Advances in Experimental Medicine and Biology 1350 Debabrata Banerjee Raj K. Tiwari   Editors Tumor Microenvironment: Cellular, Metabolic and Immunologic Interactions Advances in Experimental Medicine and Biology Volume 1350 Series Editors Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux UMR 5287 Pessac Cedex, France Haidong Dong, Departments of Urology and Immunology Mayo Clinic, Rochester, MN, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital Munich, Germany Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Science, School of Life Science, Shanghai University, Shanghai, China Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. 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Tiwari Department of Pharmacology Department of Pathology, Microbiology Robert Wood Johnson Medical School and Immunology Rutgers, The State University New York Medical College of New Jersey Valhalla, NY, USA Piscataway, NJ, USA ISSN 0065-2598 ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-83281-0 ISBN 978-3-030-83282-7 (eBook) https://doi.org/10.1007/978-3-030-83282-7 © Springer Nature Switzerland AG 2021 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. 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This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Preface Appreciation of the important role of tumor microenvironment in supporting growth and dissemination of tumors has come as a result of increasing knowl- edge of the complexities of the tumor microenvironment. The field has grown from the early observations on spread of tumors to our current knowledge regarding the wide array of host cells and how they contribute to many aspects of tumor growth and spread. In 1889, the English surgeon Stephen Paget proposed the “seed and soil” hypothesis, based on his study of autopsy records of hundreds of breast can- cer patients, which basically suggested that tumors or “seeds” are dispersed in all directions but can thrive only if they fall on the right “soil.” This view was subsequently challenged by James Ewing who suggested that metastasis is determined by purely mechanical means and had little to do with the “soil.” Thanks to the pioneering work of Prof. Isaah Fidler and colleagues we now have an improved understanding of cancer metastasis; the soil is now termed the “tumor microenvironment” and is widely accepted as an equal partner in determining metastasis outcome [1–6]. Tumor microenvironment has many different facets, and each are impor- tant in their own right in shaping tissue homeostasis if one recognizes that the tumor is an externally growing immunologically tolerized organ constantly trying to adapt itself in the host. Immunologically it survives by invoking the classical tolerance mechanism and physically by interception of survival mechanism be it metabolic or oxidative respiration. In the present series, our aim has been to decipher the immediate cellular milieu and how it shapes tumor phenotype, specifically the metastases phenotype. This cutting-edge analysis is expected to give the highest return in terms of therapeutic inter- vention and our ability to manage cancer as it can impact metastases directly. It is our contention that if metastasis is preventable, then cancer becomes a manageable chronic disease. In this context updates on evasion of immune surveillance (Tiwari chap- ter), metabolic support (Mishra chapter), growth factor and cytokine signal- ing, provide structural support to the tumor enabling invasive behavior. We have just begun to understand how cell–cell interaction in the TME is not limited to released factors from one cell that then reaches a target cell and produces an effect but can also occur via exosomes which carry a number of molecules that evoke a variety responses in the target cell (Rameshwar chap- ter). It is well known that hematopoiesis is sustained in the bone marrow (BM) via mechanisms involving the microenvironment which includes sev- v vi Preface eral cell types, neurotransmitters from innervated fibers, growth factors, extracellular matrix proteins, as well as extracellular vesicles. Besides its hematopoietic function, the BM microenvironment can also accommodate survival of malignant cells that communicate with cells of the BM microen- vironment through exchange of exosomes, a subset of extracellular vesicles that deliver molecular signals between cells. A better understanding of exo- somal packaging, cargo, and production can be leveraged therapeutically to impede cancer progression. The crucial role of exosomes in the development and progression of BM-associated cancers, such as hematologic malignan- cies and marrow-metastatic breast cancer, are presented. Attention is also paid to exosome-based therapeutic strategies and their limitations. Mishra and Banerjee reflect on tumor stroma metabolic interaction. Tumor microenvironment (TME) contains stromal cell of different types including fibroblasts, immune cells, and endothelial cells having varied influence on the local metabolic activity. Recent advances in the understanding of complex tumor microenvironment have revealed that a multifaceted interaction between tumor cells with their neighboring stroma is essential for tumor growth and metastasis. The tumor stroma presents distinctive features which enhance tumor growth such as recruitment and ultimate activation of bone marrow–derived mesenchymal stem cells to cancer-associated fibroblasts (CAFs). This underscores molecular interactions in the tumor microenviron- ment and allows reciprocal exchange of nutrients, secretory molecules, and other signals between tumor and stromal cells. The reciprocated molecular interactions between tumor cells and non-malignant stromal cells in the tumor microenvironment not only promote tumor development and progres- sion, but largely control most of the characteristic hallmarks of tumorigenesis and stimulate chemotherapeutic drug resistance. Shared interactions between tumor and stromal cells facilitated either directly by cell-to-cell contact or via the release of secretory molecules including, cytokines, chemokines and extra cellular matrix (ECM) aid in remodeling proteins to activate signaling pathways that encourage cell growth, survival and overall development. The secretory molecules shared among tumor cells and neighboring cells instigate epithelial-m esenchymal transition (EMT), tumor cells migration, invasion, and dissemination to secondary sites. The metabolic activities in the microen- vironment are influenced by tumor and stromal factors present in the micro- environment. Metabolic reprogramming allows fulfillment of demands of growing tumor cells. Different stromal components in the tumor microenvi- ronment provide additional nutrients that supplement local nutrient pool. Stromal cells present in the immediate proximity of tumor cells are inevitably most affected by the metabolic alterations caused by neighboring cancerous cells. Stromal fibroblasts present in the tumor microenvironment also known as cancer- associated fibroblasts (CAFs) play a key role in metabolic repro- gramming. CAFs are predominantly resident mesenchymal cells in origin that get activated and reprogrammed in response to signals from cancer cells. Tumor cells display heightened glucose uptake and even under normoxic conditions display increased generation of lactate from pyruvate by aerobic glycolysis, also known as the Warburg effect. This adaptation not only allows generation of biosynthetic precursors for added nutritional demands of tumor Preface vii cells but also directs the metabolic reprogramming of neighboring stromal cells. The lactate generated as a result of metabolic reprogramming of tumor cells and stromal cells play diverse role in the tumor microenvironment. Both tumor and stromal CAFs consume and secrete lactate differently, which makes it an integral modulating factor in tumor microenvironment. Molecular evidences collected over the last several years have prompted deeper exami- nation of tumor stroma interactions. Mishra and Banerjee have covered role of cytokines, chemokines, and lactate in driving tumor-stroma interactions in the microenvironment. Pro-tumorigenic molecular interactions between tumor cells and CAFs mediated via altered signaling pathways, cytokines, chemokines, and lactate in the tumor vicinity are discussed. A better under- standing of the complex cancer cell–CAF interactions will help in designing successful therapeutic strategies targeting the stromal rich tumors in the clinic. Freeman writes on the structural aspects of the tumor microenvironment. Cancers can be described as “rogue organs” because they are composed of multiple cell types and tissues and appear to be independent of control mech- anisms operative in normal organs and tissues. The transformed cells can recruit and alter healthy cells from surrounding tissues for their own benefit. It is these interactions that create the tumor microenvironment (TME). The TME describes the cells, factors, and extracellular matrix proteins of the tumor and the area around it. Alterations in the TME can lead to growth and development of the tumor, the death of the tumor, or tumor metastasis, a pro- cess by which cancer spreads from its initial site to different sites. Metastasis occurs when cancer cells enter the circulatory system or lymphatic system after breaking away from a tumor. Once the cells reach the circulatory sys- tem, they can travel to a different part of the body and form new tumors. Understanding the TME therefore becomes critical to fully understand cancer and develop strategies to control it. Knowledge of the TME can better inform researchers of the ability of potential therapies to reach tumor cells. It can also identify potential targets within the tumor. Instead of directly killing the cancer cells, therapies can target an aspect of the TME which could then halt tumor development or lead to tumor death. In other cases, targeting another aspect of the TME could make it easier for another therapy to kill the cancer cells. The TME can be split simply into cells and the structural matrix and include fibroblasts, structural proteins, immune cells, lymphocytes, bone marrow-derived inflammatory cells, blood vessels, and signaling molecules. From structure scaffolds to providing nutrients for growth, each of these com- ponents impacts cancer growth, development, and resistance to therapies. This chapter describes the TME and underscores the importance of cellular and structural elements of the TME. Gene expression analyses have also brought to light the emerging role of long non-coding RNAs in cellular communication in the TME (Geliebter chapter). The idea of regulating gene expression was prominent in tumor cells and has contributed to a wealth of information in the discovery of all the tran- scriptional factors and their role in oncogenesis and metastases. Although transcriptional factors have been difficult target, they have been of immense benefit. Recent advances in regulators of gene expression include long non- viii Preface coding RNAs which may be more amenable to novel therapies that use gene deletion techniques such CRISPR but not limited to one technique. Linking these molecules with cancer differentiation phenotype is a foundational dis- covery in carcinogenesis that brings gene expression and genotype variation with observed cancer phenotype in the mainstream of cancer biology. This is indeed cutting-edge discovery in our battle to understand the heterogeneity of cancer and devise personalized therapy. Dr. Maniyar furthers the concept of linking cancer phenotype resulting from genetic lesions with the cellular environment of the tumor microenvi- ronment specifically as dictated by the immune cells. This is an attempt to define the negative and positive regulators of immune activation and check- points so as to advance a combinatorial therapy that can target the genetic lesion–based signal transduction pathway together with the checkpoint inhib- itor–based immunotherapy. Taking a stock of the immune cells in the tumor microenvironment one characterizes the exhausted effector tumor cells that have now become signals of preexisting immune activation and the various modalities to specifically use the characteristics of these exhausted effectors cells to tailor-made novel combinatorial dual targeted cancer therapies. One directed against the driver mutations and the other to boost immune effector cells. The emphasis in this chapter is also in the need to characterize the expression of checkpoint molecules on the tumor cells itself so that the ther- apy can be tailored to thwart the tumor’s attempt to propagate an immunosup- pressive environment. These finding and the concepts put forward in the context of melanoma by Maniyar et al apply to other cancers as well. A very important piece of the carcinogenesis puzzle is to identify a set of markers that can define the phenotype of the growing tumor preferably in biological fluids. Almost 70 years of research has resulted in tumor-a ssociated markers. Most of these markers reflect our technical advances to compare tumor versus normal in regard to macro molecules, enzymes and proteins, lipids, and cell-surface carbohydrates culminated into genes and regulators of gene expression. Tumor-specific markers remain elusive though tumor- associated markers have contributed to defining subsets of tumors. Cell-cell communication in the evolution of tumorigenic phenotype is a recent discov- ery, and more significantly, we have been characterizing the template of this communication where secretory exosomes play a significant role. It is the contention of Jarboe et al that these exosomes have defined cargo and reflect both the inflammatory cell phenotype and the evolving cancer using anaplas- tic thyroid cancer as a model system and the deregulated miRNAs in ATC tissues they propose a novel category of biomarker(s) that could define meta- static propensity. The use of these miRNA markers in secreted fluids remains to be analyzed; however, such analysis and categorization of inflammation promoting markers especially in secretory exosomes in serum can provide us important clues on tissue tumor evolution. The success of immunotherapy, specifically checkpoint inhibitor, is at least partly dependent on the selection of the right target that thwarts the tumor-induced immune suppression. Chakraborty et al promote the conten- tion, using anaplastic thyroid cancer as a model, that there are several tumor- intrinsic and tumor-extrinsic factors that shape the final response. Extrinsic Preface ix factors include quality of T cell infiltrates, composition of cytokines, and percentage of immune suppressor cells such as MDSCs. All these eventually shape the immune response in a highly individualized manner. High percent- age of tumor-associated macrophages and immune suppressive cytokines are well established features of the immune landscape in ATC.  In the three-way cell-cell communication, antigen presentation cells, T cells, and tumor cells, the cytokine milieu is of major significance that can be measured in serum as end point markers. These inflammatory markers can provide important clues to ATC evolution, and the characterization of the expression of positive and negative regulators on tumor cell surface leads to additional immunothera- peutic targets as identified by Chakraborty et. al. The importance of cellular components of the tumor microenvironment in promoting tumor cell growth and dissemination is now well accepted in the field of cancer biology. The interaction between these components and the tumor cells is becoming an area of intense research, and chapters in this series have touched upon various aspects of these interactions. An increased under- standing of these interactions will likely result in improved therapeutic strate- gies to control growth and spread of tumor cells from the primary site. Comprehension of these complex interactions is limited due to studies being conducted in isolated systems under restrictive experimental conditions. We regret that other important aspects of TME such as innervation of tumors and cancer stem cells in the TME could not be included in this volume. References for general reading on history of Tumor Microenvironment: 1. Langley, R. R., & Fidler, I. J. (2007). Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis. Endocrine Reviews, 28, 297–321. [PubMed: 17409287]. 2. Fidler, I. J. (2003). The pathogenesis of cancer metastasis: The ‘seed and soil’ hypothesis revisited. Nature Reviews. Cancer, 3, 453–458. [PubMed: 12778135]. 3. Talmadge, J.  E., Benedict, K., Madsen, J., & Fidler, I.  J. (1984). Development of biological diversity and susceptibility to chemotherapy in murine cancer metastases. Cancer Research, 44, 3801–3805. [PubMed: 6744297]. 4. Paget, S. (1889). The distribution of secondary growths in cancer of the breast. The Lancet, 133(3421), 571–573. 5. Fidler, I. J., & Poste, G. (2008). The “seed and soil” hypothesis revisited. The Lancet Oncology, 9(8), 808. 6. Ewing, J. (1928). Metastasis, neoplastic disease: A treatise on tumors. Philadelphia and London. Valhalla, NY, USA Raj K. Tiwari Piscataway, NJ, USA Debabrata Banerjee