapeutic strategy for bone metastatic disease.

5.2.1.3 Bone Marrow Adipocytes Bone marrow is known to contain significant numbers of adipocytes. Bone marrow adipocytes take up about 70% of marrow space in adults [51] and serve as one of the major components of the bone marrow microenvironment for hematopoietic stem cells [9, 10]. However, the effects of bone marrow adipocyte on bone metastatic progression have been only recently uncovered [52– 59]. Direct contact between human primary bone marrow mesenchymal stem cell (MSC)-derived

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adipocytes (BMMAs) and promyelocytic leukemia protein-retinoic acid receptor α (PML-­ RARα)-expressing human acute promyelocytic leukemia cell line U937/PR9 cells protected U937/PR9 cells from apoptosis through the leptin (adipocytes)/leptin receptor (leukemia) axis [60]. Additionally, BMMAs activated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and ERK mitogen-activated protein kinase (MAPK) in U937/PR9 cells [60]. Likewise, coculture with human primary BMMAs protected human acute monocytic leukemia cell line U937 cells from spontaneous apoptosis by increasing leukemia cells’ free fatty acid intake and consequent fatty acid β-oxidation [61]. This is in part due to the upregulation of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4), CD36, and B-cell lymphoma 2 (BCl2) genes; the phosphorylation of AMP-activated protein kinase (AMPK) and p38 MAPK; and the upregulation of antiapoptotic chaperone 70-kDa heat-shock protein (HSP70) [61]. Human primary BMMAs also supported the proliferation of human primary acute myeloid leukemia (AML) blasts by upregulating FABP4  in adipocytes and enhancing lipids intake and fatty acid β-oxidation in AML cells [62]. When connective tissue growth factor (CTGF) was downregulated in human bone marrow MSCs, these MSCs differentiated into adipocyte lineage [63]. When CTGF-­ downregulated MSCs were implanted with endothelial cells into the flank of nonobese diabetic/ severe combined immunodeficiency (NOD/ SCID) IL-2rγnull (NSG) mice, they contained more adipocytes than the implants of CTGF-­ normal MSCs with endothelial cells [63]. Intravenously inoculated human AML cell line Molm13 cells and acute lymphoblastic leukemia cell line Nalm6 cells homed better to CTGF-­ downregulated MSC implants compared to those with CTGF-normal MSCs since adipocytic CTGF-downregulated MSC implants expressed higher levels of C-X-C motif chemokine ligand 12 (CXCL12) and leptin [63]. Moreover, bone marrow of multiple myeloma patients contained more adipocytes than that of healthy counterparts [64]. The 5TGM1 cells migrated toward CM

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from murine pre-adipocyte cell line 3T3-L1 cells higher levels of heme oxygenase 1 (HO-1; a prothrough CCL2 and CXCL12 [64]. When 5TGM1 tector from oxidative cell damage) compared to cells were cocultured with 3T3-L1 cells, 3T3-L1 primary prostate tumors or samples from other cells enhances the viability of 5TGM1 cells by metastatic sites [69]. When PC-3 and ARCaP(M) activating β-catenin and inhibiting caspase3  in cells were cocultured with murine primary 5TGM1 cells [64]. When 5TGM1 cells primed BMMAs, the expression of HO-1 and endoplaswith 3T3-L1 cells were intravenously injected mic reticulum (ER) stress chaperone glucose-­ into C57BL/KaLwRij mice, these 3T3-L1-­ regulated protein 78 (GRP78; another protector primed 5TGM1 cells grew greater in the bone from oxidative cell damage) was enhanced [69]. than those without 3T3-L1 priming [64]. These Adipocyte-induced oxidative stress and overexfindings suggest that bone marrow adipocytes are pression of HO-1 in PC-3 and ARCaP(M) cells also crucial components of microenvironment for promoted the invasion of these cells [69]. hematopoietic malignancies, similar to their roles Similarly, CM of murine primary BMMAs for hematopoietic stem cell [9, 10]. induced the invasion of PC-3 cells by upregulatRecent studies have also revealed the impact ing HO-1, IL-1β, and FABP4 but downregulating of bone marrow adipocytes on bone metastatic PPARγ in PC-3 cells [70]. Overexpression of progression on solid tumors. When MDA-MB-231 HO-1 in PC-3 and ARCaP(M) cells also induced cells were cocultured with bone fragments the enhanced levels of GRP78 and pro-survival obtained from patients who undergo hip replace- factors Bcl-xl and survivin in prostate cancer ment, MDA-MB-231 cells migrated toward bone cells. HO-1 overexpression significantly fragments and extensively colonized within the increased growth in the bone of intratibially bone marrow adipose tissue compartment [65]. injected PC-3 and ARCaP(M) cells compared to When human bone marrow stromal cells were those with control vector infection [69]. transformed into adipocytic lineage with omega-6 Intratibial injections of PC-3 and ARCaP(M) polyunsaturated fatty acid (PUFA) treatments, cells into mice with high-fat diet (60 kcal% fat) more PC-3 cells migrated toward these omega-6 showed the enhanced numbers of bone marrow PUFA-treated human bone marrow stromal cells adipocytes osteolytic lesions [71]. CM from [66, 67]. Additionally, more lipid intake of PC-3 PC-3 and ARCaP(M) cells stimulated the secrecells from bone marrow stromal cells was tion of CXCL1 and CXCL2 from murine primary observed [66, 67]. Murine primary BMMAs also BMMAs, and BMMA-derived CXCL1 and induced glycolytic shift in human prostate cancer CXCL2 induced osteoclastogenesis of murine cell lines PC-3 and ARCaP(M) cells by upregu- bone marrow macrophages [71]. Along with this lating glycolytic markers [enolase 2 (ENO2), lac- notion, when murine melanoma cell line B16-­ tate dehydrogenase (LDHa), hexokinase 2 F10 cells were directly injected into the tibia of (HK2), pyruvate dehydrogenase kinase 1 high-fat diet-fed C57BL/6 N mice, more growth (PDK1), and phosphorylated pyruvate dehydro- of B16-F10 cells in the bone was observed along genase (p-PDH)], increasing lactate release, with the increased numbers of bone marrow adireducing oxygen consumption rate, and decreas- pocytes and osteoclasts, and enhanced osteolytic ing ATP levels [68]. This was due to the upregu- lesions, compared to those in the bone of lation of HIF-1α target genes [carbonic anhydrase C57BL/6 N mice with normal diet (10 kcal% fat) 9 (CA9), vascular endothelial growth factor [72]. Additionally, serums of tumor-bearing mice (VEGF), and glucose transporter 1 (GULT1)] with high-fat diet, and macrophages and osteo[68]. Conversely, CM from PC-3 and ARCaP(M) clasts found in their bone expressed higher levels cells promoted lipolysis in murine primary of osteopontin than those from animals with norBMMAs by increasing free glycerol release, mal diet [72]. Murine primary BMMAs induced which was uptaken by prostate cancer cells [68]. IL-6 mRNA expression in B16-F10 cells, and Bone metastatic samples obtained from prostate higher serum IL-6 concentration was observed in cancer patients with bone metastases showed of tumor-bearing mice with high-fat diet [72].

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Neutralizing anti-osteopontin antibody and neutralizing anti-IL-6 antibody or Janus kinase 2 (JAK2; a downstream of IL-6) inhibitor AG490 reduced the enhanced B16-F10 growth in the bone of tumor-bearing mice with high-fat diet [72]. When B16-F10 cells were injected into C57BL/6 mice by intracardiac injection, the numbers of bone marrow adipocytes and the serum levels of leptin increased at day 7 of injection, and these increased adipocytes and leptin levels decreased by day 10 [73]. Similar trend was observed in osteoclast activities [73]. Interestingly, B16-F10 cells localized around bone marrow adipocytes [73]. Consistent with the observations in in  vivo, CM from B16-F10 cells induced differentiation of murine primary BMMAs while mature BMMAs were dedifferentiated into immature BMMAs when they were cocultured with B16-F10 cells [73]. On the other hands, CM from murine primary BMMAs induced proliferation and accelerated cell cycle of B16-F10 cells [73]. Similarly, CM from murine primary BMMAs enhanced DNA synthesis of murine multiple myeloma cell line 5T33MM cells, stimulated their migration toward adipocyte CM, and protected from apoptosis [74]. These data suggest that bone marrow adipocytes also play an important role in the regulation of bone metastatic progression of solid tumors.

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tion [76]. Osteoclasts promote extracellular calcium release and create extracellular acidic environments during bone resorption. Higher levels of calcium-sensing receptor were observed in bone metastatic tumor samples of prostate cancer [77] and renal cell carcinoma patients [78] compared to primary tumor samples. Extracellular calcium stimulated the proliferation of human breast cancer cell lines BT474 and MDA-MB-231-1833 cells [79] and PC-3 cells [80] through calcium-sensing receptor. Extracellular pH promoted the spheroid and colony formation of PC-3 cells [81]. Although there is no direct evidence yet, these findings suggest that osteoclasts may also play a crucial role as a bone marrow tumor microenvironment in an indirect manner by developing extracellular high-­ calcium and extracellular acidic environments.

5.2.2.2 Immune Cells It has been extensively studied antitumor or pro-­ tumor effects of immune system at primary tumor site. Although bone marrow contains various immune cells, the cross talk between bone metastatic cancer cells and bone marrow immune cells remains largely unexplored. Higher numbers of CD4-positive/Foxp3-positive regulatory T (Treg) cells were found in the bone marrow of prostate cancer patients with bone metastases than that of patients without bone metastases [82]. Treg cells obtained from bone metastatic prostate cancer 5.2.2 Cells of Hematopoietic Origin patients had greater migratory and proliferative capacities and were responsible for the inhibition of osteoclasts, resulting in osteoblastic bone 5.2.2.1 Osteoclasts Osteoclasts are the multinucleated cells derived lesions [82]. Contrary, RANKL levels in CD3-­ from a monocyte/macrophage lineage and are positive T cells within the marrow of BALB/c associated with the development of osteolytic mice in which 4  T1 cells were inoculated into bone metastatic lesions. Osteoclasts are known to mammary fat pad were enhanced compared to indirectly serve as a bone marrow tumor micro- those of mice inoculated with nonmetastatic environment. Osteoclasts create a sufficient space 4T1subline, 67NR cells [83]. However, there for bone metastatic cancer cells to grow by were no changes in the numbers of CD3-, CD4-, resorbing bone matrix [75]. Indeed, osteolytic and CD8-positive T cells between 4T1- and bone metastatic lesions in athymic nude mice 67NR-bearing mice [83]. Consequently, the inoculated with SCP28 cells (intracardiac injec- enhancement of RANKL in T cells increased tion) were prevented by treatment of microRNA osteoclastic activities in the marrow, leading to (miR)-141 and miR-219 which downregulated in bone metastatic colonization of 4 T1 cells [83]. activated osteoclasts, while miR-141 and miR-­ Intracardiac injection of 4 T1 cells into BALB/c 219 did not directly affect SCP28 cells prolifera- mice increased the numbers of B220-positive/

5  The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis

CD11c-positive plasmacytoid dendritic cells (pDCs) and the increased CD4-positive T cells in their marrow [84]. pDCs and CD4-positive T cells produced osteolytic bone lesions and subsequently increased tumor burden and bone damage [84]. Depletion of pDCs with PDCA-1 antibody led to decreased tumor burden and bone damage by reducing the numbers of CD4-positive T cells and CD11b/Gr-1 double-positive myeloid-­ derived suppressor cells (MDSCs) and activating cytotoxic CD8-positive T cells in the marrow [84]. More number of CD11b/Gr-1 double-positive MDSCs were observed in the marrow of C57BL6/ KaLwRij mice intravenously injected with 5TGM1 cells compared to non-tumor-bearing mice [85]. The MDSCs from 5TGM1-bearing mice showed greater potential to differentiate into mature and functional osteoclasts than those from control mice [85]. When 5TGM1-bearing MDSCs were delivered to 5TGM1-bearing mice, osteolytic bone lesions were further enhanced compared to 5TGM1-bearing mice without MDSC treatments [85]. Additionally, the enhancement of osteoclastic activities mediated by 5TGM1-bearing MDSCs both in  vitro and in  vivo were inhibited by ZOL treatments [85]. Likewise, CD11b/Gr-1 double-positive MDSCs obtained from bone marrow cells of BALB/c mice which were intracardially inoculated with 4T1 cells and developed bone metastases differentiated into osteoclasts by increasing NO levels, but not MDSC from lung of 4T1-bearing mice with bone metastases or from bone marrow of 4 T1-bearing mice without bone metastases [86]. In bone samples obtained from prostate cancer patients with bone metastases, more CD68-­ positive macrophages were observed near tumor mass and pathological woven bone [87]. When F4/80-positive macrophages were depleted in the macrophage Fas-induced apoptosis (MAFIA) mice with AP20187 treatments, the growth of murine prostate cancer cell line RM-1 cells (intratibial inoculation) in their bone and resultant osteolytic lesions were significantly reduced [88]. Similarly, F4/80-positive macrophage depletion by clodronate liposome treatments resulted in the reduction of the growth of intrati-

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bially injected RM-1 cells (into C57BL6 mice) and PC-3 cells (into athymic nude mice) [88]. However, contrarily to MAFIA mice, depletion of macrophages by clodronate liposome treatments increased bone volume [88] but reduced pathological woven bone development [87]. When CD169-positive macrophages were depleted in the CD169-diphtheria toxin receptor (DTR) mice with DT treatments, the woven bone development in RM-1-bearing bone (intratibial injection) were attenuated, while CD169 macrophage depletion did not affect the tumor growth in the bone [87]. The treatments of anti-mouse CD115 monoclonal antibody which only target murine myeloid cells also inhibited osteolytic bone metastatic progression in athymic nude mice intracardially inoculated with MDA-MB-231 cells [89]. The beneficial effects of anti-mouse CD115 monoclonal antibody were comparable to ZOL [89]. When marrow-resident F4/80-positive/CD206-positive pro-tumorigenic M2-like macrophages, which enhance prostate cancer proliferation by efferocytosis, were reduced by trabectedin treatments, the growth of PC-3 cells (intratibial or intracardiac injection) in the bone of athymic nude mice was attenuated [90]. However, trabectedin treatments failed to change osteoclastic activities in the tumor-­ bearing bones [90]. These findings suggest that bone marrow immune cells also support bone metastatic progression as a bone marrow tumor microenvironment directly in an immunosuppressive manner and indirectly by influencing bone remodeling.

5.2.3

 ther Components of Bone O Marrow Microenvironment

5.2.3.1 Endothelial Cells Bone is a highly vascularized organ and subsequently supplies hematopoietic cells and cytokines produced in the marrow. Although the vasculature is known to be crucial for tumor growth and metastasis, little is known as to the roles of bone marrow endothelial cells as a tumor microenvironment. Human breast cancer cell lines MDA-MB-231 cells (orthotopic inocula-

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tion) and HMT-3522-T4-2 cells (intracardiac 5.2.3.2 Nerves inoculation) disseminated into the bones of NOD/ The importance of nerves for cancer progression SCID mice, and those disseminated tumor cells has been appreciated [93]. The sympathetic nerbecame dormant when they localized microvascu- vous system regulates the metastatic process of lar endothelium [3]. Consistently, HMT-­prostate cancer to bone [94], and denervation can 3522-­T4-2 cells stayed dormant on the organotypic suppress tumorigenesis and metastasis [94–97]. bone microvascular mimic (the mixture of Moreover, the roles of sympathetic nervous sysHUVECs and murine primary bone marrow stro- tem in bone metastatic progression have recently mal cells) [3]. More dormant HMT-3522-T4-2 begun to be revealed. Chronic immobilization cells were observed when they reside on the stress and a nonselective β1/β2 adrenergic recepthrombospondin 1 (TSP-1)-expressing mature tor agonist isoproterenol treatments enhanced the endothelium, while TGF-β1 and periostin- colonization of intracardially injected expressing immature neovascular tips accelerated MDA-MB-231 cells to the bone of athymic nude the growth of HMT-3522-T4-2 cells [3]. Murine mice through the RANK (cancer)/RANKL breast cancer cell line 4T07 cells (orthotopic inoc- (bone) axis by inducing the RANKL expression ulation) disseminated closer to vasculature com- in bones [98] However, chronic immobilization pared to megakaryocytes or osteoblasts, within stress and isoproterenol treatments did not affect the marrow of BALB/c mice, and vasculature- the growth of MDA-MB-231 cells in the bone resident 4T07 cells had more resistance to chemo- [98]. The enhancement of tumor colonization therapy (Adriamycin plus cyclophosphamide, mediated by isoproterenol treatments was also AC) [91]. Coculture between the organotypic thought to be due to the increased neovascularbone microvascular mimic and HMT-3522-T4-2 ization of tumor-bearing bones [99]. Consistently, cells treated with doxorubicin revealed that the isoproterenol stimulated the VEGF secretion of induction of chemoresistance mediated by vascu- MC3T3-E1 cells and murine primary bone marlature was not due to the cell cycle arrest but the row stromal cells [99]. The tube formation of binding with vasculature through the integrinβ1/ HUVECs was increased when they were treated von Willebrand factor axis and the integrinαvβ3/ with CM from isoproterenol-treated murine privascular cell adhesion molecule (VCAM)-1 axis mary bone marrow stromal cells [99]. The tube [91]. Indeed, the resistance of 4T07 cells to AC formation was prevented when cultures were regimen in the marrow of BALB/c mice were treated with an antibody targeting VEGF-­ reversed by (1) the downregulation of integrinβ1 A:VEGF receptor 2 signaling mcr84 [99]. and integrinαv in 4T07 cells using shRNA and (2) Isoproterenol-induced increased neovascularizathe treatments of the integrinβ1 inhibitory anti- tion and enhanced tumor colonization of body AIIB2 and integrinαvβ3 inhibitory antibody MDA-MB-231 cells were reversed in (i) LM609 [91]. More branching and sprouting of osteoblastic-­specific β2 adrenergic receptor bone marrow vessels along with tumor burden knockout mice (Rag2 background) and (ii) Rag2 and osteolytic bone lesions were observed by vol- mice treated with mcr84 [99]. Isoproterenol-­ umetric computed tomography (VCT) in the treated promoted the adhesion between murine femur of nude rat injected with MDA-MB-231 primary bone marrow endothelial cells or murine cells through femoral artery [92]. When MDA- endothelial cell line C166 cells and MDA-MB-231 MB-231-bearing rat were treated with anti-VEGF cells in  vitro [100]. The expression levels of antibody bevacizumab, branching and sprouting adhesion molecules E-selectin and P-selectin in of bone marrow vessels as well as tumor burden murine primary bone marrow endothelial cells and osteolytic bone lesions were inhibited [92]. and C166 cells were increased via β2 adrenergic These findings suggest that bone marrow endo- receptor when they were treated with CM from thelial cells are involved in the regulation of dor- isoproterenol-treated murine primary bone marmancy, chemoresistance, and outgrowth of bone row stromal cells [100]. High levels of IL-1β metastatic cancer cells. were observed in the CM of isoproterenol-treated

5  The Roles of Bone Marrow-Resident Cells as a Microenvironment for Bone Metastasis

murine primary bone marrow stromal cells, and IL-1β treatments enhanced E-selectin and P-selectin expression in C166 cells and promoted the adhesion between C166 cells and MDA-MB-231 cells [100]. Additionally, when PC-3 cells were cocultured with murine primary calvarial osteoblasts, their proliferation was slowed and most of them were in the G1 cell cycle phase [101]. Norepinephrine induced the proliferation and G2-M cell cycle of PC-3 cells cocultured with murine primary wild-type calvarial osteoblasts, while norepinephrine did not affect the proliferation and cell cycle of PC-3 cells cocultured with murine primary GAS6 knockout calvarial osteoblasts [101]. Since bone is a richly innervated organ, further investigations on the impact of nerves on bone metastatic progression are clearly warranted.

5.3

Conclusions and Future Directions

When cancer patients develop bone metastases, their prognosis is very poor. Current treatments for bone metastases mainly target bone remodeling. Denosumab (a human monoclonal antibody against RANKL) and bisphosphonates (which suppress osteoclast activity) are established treatments for bone metastases and are thought to work by decreasing bone resorption. However, they ultimately fail to improve overall survival [102, 103]. Only a new α-emitting radiopharmaceutical radium-223, which binds to hydroxyapatite in the marrow, can extend overall survival and then only by three months [104]. Thus, approaches that target factors other than bone remodeling are needed to lower mortality of cancer patients with bone metastases. In recent years, the supportive roles of immune cells, adipocytes, and nerves as a tumor microenvironment have been appreciated, and in the bone marrow, as described above, the interactions between these cells and bone metastatic cancer cells also seem to influence disease processes. Although further studies are clearly warranted, treatments targeting these bone marrow-resident cells, or in combination with bone-targeted therapies, may be new thera-

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peutic strategies for bone metastatic disease. A better understanding of how bone metastatic cancer and bone marrow microenvironment influence one another to worsen bone metastatic disease progression will aid in discovering new therapeutic targets for bone metastatic cancer— area in which current therapies are wanting—to decrease suffering and improve the survival of cancer patients with bone metastases. Acknowledgments This work is directly supported by Department of Defense (W81XWH-14-1-0403; W81XWH-17-1-0541; and W81XWH-19-1-0045) and the Wake Forest Baptist Comprehensive Cancer Center Internal Pilot Funding. This work is also supported by the National Cancer Institute’s Cancer Center Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. Conflict of Interests: The authors declare that they have no conflict of interests.

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Adipose Tumor Microenvironment Abbie Zewdu, Lucia Casadei, Raphael E. Pollock, and Danielle Braggio

Abstract

Keywords

The term “adipose tissue” represents a multicellular and multifunctional organ involved in lipid storage, in hormone and temperature regulation, and in the protection of bones and vital organs from impact-based damage. Emerging evidence now suggests a more malignant role of adipose tissue in promoting cancer onset and progression via the release of secreted factors such as interleukin-6 (IL6) and extracellular vesicles (EVs). These adipose-­ source factors subsequently affect various aspects of tumorigenesis and/or cancer progression by either directly enhancing the tumor cell oncogenic phenotype or indirectly by the stimulating adjacent normal cells to adopt a more pro-cancer phenotype. Due to the recent growing interest in the role of IL6 and EVs released by adipose tissue in cancer promotion and progression, we are focusing on the protumorigenic impact of fat tissue via IL6 and EV secretion.

Adipose tumor · Tumor microenvironment · Adipokines · Inflammation · Interleukin-6 · Glycoprotein 130 · Signal transduction · Extracellular vesicles · Cell-to-cell communication · Cancer · Liposarcoma · Adipocytes · Preadipocytes · Macrophages · Fibroblasts

A. Zewdu · L. Casadei (*) · R. E. Pollock D. Braggio Program in Translational Therapeutics, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA Department of Surgery, The Ohio State University, Columbus, OH, USA e-mail: [email protected]

6.1

Adipose Tissue as a Multifunctional Organ

Adipose is a dynamic tissue of mesenchymal origin, comprised of adipocytes, pericytes, endothelial cells, and preadipocytes, the adipocyte precursor cell type. Although formerly characterized as having limited physiological roles, mainly pertaining to the functions of energy storage and insulation, more recent studies have shown that adipose tissue is a complex organ capable of biological functions such as the synthesis and secretion of hormones, regulation of metabolism, immune cell function, and satiety [1, 2]. Adipose tissue functions vary greatly depending on anatomical location. For instance, omental fat releases greater levels of IL6 than subcutaneous fat [3]; similarly, visceral adipocytes are more lipolytically active than their subcutaneous counterparts [4]. The adipose organ is comprised of both white adipose tissue (WAT) and brown adipose tissue

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_6

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(BAT). While both tissue types are derived from a similar precursor, they are biologically and functionally distinct. BAT is a thermodynamic organ with the primary function of maintaining body temperature significantly higher than ambient temperatures through heat generation. Brown adipocytes are able to quickly oxidize their own fat stores and circulating substrates, resulting in heat production and metabolic rate increase [5, 6]. It is mainly present in newborns, although it is found in the neck, above the claviculae, and around the spine in adults [7–9]. Interestingly, BAT amount and activity were reported to be higher in lean individuals compared to overweight/obese individuals, suggesting a role of BAT in weight control [8]. In contrast, WAT is the most predominant form of adipose seen in adults [10], found throughout the body in different subcutaneous and visceral depots [11]. WAT is the main energy reservoir of the body via storage of triglycerides derived from ingested fatty acids and stored in the adipocytes. It is now clear that WAT is also a highly active metabolic and endocrine organ controlling essential metabolic processes, such as lipid and glucose homeostasis [12]. Excess of WAT, as in the case of obesity, has been associated with metabolic complications and several other diseases [13–16]. The adipose tissue described in this book chapter refers exclusively to WAT.

6.2

 Role for Adipose Tissue A in Cancer

Due to the modern obesogenic diet, adipose tissue is emerging as the largest organ in the human body. From 1980 to 2013, the worldwide overweight or obese adult population (BMI ≥ 25 and ≥  30, respectively) increased by ~10% (from 28.8% to 36.9% in men, from 29.8% to 38% in women) [17] and is predicted to rise further [18]. This increase in adiposity may prove problematic given the role of fat as a complex endocrine organ, leading to homeostatic aberrancies such as hormone imbalance and chronic inflammation. Under normal conditions, fat tissue releases a repertoire of primarily adipose-related cytokines

(termed adipokines) that range in function from regulating satiety (leptin) [19, 20] to controlling insulin sensitivity (adiponectin) [2]. The presence of excessive fatty tissue in overweight and obese persons is strongly associated with the development of metabolic syndrome, which refers to a class of symptoms that includes visceral obesity, insulin resistance, hyperglycemia, and a pro-inflammatory state [21, 22]. Moreover, a study by Reeves et al. of 1.2 million women reported that increased body mass index (BMI; the weight in kilograms divided by the square of the height in meters) correlates with a significant increase in cancer risk [23]. Specifically, women with high BMI suffered an increased risk for multiple myeloma, leukemia, non-Hodgkin’s lymphoma, and pancreatic and ovarian cancers [23]. Another report demonstrated that individuals in the combined overweight and obese population (BMI ≥ 30) suffered a 52% (male) and 62% (female) increased rate of death from cancer [24, 25]. In contrast, fat loss as an outcome of bariatric surgery correlated with a reduced cancer incidence and mortality [26]. A study by Arendt et al. defines a more direct relationship between fat and tumor development. This group reported that premalignant lesions in humanized mammary fat pads of obese mice displayed markedly enhanced transformation and mammary tumor growth with concurrent induction of angiogenesis [27]. This increase in vascularization, observed in both human and murine breast cancer samples, was caused by an adipocyte-­mediated recruitment and activation of macrophages via the CCL2/IL-1β/CXCL12 signaling pathway [27]. In addition to promoting cancer onset and progression, it has been suggested that fat cells could possibly transform into cancer cells, more specifically into liposarcomas, in the context of interleukin 22 (IL-22) overexpression [28]. Mice overexpressing IL-22 in adipose tissue spontaneously developed well-differentiated liposarcoma in adipose tissue after long-term feeding with high-fat diet. Although the exact mechanisms linking adiposity with the development of cancer are not clearly defined, multiple factors potentially

6  Adipose Tumor Microenvironment

contribute to this relationship. As previously ­mentioned, obesity is often related to metabolic defects that may favor not only cancer initiation but also its progression [29]. Recently, there is growing interest regarding the role of adipose tissue-secreted molecules in the development of cancer; adipose tissue may modulate tumor behavior through the release of adipokines [30, 31]. Initially thought of as a mere fat mass storage depot, adipose tissue is now recognized as an active endocrine organ [32] secreting various adipokines that are implicated in the pathogenesis of many malignancies (see Table 6.1). Recent studies have demonstrated the ability of adipocytes to secrete extracellular vesicles (EV) carrying adipokines [33, 34]. EVs are membrane-­derived secretory vesicles released in the extracellular environment that can act as autocrine, paracrine, or endocrine messengers

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mediating intercellular cross talk; they are recoverable from many body fluids. EVs can carry and transfer their bioactive cargo in target cells, thereby contributing to numerous diseases [35]. Recently, it has been shown that adipocyte EVs were able to promote melanoma aggressiveness through fatty acid oxidation [36]. The role of EVs secreted from adipose mesenchymal stem cells and preadipocytes in regulating tumor cell behavior has also been demonstrated for breast cancer cells [37, 38]. Moreover, recent studies have shown that adipose tissue-derived EVs were able to regulate gene expression in specific distant organs, suggesting mechanisms of intercellular communication by EVs similar to those mediated by adipokines [39, 40]. Ongoing studies are focusing on the role that specific molecules on the surface of adipose-derived exosomes may play in pre-metastatic niche formation.

Table 6.1  Adipose-derived secreted factors Factor Adiponectin Adipsin Angiotensinogen ANGPTL2 Apelin ASP Chemerin Endotrophin FABP4 Glycerol IGF-1 IL6 Leptin Lipocalin 1 MCP-1 Omentin-1 Osteopontin PAI-1 RBP4 Resistin TNF-α Vaspin VEGF Visfatin

Function Modulation of glucose levels and fatty acid breakdown Regulation of B-cell function and alternative complement pathway activation Blood pressure and fluid balance regulation Angiogenesis Migration of cardiomyocyte progenitor cells and blood pressure regulation Insulin secretion and triglyceride synthesis Chemotactic regulation of dendritic cells and macrophages to the site of inflammation Pro-inflammatory Involved in fatty acid uptake, transport, and metabolism Triglyceride formation Growth promotion of a wide variety of cells Pro-inflammatory, cell survival, proliferation, and differentiation Regulation of appetite and energy expenditure Transport of hydrophobic molecules Regulation of macrophage/monocyte migration and infiltration Maintenance of body metabolism and insulin sensitivity Bone remodeling and immune modulation Inhibition of fibrinolysis Retinol transportation regulation Regulation of insulin resistance Pro-inflammatory, cell survival, proliferation, and apoptotic induction Insulin sensitization Vasculogenesis and angiogenesis Involved in NAD+ biosynthesis and B-cell precursor maturation

ANGPTL2 angiopoietin-like 2, ASP acylation-stimulating protein, FABP4 fatty acid-binding protein 4, IGF-1 insulin-­ like growth factor 1, IL6 interleukin-6, MCP-1 monocyte chemoattractant protein-1, PAI-1 plasminogen activator inhibitor-1, RBP4 retinol-binding protein 4, TNF-α tumor necrosis factor alpha, VEGF vascular endothelial growth factor

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Due to the recent growing interest in the role of interleukin-6 (IL6) and extracellular vesicles secreted by adipose tissue in cancer promotion and progression, we will focus on the protumorigenic impact of fat tissue via IL6 and EV secretion.

alpha (IL6Rα) prior to binding to membranebound GP130, is necessary for normal homeostatic functions including B-cell differentiation into antibody-producing plasma cells, T-cell growth and differentiation, hematopoiesis, and osteoclast development (Fig.  6.1a) [47–53]. In contrast, the trans-activation signaling model involves the formation of an IL6:soluble IL6Rα 6.3 IL6: The Cross Talk Mediator complex prior to binding with the GP130 homodimer [54]. IL6 trans-activation of GP130 is a key The protumorigenic impact of fat tissue on can- process in promoting disease onset [55, 56] and cer onset and progression is exemplified by inter- is critically involved in chronic diseases with an leukin-­6 (IL6) secretion. Elevation of IL6 and/or inflammatory component (Fig. 6.1b) [56–59]. glycoprotein 130 (GP130) expression is the IL6 binding to GP130 initiates the activation signal-­transducing receptor to which IL6 exclu- of downstream disease-promoting signal transsively binds [3, 41–46], leading to GP130 func- ducer and activator of transcription 1 (STAT1) tional activation. There are several types of IL6 and 3 (STAT3) [60–62], phosphoinositide signaling. Classical signaling, in which extracel- 3-kinase/AKT (PI3K/AKT), and also extracellulular IL6 heterodimerizes with a membrane-­ lar signal-regulated kinase/mitogen-activated bound version of the IL6 coactivator IL6 receptor protein kinase (ERK/MAPK) pathways [60, 61].

Fig. 6.1  IL6 classical vs. trans-signaling. IL6 signal transduction occurs in a (a) classical or (b) trans-signaling fashion. ERK, extracellular signal-regulated kinase; GP130, glycoprotein 130; Grb2, growth factor receptor-­ bound protein 2; IKB kinase; inhibitor of kappa B; IKK, IKB kinase; IL6, interleukin-6; IL6Rα, interleukin-6

receptor alpha; JAK, Janus kinase; MEK, mitogen-­ activated protein kinase kinase; PI3K, phosphoinositide 3-kinase; Raf, rapidly accelerated fibrosarcoma; SHC, Src homology 2 domain containing protein; SOS, Son of Sevenless; STAT, signal transducer and activator of transcription; Tyk2, tyrosine kinase 2

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ER-negative cell lines MDA-MB-231 and Hs578T67. These findings suggest that the protumor effects of CAAs may be specific to breast cancer subtype. The differentiated murine preadipocytes cultured with BCs acquired a dedifferentiated morphology (i.e., smaller size, reduced lipid droplet formation), an altered gene signature (i.e., decreased CpebI, C/EBP-α, PPAR-γ, and FABP4 gene expression), and increased mmu-miR-5112 production [67]. Moreover, these CAAs expressed higher levels of IL6 when cocultured with breast cancer cells, suggesting 6.3.1 Adipocytes the ability of cancer cells to transform normal adipocytes into tumor-promoting cell types [67]. The role of adipocytes (the major cell type within These findings underscore the complex nature of adipose tissue) in tumor progression has been the non-tumor:tumor cell interaction in the TME extensively studied, and mounting evidence sug- and suggest the importance of considering facgests that tumor-adjacent adipocytes may act in a tors extending beyond the cellular source of the cancer-promoting fashion [64–70]. In 2016, IL6, including other tumor-specific biological Bougaret et al. reported that supernatant derived characteristics (e.g., ER status in the case of from adipocytes isolated from obese persons sig- breast cancer) as determinants of the cancer nificantly increased breast cancer angiogenesis impact conveyed by adipocyte-source IL6. by enhancing endothelial cell proliferation and Remarkably, the effects of CAA-released IL6 migration as compared to adipocytes from non- are not limited to the promotion of cancer prolifobese individuals [30]. The obese individual-­ eration. Multiple recent studies have revealed that derived adipocyte effects on tumor vascularization STAT3 activation by CAA-origin IL6 promotes may have been driven, at least in part, by IL6 sig- cancer cell induction of epithelial-to-­mesenchymal naling in that the levels of IL6 secreted by obese transition (EMT) [74, 75]. The EMT phenotype is patient-isolated adipocytes increased more than associated with enhanced chemoresistance, the levels of other secreted factors [30]. Likewise, heightened cell mobility, invasiveness, and metaadipocyte-secreted IL6 has also been shown to static potential in breast cancer [76–78]. Gyamfi play a role in cancer growth and metastasis in and colleagues demonstrated that adipocytes culbreast cancer [66, 71]. tured in the presence of breast cancer cells disIncreasing evidence now also suggests that played increased release of IL6 and TGFβ cancer cells may directly induce the conversion compared to fat cells cultured alone, regardless of of normal peritumoral cells into tumor-­promoting ER status [74]. The addition of CAA-derived conentities [43, 67, 72, 73]. Lee et al. recently dem- ditioned media (CM) activated STAT3 in an IL6onstrated that IL6 is released from cancer-­ dependent manner; IL6 loss in CAAs or use of an associated adipocytes (CAAs) due to CpebI gene anti-IL6-neutralizing antibody severely reduced expression mediated by mmu-miR-5112, thereby the CAA CM-induced activation of breast cancer promoting ER-positive breast cancer aggressive- cell STAT3. Moreover, breast cancer cell treatment ness [67]. In this study, murine preadipocyte cells with CAA CM induced an EMT phenotype were differentiated into mature adipocytes prior (marked by an enhanced mesenchymal morpholto indirect coculture with breast cancer cells. ogy, elevated vimentin, reduced E-cadherin, and Luminescent STP quantification demonstrated increased migration and invasion) that was greater CAA-mediated disease-promoting effects reversed by IL6 blockade [74]. in estrogen receptor (ER)-positive MCF7 and In addition to their role in tumor promotion ZR75–1 cells but minimal impact in the via tumor growth, angiogenesis, and metastasis, Roughly 10–35% of the IL6 in the body is supplied by adipose tissue, and circulating IL6 levels have been shown to decrease with weight loss, further implicating adipose tissue as a key source of this cytokine [63]. Interestingly, IL6 has been shown to mediate cross talk between residential non-tumor and tumor cells within the tumor microenvironment (TME) as well as communication between different non-cancer cell populations involved in tumor promotion.

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adipocytes may also attenuate cancer chemotherapeutic responses [68–70]. This adipocyte role was first reported by Sheng et  al., who demonstrated that obese mice injected with acute lymphoblastic leukemia (ALL) displayed increased relapse rates following treatment with vincristine compared to nonobese mice [69]. ALL cells cocultured with adipocytes demonstrated greater resistance to vincristine than ALL cells either grown alone or with fibroblasts [69]. More recently, Sheng and colleagues demonstrated that mature fat cells may confer chemoprotection via the sequestration and metabolism of chemotherapeutic agents. Adipocytes exposed to the anthracycline daunorubicin, a drug commonly used to treat ALL, displayed higher absorption of daunorubicin and more elevated expression of drug-­ metabolizing enzymes [68]. Taken together, these data suggest that adipocytes, through mechanisms such as IL6 secretion and drug sequestration, may help to create a TME that favors tumor progression.

6.3.2 Preadipocytes Preadipocytes, the adipocyte precursor and second most common cell type in adipose, secrete higher levels of IL6 compared to more differentiated adipocytes [79]. As seen with adipocytes, tumor-adjacent preadipocytes may also undergo reprogramming into a more pro-cancer phenotype. A study by Kim et al. evaluated the role of preadipocytes in breast cancer progression, demonstrating that ducal carcinoma in situ (DCIS) cells cultured in preadipocyte-derived CM acquired a more aggressive phenotype characterized by increased migration and invasion capabilities [80]. Use of an IL6-neutralizing antibody abrogated the in  vitro and in  vivo protumor effects derived from coculturing the DCIS cells with preadipocytes [80]. Preadipocyte differentiation has been shown to be greatly impaired in the presence of cancer cells; preadipocytes cocultured with cancers cells or in cancer cell-derived CM displayed increased α-SMA and FSP-1 levels, reduced LPL and

PPAR-γ expression, and decreased lipid body production [80]. These differentiation-impaired preadipocytes demonstrate enhanced IL6 production and secretion [80]. Given the supporting evidence that preadipocyte cells secrete much higher levels of IL6 than adipocytes [79], it is possible that preadipocyte differentiation within the TME may be stymied by cancer cells as a means of maintaining high levels of IL6.

6.3.3 Macrophages Adipose tissue resident and infiltrating macrophages have also demonstrated a protumor role via release of secreted factors, including IL6 [81, 82]. A study comparing adipose tissue macrophages (ATMs) to monocyte-derived macrophages (MDMs) not only revealed disparate gene signatures between these two macrophage types but also demonstrated that ATMs resembled a more tumor-associated macrophage (TAM)-like genotype and phenotype [81]. These characteristics included the ability to induce lipid accumulation in human breast cancer cells (a phenotype associated with poor prognosis [81, 83–85]) as well as the expression of angiogenesis- and inflammation-linked genes such as VEGFα, TGFβ, ICAM-1, IL6, and MCP-1 [81]. ATMs also exhibited high IL6 expression [81]. Interestingly, the observed ATM genotype and behavior may develop due to adjacent preadipocytes; conditioned media from preadipocytes isolated from obese patients induced the ATM phenotype in MDM cells derived from lean patients [81], suggesting that preadipocyte-origin secreted factors may play an important role in driving macrophage behaviors. Our research exploring the retroperitoneal dedifferentiated liposarcoma (DDLPS) TME revealed a novel TAM:DDLPS axis in which tumor-derived exosomes bearing microRNAs (miR-25-3p and miR-92a-3p) induce the release of IL6 from TME macrophages [82]. The DDLPS response to TAM-derived IL6-rich media included increased growth, migration, and invasiveness [82].

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6.3.4 Fibroblasts

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are emerging as a possible mediator of cell-tocell communication [90]. As with fat-lineage cells and macrophages, fibroExtracellular vesicles are produced by differblasts have also displayed a protumorigenic role ent cell types and can be formed either by cell via their release of IL6 [86, 87]. In a study exam- membrane budding or from endosomes that can ining estrogen receptor (ER)-negative breast form multivesicular bodies (MVB) fused either ­cancer, Bhat-Nakshatri et  al. demonstrated the with lysosomes or with the plasma membrane release of interleukin-1α (IL1α) by ER-negative [91, 92]. EVs can contain genetic material such (but not ER-positive) breast cancer cells (BCs), as mRNA, miRNA or noncoding RNA, DNA, leading to IL1α-mediated activation of nuclear mitochondrial DNA, as well as proteins, growth factor-κB (NF-κB) in neighboring fibroblasts. factors, and cytokines as well as metabolites, Stimulation of NF-κB signaling resulted in ele- organic acids, nucleotides, sugar, and amines vated expression of fibroblast IL6, leading to the [93–105]. When host cell origin EVs reach a tartransformation of these normal cells into cancer-­ get cell, they can be internalized in several ways, associated fibroblasts (CAFs) through the adop- i.e., either they can fuse directly to the plasma tion of an “activated” tumor-promoting phenotype membrane of recipient cells, or they can be taken [87]. The addition of IL6 to ER-negative BCs up by cadherin-mediated or caveolin-mediated resulted in elevated proliferation [88]. endocytosis [95, 106, 107]. Interestingly, obese donor-isolated adipose-­ Interestingly, EV cargo can be transferred derived stem cells (ASCs) exposed to breast either to tumor recipient or to TME cells and so cancer cells displayed altered genome and sec- can induce significant changes. For example, retome signatures that resembled those of CAFs EVs are able to induce an increase in the meta[89]. This genomic profile included increased static potential of indolent tumor cells [108]. expression of the CAF markers NG2, α-SMA, Tumor-derived EVs can induce tumor cell prolifVEGF, FAP, and FSP, as well as abundant eration in several human cancer types, including expression of IL6 [89]. Upon coculture with gastric cancer, bladder cancer, glioblastoma, and these modified ASCs, breast cancer cells dis- melanoma [105, 109–112]. Tumor EVs may also played enhanced proliferation and invasiveness modify the migratory capacity of recipient cells, [89]. These findings suggest that cancer cells as seen in nasopharyngeal carcinoma and prosmay help promote the pro-cancer behavior of tate cancer [113, 114]. peritumoral stromal cells, possibly by regulatTumor EVs may also affect cells in the adipose ing stem cell differentiation. tumor microenvironment such as macrophages, fibroblasts, endothelial cells, and stem/progenitor cells as well as in adipocyte cells per se. The role 6.4 Extracellular Vesicles of CAF and TAM vascular endothelial cells in tumor progression is being extensively studied as a Possible Mediator [115, 116]. Many cells, when introduced into the of Cell-Cell Communication tumor microenvironment, undergo transformain the Adipose Tumor tions that render them as tumor supportive. These Microenvironment cells can be “educated” as tumor-associated cells Tumors are composed of both tumor cells and to promote angiogenesis and release protumorinormal cells in the tumor microenvironment; genic growth factors, chemokines, and cytokines. communication between these two cell types This process occurs in macrophages that can can facilitate tumor growth and dissemination. become TAM in the tumor microenvironment, This communication may happen through cell- fibroblasts that become CAF, and adipocytes that to-cell contact and/or via secreted factors. can become CAAs with an activated phenotype in Among secreted factors, extracellular vesicles the TME [117]. In the tumor microenvironment,

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CAAs may undergo dilapidation and acquire a fibroblast-like morphology to become adipocytederived fibroblasts [118]. EVs can also help convert fibroblasts and mesenchymal stem cells into myofibroblasts that facilitate tumor angiogenesis and metastasis [119]. Specifically, EVs may play a role in the differentiation of fibroblasts into CAFs in which tissue transglutaminase and fibronectin from different human cancer cells (e.g., breast cancer cells and glioma cells) are transferred to normal fibroblasts and epithelial cells [120, 121]. A similar process has been observed in endothelial cells where the transfer of EV EGFRvIII activates autocrine vascular endothelial growth factor (VEGF) signaling that stimulates tumor angiogenesis [122, 123]. Webber et  al. demonstrated that EVs released by prostate cancer cells can trigger the differentiation of myofibroblastic cells, thereby inducing angiogenesis in vivo and accelerating tumor growth [124, 125]. Colorectalderived EVs are able to induce tumorlike behavior in mesenchymal stroma cells, thereby promoting tumor growth and invasiveness. EVs can also affect adipose tissue-derived mesenchymal stem cells (AD-MSCs) in ovarian cancer; EVs derived from two different ovarian cancer cell lines induced phenotypic and functional transformation of AD-MSCs into a tumor-associated myofibroblastic cell phenotype [126]. EVs are often temporally connected to NF-κB activation in macrophages. For example, EVs from gastric cancer induce NF-κB activation in macrophages and consequently enhanced release of pro-inflammatory cytokines such as IL6 and TNF-α, promoting the proliferation of gastric cancer cells [127]. Furthermore, Chow et  al. showed that breast cancer-derived EVs also stimulate the NF-κB pathway in macrophages, enhancing the secretion of pro-inflammatory cytokines such as IL6, TNF-α, GCSF, and CCL2 [128]. Similarly, Fabbri et al. shown that miR-21 and miR-29a contained in lung-derived EVs were able to induce Toll-like receptor (TLR)7 and TLR8, leading to NF-κB activation and secretion of the pro-metastatic inflammatory cytokines TNF-α and IL6. In turn, these cytokines were

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shown to induce a microenvironment supportive of metastasis [129]. EVs derived from osteosarcoma cells contain high levels of transforming growth factor β (TGF-β 1) which is at least partially responsible of IL6 secretion from mesenchymal stem cells and may increase metastatic dissemination [130]. Our group has identified communication between liposarcoma cells and their microenvironment, demonstrating that this is a process involved in liposarcoma progression. EVs released from DDLPS tumor cell contain miR-25-3p and miR-92a-3p. These two miRs were found to impact the surrounding microenvironment, inducing the secretion of IL6 from TAM in a TLR7/8-dependent fashion [82]. Furthermore, in a paracrine manner, IL6 stimulates DDLPS growth-promoting cell proliferation, invasion, and migration [82]. Recent studies also show the involvement of EVs in a dialogue between tumors and adipocytes. In hepatocellular carcinoma and prostate, lung, and pancreatic cancer, tumor-derived EVs appear to be involved in adipocytes dilapidation, fatty acid (FA) transfer to cancer cells, and adipocyte lipolysis [117, 131, 132]. In hepatocellular carcinoma, tumor-derived EVs can also induce adipocytes to create a favorable microenvironment for tumor progression by causing transcriptomic alterations and an inflammatory phenotype in EV-recipient adipocytes [133]. EVs can also stimulate myofibroblast differentiation and proangiogenic behavior of adipose stem cells [134]. Another aspect of the dialogue between tumor and microenvironment is highlighted by the capacity of EVs to affect metabolism in recipient cells, including glycolysis, oxidative phosphorylation, and lipid metabolism [117]. In that EVs can reach distant organs through the circulatory system, another important EV role is promotion of pre-metastatic niche formation in different cancers including melanoma and pancreatic and breast cancer [92, 126]. It is also important to keep in mind that TME cells, after being activated by EVs, can subsequently release EVs and promote tumor progression. For example, regulatory T lymphocyte (Treg) vesicles are involved in immunosuppression by

6  Adipose Tumor Microenvironment

transferring of miRNA-containing exosomes. Treg cells have been shown to transfer miRNA to several immune cells, thereby suppressing Th1 cell proliferation and cytokine secretion [135, 136]. TAM-derived vesicles were found to affect different aspects of tumor progression including invasion and resistance to drugs [137, 138]. CAF-derived EVs can promote cancer cell motility, MSCs activated by multiple myeloma cells can secrete EVs that promote cancer cell proliferation, and CAF-derived EVs have been shown to promote breast cancer cell motility and metastasis by activating EV-mediated wnt-planar cell polarity (PCP) [139–142].

6.5

 he Future of Adipose T Research

6.5.1 R  ealizing the Role of Adipose Tissue in Cancer Onset and Progression Comprehending the potential impact that non-­ cancer adipose cells within the TME may have on tumor biology and behavior is critical, especially when considering the functional role of adipose cells in promoting cancer progression, which often involves their transition from normal cells into tumor-promoting entities when in the presence of cancer cells [67, 143, 144], as well as their emerging role in mediating tumor chemoresistance [68–70]. Thus, future studies evaluating the TME as a whole rather than cancers as independent entities need to develop a

81

more comprehensive understanding of the complex tumor:TME interaction upon which more effective future therapies might be based.

6.5.2 Targeting the IL6:GP130 Axis The disease-promoting role of IL6 signaling through GP130 renders this pathway a target for therapeutic exploitation [145–149]. Surprisingly, the clinical use of anti-IL6 and anti-IL6Rα agents has been limited to rheumatoid arthritis, Castleman’s disease, and systemic juvenile idiopathic arthritis [150–153]. Table 6.2 lists ongoing studies evaluating the cancer-mitigating impact of IL6/GP130 signaling blockade.

6.5.3 EVs: The Next-Generation Mediators of Cell-to-Cell Communication EVs provide a novel communication and exchange mechanism between cells. The transfer of EV cargo can trigger changes in several TME cell types including fibroblasts, endothelial cell, MSC, adipocytes, and preadipocytes. Following transfer of EV cargo, adipose TME cells undergo phenotypic and functional transformation, thereby becoming more capable of supporting tumor growth, invasion, and metastasis. Further elucidation of the role of EVs in the adipose components of TME appears to be a promising area of research focus, if we are to make bona fide progress against various lipid-associated malignancies.

Table 6.2  Ongoing investigations of anti-GP130 signaling therapies in cancer Agent Bazedoxifene Bazedoxifene Siltuximab Siltuximab Siltuximab Tocilizumab Tocilizumab Tocilizumab Tocilizumab Tocilizumab

Study ID NCT02448771 NCT02694809 NCT00401843 NCT01484275 NCT03315026 NCT03135171 NCT02767557 NCT02336048 NCT03075696 NCT02057770

MOA mechanism of action

Cancer HR+ breast cancer Ductal carcinoma Refractory multiple myeloma Smoldering multiple myeloma Multiple myeloma Metastatic HER2+ breast cancer Pancreatic cancer B-cell chronic lymphocytic leukemia B-cell non-Hodgkin’s lymphoma Acute myeloid leukemia

Phase I/II II II II II I II I I I

MOA Competitive IL6 inhibitor Competitive IL6 inhibitor Anti-IL6 Anti-IL6 Anti-IL6 Anti-IL6Rα Anti-IL6Rα Anti-IL6Rα Anti-IL6Rα Anti-IL6Rα

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Appendix Tumor Microenvironment Luca Roncati, Paolo Gasparri, Graziana Gallo, Giuditta Bernardelli, Giuliana Zanelli, and Antonio Manenti

posed tumor complications, such as infection or ischemia. In practice, we consider the The pathological features of the appendix appendix TME a complex framework with tumors fundamentally recall those of the more immunological, mechanic, and metabolic frequent colorectal neoplasms, although with functions, all supported by a marked a higher relative incidence of carcinoids, due neo-lymphoangiogenesis. to the abundant presence of enteroendocrine cells in the appendix wall. Moreover, different types of lymphomas, Hodgkin and non-­ Keywords Appendix · Tumor microenvironment · Hodgkin, arising from the extra-nodal Cancer · Adenocarcinoma · Signet-ring cell mucosal-­associated lymphatic tissue, can be carcinoma · Mucocele · Carcinoid · encountered. The appendix tumor microenviNeuroendocrine tumor · Tumor-infiltrating ronment (TME) consists of a cellular compolymphocytes (TILs) · Brisk · Non-brisk · nent and of a noncellular component: the Immune score · Lymphoma · High-grade former includes the immunocompetent cells, large B-cell lymphoma · MALToma while the latter represents the support stroma. Particularly in carcinoids, the immune cell reaction can be explicated by tumor-­infiltrating lymphocytes, which, in some circumstances, may arrange around and inside the tumor in a brisk fashion influencing favorably the prognosis. This active reaction has to be distin- 7.1 Introduction guished from any preexisting inflammatory condition of the appendix and from superim- The histological types of appendix malignant tumors fundamentally correspond to the ­equivalent colon counterparts, in agreement with L. Roncati (*) · P. Gasparri · G. Gallo their common embryological origin. They can be G. Bernardelli · G. Zanelli Department of Medical and Surgical Sciences, essentially subdivided into two main groups: the Institute of Pathology, University Hospital of epithelial tumors, comprehensive of carcinomas Modena, Modena (MO), Italy and carcinoids, and the lymphomas, originated A. Manenti from B- and T-cell lineages present in the mucoDepartment of Medical and Surgical Sciences, sal or submucosal layer, absolutely rare in both Section of Surgery, University Hospital of Modena, their subtypes, Hodgkin and non-Hodgkin. About Modena (MO), Italy Abstract

© Springer Nature Switzerland AG 2020 A. Birbrair (ed.), Tumor Microenvironments in Organs, Advances in Experimental Medicine and Biology 1226, https://doi.org/10.1007/978-3-030-36214-0_7

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Fig. 7.1  Panoramic view (on the left) of a not otherwise specified adenocarcinoma of the appendix: at higher magnification (on the right), the wall infiltration is well noticeable (hematoxylin and eosin)

the first group, it has to underline a higher relative incidence of carcinoids, due to the abundant presence in the appendix wall of enteroendocrine cells [1–5]. The carcinoma group includes the not otherwise specified adenocarcinoma (Fig.  7.1), the rarer signet-ring cell variant (Fig.  7.2) [6], and the primary cysto-adenocarcinoma, often presenting with the typical macroscopic and microscopic features of mucocele (Fig. 7.3).

7.2

Appendix Tumor Microenvironment (TME)

The cancer occurrence in the digestive tract usually demands an adequate microenvironment, which provides the necessary “niche” for the development of foreign newborn neoplastic cells. At this point, we have to consider two different conditions: the first, more common, concerning the tumor outset in a district free from any other pathology and a second, rarer, when it involves an organ affected by a preexisting disease, usually inflammatory. In this last case, its favoring role is today admitted; otherwise, the presence of a particular immune-like reaction around or inside the tumor can be considered as the result of a specific action. All this includes a complex cross-talking between cancer cells and host tissue, developed mainly inside the TME. In fact, once mature, TME shows a cellular and a noncellular component, with different proportions and compositions. The cellular component

includes mesenchymal elements, such as fibroblasts, adipocytes, pericytes, and endothelial cells, but also elements derived from the lymphoid or myeloid lineages. Among these, there are lymphocytes, dendritic cells, and macrophages, which respectively acquire the proper definition of tumor-­ infiltrating lymphocytes (TILs), of tumor-infiltrating dendritic cells, and of tumor-­resident macrophages [7–9]. The lymphocytes are able to replicate inside the TME, generating small lymphoid aggregates [10]. The noncellular component, corresponding to the extracellular matrix, is mainly composed by proteins, glycoproteins, and proteoglycans: it represents a stroma-like structure for the tumor, which can preserve more or less compact its shape. Inside the neoplastic mass, we consider interesting the changes in the lymphatic and blood small vessels. The precapillaries and capillaries often appear tortuous and disorganized, causing a possible blood and lymphatic congestion and a subsequent local edema. Besides, the large network of new-generated lymphatic capillaries favors the migration of neoplastic cells [11–16]. This negative phenomenon is aggravated by the action of different tumor-derived molecules, such as the vascular endothelial growth factor (VEGF) and the microRNA105, produced from exosomes of the endothelial capillaries, which increase, in a paracrine fashion, the permeability of the blood and lymphatic capillaries. At the same time, microvascular blood pooling responds to the increased demand

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Fig. 7.2 Signet-ring cell carcinoma of the appendix (left panel, hematoxylin and eosin): the immunohistochemistry for pan-keratins highlights a scantly cohesive pattern of growth (right panel, AE1/AE3/PCK26, Ventana–Roche)

Fig. 7.3  A massive mucocele in course of primary cysto-­ adenocarcinoma of the appendix is blue-stained by histochemistry for Alcian Blue

of oxygen. In some cases, the vascular density appears proportional to the tumor aggressiveness and subsequent metastatic spread [17–19]. Moreover, other features of the blood and lymphatic vascular network can be interpreted from an immunological perspective. In fact, while the simple penetration of tumor cells into the blood or lymphatic capillaries can be considered dependent only from a particular power of

spreading, their temporary “parking” and further downstream progression demand a neoplastic receptive attitude and an immunological silence from the intrinsic mesenchymal ­cellular apparatus of the capillaries, although simply composed by pericytes and myofibrils [20]. In practice, we consider TME a complex framework with mechanic, metabolic, and immunological functions.

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7.3

TME Immune Power

neuroendocrine carcinomas and, more commonly, in carcinoid tumors, where the abundant As in colon carcinoma, the presence inside the lymphocyte population can arrive to constitute a appendix TME of multiple types of immunocom- proper “lymphoid stroma.” They are represented petent cells, such as fibroblasts, lymphocytes, by CD4+ T-helper, CD8+ T-cytotoxic, and and mainly cytotoxic and helper, dendritic cells CD20+ B lymphocytes disposed around and and macrophages, indicates their immune-­ inside the neoplastic mass, proving a real immuspecific function. In particular, we underline the nological reaction [17, 26–30]. role of fibroblasts, which, leaving their quiescent status, can actively diffuse around and inside a neoplastic mass, favored by the action of factors 7.4 Tumor Immunology derived from the tumor cells, such as the tumor growth factor β (TGFβ) and the platelet-derived The cross-talking between the cancer cells and growth factor (PDGF); encoded by proto-­ the tissue immunocompetent elements can oncogenes; and promoted by reactive oxygen develop in different ways. It has been ascertained species. These fibroblasts, through a process of that many cancers are invisible to the immune trans-differentiation [21], can be originated system, from their early onset until an advanced locally, from less-differentiated mesenchymal or stage. Interestingly, this occurs also when a endothelial cells, or can be derived from bone micro-skip metastasis, consisting of a small marrow stem cells, recruited at distance, and number of neoplastic cells, spreads to an aggreaddressed toward an immunocompetent lineage. gate of B/T lymphocytes, where they can be harThey are provided by a great power of replicat- bored and develop without any secondary ing, producing also large amounts of proteins, adverse reaction [31, 32]. In other cases, the among which some further transform in growth immunological reaction, although morphologifactors, or aggregate to form the mature collagen cally detected, remains ineffective and without and elastic fibers of the extracellular matrix. All any impact on the patients’ survival. This is the this can differently interact with the cancer cells, case of lymphocytes present in the TME, but not as tumor-promoting or even suppressor agents, aggregating around and, especially, not penetratsometimes leading to the paradox that the TME ing inside the tumor mass. Under these aspects, fibrotic component, produced by fibroblasts, the tissue immunological reaction seems dependirectly correlates with the tumor spread, favor- dent from many factors. At first, this paradoxiing the epithelial-mesenchymal transition [22– cally correlates with the proper characteristics of 25]. Interestingly, colon cancers can follow the tumor, which sometimes appears compatible preexisting chronic pathologies, like inflamma- with the host tissues, notwithstanding its poor tory bowel diseases, which can be considered grade of differentiation and deep cytological precancerous lesions, although nonobligatory. abnormalities. It is typically observed in some Otherwise, a common inflammatory reaction can small-cell neuroendocrine carcinomas [33–35]. superimpose to these tumors, as the result of dif- When histological evident, the immune reaction ferent processes, such as local infection, isch- involves a cohort of immunocompetent cells, as emic necrosis, or accelerated apoptosis, promoted ­macrophages, plasma cells, and different types by different mechanisms, also immune related. In of B and T lymphocytes, sometimes with an these cases, common inflammatory mediators, as increased cytotoxic power, directly promoted by nitric oxide, prostaglandins, cytokines, and che- the tumor itself. The final results of all these promokines, can have an important role. Besides, the cesses can vary from a good immunological presence of a great number of lymphocytes sur- response determined by TILs, as observed in rounding or penetrating the tumor indicates a some carcinoids, to a complete immunological particular expression of immune power. This has silence. Between these two extremes, intermedibeen recently observed in colorectal large cell ate conditions are represented by an immune

7  Appendix Tumor Microenvironment

action only against particular neoplastic clones, with subsequent selection of others invisible to the immune system, and by an acquired equilibrium between the neoplastic cells replication and their increased apoptosis. This last mechanism explicates the development of metastases greater than the original tumor. Any tissue immune response demands to be supported, also at long term, by an adequate host humoral power, in order to avoid its progressive weakening. Besides, some observations would suggest that the immunological action can manifest not directly against the neoplastic cells but mainly against the extracellular matrix of the tumor, hindering their replication. This mechanism, usually weak and not directly effective on the neoplastic cell replication, cannot involve the metastases, which offer a better field of tumor development. Of note, the immune response is cell-specific and pathophysiologically different from the common inflammatory processes; preexisting, as in case of inflammatory bowel diseases; or subsequent to the tumor outset. This can be promoted by two different mechanism: a common superimposed infection, typical of hollow bowel, or an aseptic necrosis, following a condition of tumor ischemia or sub-ischemia [32–34].

7.5

Appendix Tumor Immunology

Referring to appendix cancers, a deep distinction has to be made between adenocarcinomas and carcinoids. The first tumors repeat the immunological behavior of the more common colon cancers, while the others merit a greater specific attention. In appendix carcinoma, the profile of immunological reaction is generically correlated to different biology and morphological characteristics of the neoplastic cells as their origin from different clones, gene and molecular expression, mutation status, etc. It can be expressed through the “in situ” presence of various immunocompe-

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tent cells, such as B and T lymphocytes and their subgroups, plasma cells, macrophages, and fibroblasts. They can dispose around the tumor mass but also penetrate inside, delineating a more active behavior. However, the low incidence of these findings indicate that rarely an appendix carcinoma can develop a proper immune reaction, exactly as observed in the more common colon tumors. On the contrary, in case of carcinoids (Fig.  7.4), we observed a tissue-specific immune reaction, mainly expressing with a population of B and T lymphocytes, which may dispose around the neoplasia but also penetrate inside it (Fig. 7.5). This last lymphocytic arrangement can be considered expression of a brisk reaction (brisk TILs), while the first only a weak non-brisk reaction (non-brisk TILs). Clearly, the absence of a lymphocytic reaction indicates an immunological silence, as we noted also in small-­ cell lung cancers, histogenetically correlated to carcinoids, in which, on the contrary, a possible active reaction usually correlates with a better prognosis [32]. These findings would suggest to add an Immunoscore to the current TNM classifications; today, it is simply admitted that a high presence of T lymphocytes, in particular cytotoxic, is a good predictor of survival [34–40]. Moreover, they could also give important indications for a complementary immunotherapy [26, 41, 42]. We recognize the technical difficulties in assessing all the immunological aspects of carcinomas and carcinoids of the appendix. However, we underline that the proper appendix mesentery, but also that of the cecum and of the last ileal loop, represents the preferential site where to expect a possible immune response, involving the already mature lymph nodes but also the small lymphoid aggregates normally present in the peritoneal leaves and in the mesenteries; these aggregates can develop into new tertiary lymphatic organs more rapidly and in greater number under the action of immunological stimuli [43]. This basal mechanism can explicate the great increase of lymph nodes observed in inflammatory pathologies, like the Crohn disease.

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Fig. 7.4  Typical carcinoid/grade 1 neuroendocrine tumor of the appendix fundus (left panel, hematoxylin and eosin) strongly immunoreactive for chromogranin (middle panel,

LK2H10, Ventana–Roche): a very low Ki67 index (