Tumor Microenvironment Tonya C. Walser, Jane Yanagawa, Edward Garon, Jay M. Lee, and Steven M. Dubinett

Abstract  While genetic changes are critical for the malignant transformation of epithelial cells, the microenvironment in which the cells reside also governs carcinogenesis. Most tumors arise within a cellular microenvironment characterized by suppressed host immunity, dysregulated inflammation, and increased production of cellular growth and survival factors that induce angiogenesis and inhibit apoptosis. The studies highlighted in this chapter indicate that the lung tumor and its microenvironment interact, together informing the process of carcinogenesis. Understanding the molecular mechanisms driving the contributions of the tumor microenvironment to lung carcinogenesis may afford us the opportunity to develop new drugs that target these reversible nonmutational events in the prevention and treatment of lung cancer. Findings from recent microenvironment-related clinical studies have implications for understanding the immunopathobiology of lung cancer, for targeting surgery and adjuvant therapy, and for designing future trials of adjuvant therapy. If the field is to progress and promising leads in the laboratory are to translate into anticancer therapeutics, future trials targeting the tumor microenvironment must incorporate improved patient risk assessment and selection, in addition to the continued evaluation of combination therapies using the optimal biological dose of each compound being tested. Appropriately targeting the tumor microenvironment in a highly selected patient population is a newly emerging strategy that holds unique potential for advancing the current state of lung cancer prevention and treatment. Keywords  Tumor microenvironment • NSCLC prognosis • Mast cells • Macrophage • Dendritic cells • Ectopic lymph nodes • T regulatory cells • MMP • COX-2 • PGE2 • PPARg • 15-PGDH • Inflammation • EMT • NF-kB • HGF • c-MET • Angiogenesis • Molecular signatures

S.M. Dubinett (*) Division of Pulmonary & Critical Care Medicine, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA e-mail: [email protected]

D.J. Stewart (ed.), Lung Cancer: Prevention, Management, and Emerging Therapies, Current Clinical Oncology, DOI 10.1007/978-1-60761-524-8_2, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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Introduction While genetic changes are critical for the malignant transformation of epithelial cells, the microenvironment in which the cells reside also governs carcinogenesis. Most tumors arise within a cellular microenvironment characterized by suppressed host immunity, dysregulated inflammation, and increased production of cellular growth and survival factors that induce angiogenesis and inhibit apoptosis. The pulmonary microenvironment, in particular, represents a unique milieu in which lung carcinogenesis proceeds in complicity with the structural (extracellular matrix or ECM), soluble (cytokines, proteases, hormones, etc.), and cellular (fibroblasts, inflammatory cells, endothelial cells, etc.) components of the microenvironment. Understanding the molecular mechanisms driving the contributions of the tumor microenvironment to lung carcinogenesis may afford us the opportunity to develop new drugs that target these reversible nonmutational events in the prevention and treatment of lung cancer. In recent years, gene expression profiling studies of several tumor types have described molecular signatures associated with progression. Identification of robust biomarkers predictive of cancer progression and prognosis could have a clinically significant impact on non-small cell lung cancer (NSCLC) management, as these biomarkers would aid in the appropriate selection of patients who would benefit from therapy beyond surgery. The molecular signatures that have emerged from these progression-associated gene sets are composed mainly of cytokine genes involved in inflammatory and immune responses. In one such study by Bhattacharjee and colleagues in 2001, microarray-based expression profiling of 139 resected adenocarcinoma specimens allowed the investigators to discriminate between biologically distinct subclasses of adenocarcinomas, as well as primary lung adenocarcinomas versus metastases of nonlung origin (1). A gene expression profiling study that closely followed came from Beer et al. (2). This group used expression profiling to predict survival among patients with early stage lung adenocarcinomas. Using the top 50 differentially expressed genes, the investigators developed a survival-based risk index, whereby patients were determined to have high-risk or low-risk stage I adenocarcinomas and poor or favorable predicted survival, respectively, based on their molecular signature. Novel survival-associated genes were identified, but, more importantly, the molecular profile that emerged predicted survival of the patient population. Likewise, the high hsa-mir-155 and low hsa-let7a-2 miRNA expression signature described by Yanaihara and colleagues correctly predicted the poor survival of patients with stage I adenocarcinomas (3). And finally, an mRNA expression profile described by Potti and colleagues identified a subset of stage IA NSCLC patients at high risk of recurrence (4). Together, these studies provided an early indication of the diagnostic potential of expression profiling and clear evidence that molecular signatures composed mainly of inflammation- and immune-related cytokines correlate with important clinical parameters. A recent investigation of the role of the lung tumor microenvironment in promoting carcinogenesis was conducted by Seike et al. (5). To inquire whether gene

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expression changes in the noncancerous tissue surrounding tumors could be used as a biomarker to predict cancer progression and prognosis, this group conducted a molecular profiling study of paired noncancerous and tumor tissues from 80 patients with adenocarcinoma. Many of the genes identified were part of a unique inflammatory and immune response signature that this group previously observed in noncancerous hepatic tissue from HCC patients (6). Ultimately, however, they identified an 11-gene signature, called Cytokine Lung Adenocarcinoma Survival Signature of 11 genes (CLASS-11), which predicted lymph node status and disease prognosis. The results of this well designed trial demonstrate that molecular signatures associated with the tumor microenvironment can serve as robust biomarkers predictive of cancer progression and prognosis. Though not in lung cancer, a recent publication by Farmer and colleagues was the first to report a major contribution of stromal genes to drug sensitivity in the context of a randomized clinical trial (7). Using 63 tumor biopsies from individuals in the EORTC 10994/BIG 00-01 trail with estrogen receptor-negative breast cancer treated with 5-fluorouracil, epirubicin, and cyclophosphamide (FET), the Farmer group described a stromal gene signature that predicts resistance to preoperative chemotherapy. This study expands the clinical significance of the identification of tumor microenvironment-associated gene signatures, and it encourages the development of antistromal agents as a new method by which to overcome resistance to chemotherapy. A translational study by the Kurie Laboratory in 2008 also defined tumor cell and stromal cell interactions that inform the course of NSCLC progression (8). By coculturing a K-ras mutant lung adenocarcinoma cell line with one of three lung stromal cells lines (macrophage, endothelial cell, or fibroblast) and subsequently profiling the secreted proteins, they developed an in vitro model for evaluating the mechanisms by which stromal cells regulate the biological properties of lung adenocarcinoma cells. The group confirmed that the in vitro model robustly recapitulates many of the features of their K-ras mutant murine model and, most importantly, NSCLC, suggesting that it can serve as a useful model of the NSCLC tumor microenvironment. By two different proteomic approaches, the investigators profiled the secretome of the tumor cells and evaluated its regulation by the stromal cells. They concluded that stromal cells in the tumor microenvironment do alter the tumor cell secretome, including proteins required for tumor growth and dissemination. Specifically, enhanced stromal cell migration, induced endothelial tube formation, increased tumor cell proliferation, and differentially expressed of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial–mesenchymal transition (EMT) were all observed when tumor cells were cocultured with stromal cells. These findings suggest that stromal cells drive the aggressiveness of tumor cells via their effect on the tumor cell secretome. By extension, inhibition of specific interactions between tumor cells and the tumor-adjacent stroma holds significant potential in the search for novel cancer therapeutics. Cancer progression depends on both genetic and epigenetic changes that affect gene expression by the tumor and surrounding stroma, and it depends on the immunologic status of the host. The studies highlighted in this chapter indicate that the

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lung tumor and its microenvironment interact, together informing the process of carcinogenesis. Appropriately targeting the tumor microenvironment in a highly selected patient population is a newly emerging strategy that holds unique potential for advancing the current state of lung cancer prevention and treatment.

Macrophages and Mast Cells Macrophages and mast cells are components of the innate immune cell infiltrate present in nearly every malignancy. In basic, translational, and clinical research investigations, these particular immune effector cells have been found to both thwart and support tumor growth depending upon their microenvironmental context (9–12). While studies correlating macrophage and mast cell infiltrates with NSCLC prognosis are relatively few, the data that exist are sharply divided between support for a correlation to favorable prognosis and support for a correlation to poor prognosis (13–23). Several recent publications suggest that these discrepancies may reflect differences in the number, grade, stage, and size of tumors included in each study, all of which varied considerably across the studies. The lack of consensus both within and between tumor types may also be related to the diverse approaches used to assess the infiltrates. For example, in two publications by Chen and colleagues, tumor-associated macrophage density correlated with poor prognosis in NSCLC, but macrophages within the tumor and those in the adjacent stroma were counted together (13, 14). Toomey and colleagues found no association between macrophage counts and NSCLC outcome, but macrophages within the tumor and those in the adjacent stroma were again counted together 23, as in Chen et al. (13,14). Johnson and colleagues found no correlation between tumor- or stroma-associated macrophages and NSCLC prognosis, however, their assessment was semiquantitative, and the number of cases evaluated was relatively small (16). The original study to demonstrate the importance of the microanatomical location of macrophages as related to prognosis was in gastric cancer rather than lung cancer. Ohno and colleagues specifically counted macrophages within gastric carcinoma tumor cell islets/nests and the adjacent stroma and found that tumor-infiltrating macrophages were associated with increased survival (24). The 5-year disease-free survival rate was significantly increased in patients with a high number of macrophages in the tumor islets when compared to those with a low number of macrophages in the tumor islets (87% versus 44%, respectively; p = 0.0002). In fact, the density of tumor-infiltrating macrophages was an independent predictor of patient survival by Cox’s multivariate analysis (p = 0.016). When combined with additional immunohistochemical staining data, these results led the investigators to conclude that aggregation of macrophages within gastric tumors has a beneficial effect on host survival via augmented cytotoxicity and antigen presentation. One of the more recent investigations of the prognostic significance of macrophage and mast cell infiltration in NSCLC was launched by Welsh et  al. (25). Like the Ohno group, the authors of this study suggest that the microanatomical

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location of macrophages and mast cells associated with a tumor must be taken into account when considering their correlation to prognosis. Because the microenvironment is a key determinant of immune cell phenotype and function, the authors suspected that it might also influence the nature of the immunocyte–tumor interaction. Using immunohistochemistry to identify CD68+ macrophages and tryptase+ mast cells in the tumor islets and adjacent stroma of 175 patients with surgically resected NSCLC, the authors identified tumor islet CD68+ macrophage density as a powerful independent predictor of survival. Specifically, increasing tumor islet macrophage density (p