Microenvironment and Lung Cancer

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Microenvironment and Lung Cancer Tonya C. Walser, Elvira L. Liclican, Kenneth J. O’Byrne, William C.S. Cho, and Steven M. Dubinett

SUMMARY OF KEY POINTS

















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• There is untapped potential for targeted lung cancer prevention and therapy that requires, as a first step, a more clear delineation of the biology underlying the lung carcinogenesis process. • The pulmonary microenvironment represents a unique milieu in which lung carcinogenesis proceeds in complicity with the four main components of the tumor microenvironment (TME): the field, cellular, soluble, and structural components. • The literature now suggests that the adjacent histologically normal-appearing epithelium is a participant in the dynamic process of lung tumor initiation and carcinogenesis. • Evidence continues to mount in support of the stromal compartment of the TME as an active participant in carcinogenesis, often driving the aggressiveness of tumors via its impact on the tumor cell secretome. • Molecular signatures composed mainly of immuneand inflammation-related cytokines characterizing the cellular and soluble components of the TME correlate with important clinical parameters. • The developing lung TME is populated by diverse cell types with both immune-protective and immunesuppressive potential—it is the balance of these effectors and their secretory products, along with their spatial and temporal context (i.e., the immune contexture), that often dictates clinical outcomes. • One of the consequences of the inflammatory TME is suppression of antitumor immunity, thus recent strategies have been designed to specifically target the immune system. • Dendritic cells are one of the cellular components of the TME that can be successfully utilized to redistribute soluble components of the TME (e.g., CCL21), ultimately redirecting the trafficking of immune cells into the tumor and enhancing immune activation. • Two families of drugs directed at the immune system include pattern recognition receptor agonists (PRRago) and immunostimulatory monoclonal antibodies (immune checkpoint inhibitors).

Many questions central to a discussion of the influence of the lung tumor microenvironment (TME) on tumorigenesis and progression persist: the cells of origin for cancers arising in the proximal versus distal airways; the identities of key driver versus passenger mutations distinguishing histologically diverse tumors; the critical mass or combination of molecular and environmental events tipping the balance in favor of malignant conversion of the airway; and the order of events characterizing tumor initiation and systematic progression. Regardless of the answers to these questions, there is undoubtedly untapped potential for targeted lung cancer prevention and therapy that requires, as a first step,

a clearer delineation of the biology underlying the lung carcinogenesis process. Perhaps no clinical approach holds more potential than targeting the molecular underpinnings of the interplay between premalignant lesions and the developing lung TME. The opportunities for combination approaches that target multiple components of the TME simultaneously also abound and are extremely promising clinically. Past and present attempts to molecularly delineate lung carcinogenesis and to target the epithelial-TME interface are discussed in this chapter.

LUNG CARCINOGENESIS The link between premalignancy and subsequent development of cancer is well established for some organ systems, but not for the lung.1 For example, removal of premalignant lesions is the standard of care and has been shown to decrease cancer incidence and mortality in the case of cervical dysplasia and colorectal polyps. However, it has been difficult to demonstrate the link between premalignant histologic airway abnormalities and subsequent development of lung cancer.2 Uncertainties about the clinical behavior of a premalignant lung lesion can lead to either inappropriate inaction or inappropriate aggressive treatment, both of which can result in harm to the patient. The seminal autopsy studies of Auerbach et al.3 from the early 1960s demonstrated multiple histologic abnormalities in nonmalignant bronchial epithelia of smokers with and without lung cancer. Because progressive sputum abnormalities have been shown to precede the development of lung cancer,4 it has been suggested that the development of lung cancer proceeds in an orderly fashion through increasing grades of histologic abnormalities that culminate in metastatic carcinoma, as in cervical and colorectal cancer. Recent molecular findings support this stepwise lung tumor initiation model in which injury or inflammation leads to dysregulated repair by stem cells.2 Tobacco smoking is a leading source of chronic injury and inflammation; thus, the majority of heavy smokers bear regions of airway epithelial dysplasia that are classified as premalignant lesions.5 Additional genetic and epigenetic alterations prevent normal differentiation of cells in these lesions and facilitate proliferation and expansion of the field, gradually displacing the normal epithelium and giving rise to full-blown malignancy and metastatic behavior. The initiation and expansion of this premalignant field (i.e., field cancerization) appear to be critical steps in lung carcinogenesis that can persist even after smoking cessation.6,7 The originally proposed and still prevailing model of lung cancer progression, termed the linear progression model, places the focus on the fully malignant primary tumor and its size, and metastatic dissemination is conditional on both.8,9 Conversely, the more recently posited parallel progression model proposes that metastases may also arise from the early dissemination of premalignant epithelial cells before their full malignant conversion or collective growth into a large primary tumor.8,9 Cell invasion and metastasis are hallmarks of cancer that are mediated by epithelial-to-mesenchymal transition (EMT) and typically are associated with late-stage disease.9–11 As per the linear progression model, EMT only occurs in rare cells at the leading invasive edge of advanced cancers, facilitating the final step (i.e.,

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metastasis) in tumor progression. However, many groups have now demonstrated that EMT also drives malignant transformation and early dissemination of epithelial malignancies, including tobacco-related cancers.9,12,13 In addition, consistent with the parallel progression model, it was recently proposed that EMT promotes dissemination of lung epithelial cells prior to, or concomitant with, their malignant conversion. These alternate models of tumor initiation and progression were highlighted by Sanchez-Garcia9 in 2009, because they represented paradigm shifts in terms of our understanding of the protracted process of epithelial cell conversion from normal to cancer. Importantly, the parallel progression model may represent a more accurate model of lung cancer progression, given the clinical observation that 30% of patients with early-stage lung cancer who have surgery subsequently have metastatic disease, an indication that undetected micrometastatic disease may have already been present at the time of surgery.14 

THE DEVELOPING LUNG TUMOR MICROENVIRONMENT In the not-so-distant past, malignant epithelial cells were considered the tumor, and the adjacent histologically normal appearing epithelium, immune effector cells, inflammatory mediators, and the stroma were all considered irrelevant bystanders. Although genetic changes are critical for the malignant transformation of epithelial cells, we now understand that all components of the developing lung TME are active participants in the events precipitating lung cancer development. In fact, most tumors arise within, and are dependent on, 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 each of what we consider the four main components of the TME: the field, cellular, soluble, and structural components.

TME Field Component: Adjacent Histologically Normal-Appearing Epithelium Slaughter et al.15 initially coined the term “field cancerization” in 1953 to describe the histologically normal-appearing tissue adjacent to a neoplastic lesion that displays molecular abnormalities often identical to those in the tumor. The concept was seemingly rediscovered more than four decades later, when investigators renewed the effort to define the molecular mechanisms precipitating the development of an array of epithelial malignancies, including lung cancer.2,16–18 In contrast to other common epithelial malignancies, there is not yet a clinical rationale to evaluate potential premalignant lesions in people at risk for lung cancer. Thus, carefully designed clinical investigations are required to harvest these clinical specimens that would not otherwise be collected from these individuals. Although knowledge regarding the molecular changes that occur in the airway in the setting of lung carcinogenesis is only fragmentary at present, it is generally accepted that there are alterations in the airway epithelium that mirror many of the changes seen in the primary lung tumor. For example, in lung cancer, mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene were described in nonmalignant histologically normal-appearing lung tissue adjacent to lung tumor.16,19 Moreover, loss of heterozygosity events were frequent in cells obtained from bronchial brushings of normal and abnormal lungs from patients undergoing diagnostic bronchoscopy and were detected in cells from the ipsilateral and contralateral lungs.20 Likewise, mutations in the epidermal growth factor receptor (EGFR) oncogene were reported in normal-appearing tissue adjacent to EGFR-mutant

lung adenocarcinoma and also occurred at a higher frequency at sites more proximal to the adenocarcinomas than at more distant regions.21,22 Global mRNA and microRNA expression profiles were also described in the normal-appearing bronchial epithelium of healthy smokers,23,24 and a cancer-specific gene expression biomarker was developed from the mainstem bronchus that can distinguish smokers with and without lung cancer.25,26 In addition, modulation of global gene expression in the normal bronchial epithelium in healthy smokers was similar in the large and small airways, and the smoking-induced alterations were mirrored in the epithelia of the mainstem bronchus and the buccal and nasal cavities.17,27,28 Kadara et al.29,30 advanced the field in 2013 with their investigation of the spatial and temporal molecular field of injury in individuals with early-stage nonsmall cell lung cancer (NSCLC), as determined by expression profiling of the large airways after definitive surgery. The normal airway epithelia were collected by endoscopic bronchoscopy brushings 12 months after surgical removal of the tumors, then every 12 months thereafter for up to 36 months. Although the study had key limitations, gene networks mediated by the phosphoinositide 3-kinase (PI3K) and ERK gene networks were upregulated in the airways adjacent to the resected tumor, suggesting that PI3K pathway dysregulation in the field of cancerization represents an early event in lung carcinogenesis that may persist even after resection of the primary tumor. In a follow-up study, the same researchers performed expression profiling of multiple normal-appearing airways various distances from tumors in conjunction with paired NSCLC tumors and normal lung tissues that were still in situ at the time of airway epithelial cell collection.31 Site-independent profiles, as well as gradient and localized airway expression patterns, characterized the adjacent airway field of cancerization, suggesting they may be useful for distinguishing the large airways of people with lung cancer from those of cancer-free smokers. Such studies of the field of cancerization enrich our understanding of the molecular pathogenesis of lung cancer and have transformative clinical potential. Biomarker signatures within the field could be used for risk assessment, diagnosis, monitoring progression of disease during active surveillance, and predicting the efficacy of adjuvant therapies following surgery. 

TME Cellular and Soluble Components: Immune Effector Cells and Cell-Secreted Inflammatory Mediators Since the early 2000s, the authors of gene expression profiling studies of several tumor types have described molecular signatures associated with carcinogenesis and progression. The molecular signatures that emerged from the original gene sets were composed mainly of cytokine genes involved in immune and inflammatory responses. In a seminal study by Bhattacharjee et al.32 in 2001, microarray-based expression profiling of resected tumor specimens allowed the investigators to discriminate between biologically distinct subclasses of adenocarcinomas, as well as primary lung adenocarcinomas and metastases of nonlung origin. Soon thereafter, Beer et al.33 used expression profiling to predict survival among patients with early-stage lung adenocarcinomas. Likewise, an mRNA expression profile developed by Potti et al.34 identified a subset of patients with early-stage NSCLC at high risk of recurrence. More recently, to inquire whether gene expression changes in the noncancerous tissue surrounding tumors could be used as a biomarker to predict cancer progression and prognosis, Seike et al.35 conducted a molecular profiling study of paired noncancerous and tumor tissues from patients with adenocarcinoma. Many of the genes identified were part of an immune and inflammatory response signature previously reported in other cancers, but a unique subset of the genes was also predictive of lymph node status and disease prognosis

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among patients with NSCLC.36 Together, these studies provided the earliest indication of the potential for expression profiling and clear evidence that molecular signatures composed mainly of immune- and inflammation-related cytokines characterizing the cellular and soluble components of the TME correlate with important clinical parameters. 

TME Structural Component: Stroma As mentioned previously, the stroma was long thought to be the inert framework of the lung and irrelevant to the carcinogenesis process. Farmer et al.37 were the first to report a major contribution of stromal genes to drug sensitivity, although not in the lung, in the context of a randomized clinical trial. These researchers used tumor biopsy specimens from individuals in the European Organization for Research and Treatment of Cancer 10994/ BIG 00-01 trial with estrogen receptor-negative breast cancer treated with 5-fluorouracil, epirubicin, and cyclophosphamide and described a stromal gene signature that predicted resistance to preoperative chemotherapy. This study expanded the clinical significance of the identification of TME stroma-associated gene signatures, and it encouraged the development of antistromal agents as a new approach to overcome chemotherapy resistance. An important translational study by Zhong et al.38 also defined tumor cell and stromal cell interactions that inform the course of NSCLC progression. By coculturing a KRAS-mutant lung adenocarcinoma cell line with one of three lung stromal cells lines (macrophage, endothelial, or fibroblast) and subsequently profiling the secreted proteins, the group developed an in vitro model for evaluating the mechanisms by which stromal cells regulate the biologic properties of lung cancer cells. By two different proteomic approaches, the investigators concluded that stromal cells in the TME alter the tumor cell secretome, including proteins required for tumor growth and dissemination. Furthermore, they confirmed that the in vitro model robustly recapitulated many of the features of their KRAS-mutant murine model and human NSCLC specimens, suggesting its usefulness as a model of the lung TME. Still more recently, Li et al.39 demonstrated that mesenchymal stem cells (MSCs) recruited to the tumor stroma influence the phenotype of the tumor cells. Specifically, tumor cell-derived interleukin (IL)-1 induces prostaglandin E2 (PGE2) secretion by MSCs recruited to the tumor-associated stroma, which then acts in an autocrine manner to induce cytokine expression by the MSCs. The MSC-derived cytokines and PGE2 subsequently elicit a mesenchymal or stem cell–like phenotype in the tumor cells through activation of β-catenin signaling. Collectively, the results of these studies suggest that the stromal compartment of the TME is an active participant in carcinogenesis, often driving the aggressiveness of tumors via its impact on the tumor cell secretome. By extension, inhibition of specific interactions between tumor cells and the tumor-adjacent stroma holds significant potential in our search for novel lung cancer preventives and therapeutics. 

PROTOTYPICAL CELL TYPES COMPRISING THE CELLULAR COMPONENT OF THE DEVELOPING LUNG TUMOR MICROENVIRONMENT The developing lung TME is a unique and ever-changing milieu populated by diverse cell types with both immune-protective and immune-suppressive potential. The cell types are too numerous to describe in detail in the pages that follow. Thus, we discuss the induction, targeting, and potential pitfalls associated with attempting to harness three prototypical cell types characterizing the cellular component of the developing lung TME: cytotoxic and helper T cells, T regulatory cells (T regs), and dendritic cells.

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Cytotoxic and Helper T Cells The presence of tumor-infiltrating lymphocytes (TILs) has long been considered a manifestation of antitumor immunity. However, the prognostic significance of TILs was only appreciated after the development of markers that define the individual subsets of TILs.40,41 Traditionally identified as a component of the cellular immune response to bacterial and viral infections, the integral role of cytotoxic CD8+ T cells (CTLs) in cell-mediated antitumor immune responses is now recognized. The reduced infiltration of CTLs, along with their reduced proliferation rate, increased susceptibility to spontaneous apoptosis, and impaired cytolytic activity against tumor cells, contributes to the immunosuppressive milieu that characterizes the developing lung TME.42,43 Accordingly, a high infiltration of CTLs expressing granzyme B, the classic effector of CTL cytolytic activity, as well as the location of TILs in tumor cell nests, is associated with a good clinical outcome in several types of cancer, including colorectal cancer, ovarian cancer, and lung cancer.44–50 Several reports have demonstrated that CTLs are associated with prolonged survival in lung cancer and positively correlated with favorable prognosis in patients with lung cancer.43,46,51,52 However, more important than the number of CTLs present is the ratio of effector to regulatory TILs. In recent studies of patients with hepatocellular and ovarian cancer, it was shown that the ratio of CD8+ TIL:T regs was an independent prognostic factor, whereas the numbers of T regs and CD8+ TILs by themselves had lower or no predictive value, respectively.53,54 It is becoming increasingly clear that CD4+ helper T cells are also a critical determinant of effective antitumor immune responses. On stimulation, naïve CD4+ T cells differentiate into effector cells known as T helper (Th) cells, of which there are four subsets: Th1, Th2, Th17, and T regs. While T regs dampen antitumor immunity (discussed later), Th1 cells, characterized by production of interferon gamma and tumor necrosis factoralpha, often lead to enhanced activation of CTLs, dendritic cells, and macrophages and beneficial downstream antitumor effects. In addition to assisting with the activation of other innate and adaptive immune cells, CD4+ helper T cells can induce apoptosis in tumor cells through Fas cell surface death receptor (FAS)- or tumor necrosis factor-related apoptosis-inducing ligand-dependent pathways.55,56 Accumulating evidence also suggests that CD4+ helper T cells can acquire cytolytic activity.57,58 As with CTLs, tumor-driven aberrant CD4+ T-cell differentiation and apoptosis, as well as Th dysfunction characterized by increased expression of the immune checkpoint molecule programmed death-1 (PD-1), contribute to the tolerogenic nature of the developing lung TME.59 In this regard, immunotherapy strategies that aim to enhance the infiltration and/or activity of both CTLs and CD4+ helper T cells are found to have a synergistic effect in boosting antitumor immunity. 

T Regulatory Cells One major impediment to our efforts in both the prevention and treatment of lung cancer is our inadequate understanding of how lung cancer cells escape immune surveillance and inhibit antitumor immunity. Thus, identification of T regs in patients with cancer was a finding of great clinical importance. June et al.60,61 were the first to document increased CD4+CD25+ T reg populations at the tumor site in patients with lung cancer. A subsequent examination of normal and tumor tissue from patients with NSCLC also indicated that tumor tissues have significantly higher expression of FOXP3 mRNA than normal tissues, rendering CD4+CD25+FOXP3+ the more specific phenotypic marker of functional T regs at the time.62 Next, investigators began to note increased numbers of T regs in the peripheral blood of patients with lung cancer relative to healthy volunteers and even patients with breast cancer.63 Zhang et al.64 made another key finding when they discovered that among

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patients with NSCLC receiving paclitaxel-based chemotherapy, the mitotic inhibitor selectively decreased the size of the T reg cell population in the peripheral blood, but not the size of the effector T cell subsets. They went on to determine that the effect was mediated by the upregulation of the cell death receptor Fas (CD95) and selective induction of apoptosis of T regs. Although T reg cell function was significantly impaired, production of Th1 cytokines and expression of the CD44 activation marker were intact and even elevated within the helper and effector T cell subsets after treatment with paclitaxel. In addition to these studies in lung cancer, there are numerous reports of increased T regs in the peripheral blood coincident with increased TILs in the tumor bed in other malignant diseases.65–68 These seminal findings are consistent with studies in murine models demonstrating that depletion of T regs can significantly augment the efficacy of cancer vaccination.69 Together, these data suggest that T regs are selectively recruited to developing lung tumors, where they contribute to the immunosuppressive microenvironment that facilitates progression and metastasis. Likewise, the data suggest that T reg status can serve as an indicator of responsiveness to certain therapeutic regimens. One of the first studies to link T reg cell recruitment and prognosis, although not for lung cancer, came from Curiel et al.,70 who discovered that an increase in the number of tumor T regs was a significant predictor of increased risk for death and reduced survival in those with ovarian cancer. They also discovered that tumor cells and tumor-adjacent macrophages were contributors of the CCL22 chemokine that mediated trafficking of the T regs to the tumor. This was the first report of functional CCL22 within the lung TME and the earliest indication that blocking CCL22 in vivo reduces human T reg cell tumor trafficking. This report paved the way for those that followed seeking to develop novel immune-boosting strategies based on eradication of the T reg cell population in patients with cancer. Lastly, our group reported on the phenomenon of cycloxygenase-2 (COX-2) and PGE2 inhibition of immune responses in lung cancer via promotion of T reg activity. Numerous studies have now demonstrated that PGE2 enhances the in vitro inhibitory function of T regs and induces a regulatory phenotype in T helper cells.71–73 These and other basic and translational research investigations have informed our understanding of the role of CD4+CD25+ T regs in the developing lung TME, collectively suggesting that the development of clinical strategies to reduce the suppressive effects of these T regs in lung cancer is warranted. Efforts directed at ablating the suppressive activities of T regs have included clinical trials that use total lymphodepletion.74–76 Others have evaluated immunotoxins to specifically ablate the T reg population,77 and ongoing clinical investigations are assessing the role of celecoxib in controlling T reg numbers, activity, and differentiation in human NSCLC. While lymphodepletion or therapy with T reg immunotoxins may prove beneficial, COX-2/ PGE2 inhibition has additional potential benefits in the setting of NSCLC. In addition to the potential capacity to clinically decrease T reg cell function, COX-2 inhibition has been found to limit angiogenesis, decrease tumor invasiveness, and decrease tumor resistance to apoptosis in NSCLC.78–80 These pathways and malignant phenotypes may be inhibited by several different agents in the class of nonsteroidal anti-inflammatory drugs.81 Therefore, trials are evaluating COX inhibition in combination with other therapies.80 Such studies will help further define the required interventions in this pathway and lead to more specifically targeted agents to diminish T reg cell activities in cancer. These agents could then be combined with other immune-based clinical therapies in an informed manner. 

Dendritic Cells In a seminal publication, Dieu-Nosjean et al.46,82,83 identified ectopic lymph nodes or tertiary lymphoid structures within

human NSCLC specimens and correlated their cellular content with clinical outcome. Specifically, the density of mature dendritic cells within these structures was a predictor of long-term survival in patients with lung cancer.46 These findings were the first to suggest that ectopic lymph nodes participate in the host’s antitumor immune response and are consistent with now abundant preclinical and clinical data.84–88 For example, in murine tumor models, dendritic cells genetically modified to secrete CCL21 were reported to produce lymphoid cell aggregates and prime naïve T cells extranodally within a tumor mass, resulting in the generation of tumor-specific T cells and subsequent tumor regression.85,89 Thus, the intratumoral approach may achieve tumor antigen presentation by utilizing the tumor as an in vivo source of antigen for the dendritic cells. In contrast to in vitro immunization with purified peptide antigen(s), autologous tumor has the capacity to provide the activated dendritic cells administered at the tumor site access to the entire repertoire of available antigens in situ. This may increase the likelihood of a response and reduce the potential for tumor resistance due to phenotypic modulation. Dendritic cells are the most potent antigen-presenting cell capable of inducing primary immune responses.90 Dendritic cells express high levels of major histocompatibility complex and costimulatory molecules, such as CD40, CD80, and CD86. Dendritic cells also release high levels of cytokines and chemokines into the TME that attract antigen-specific T cells in vivo. These properties, combined with efficient capture of antigens by immature dendritic cells, allow them to efficiently present antigenic peptides and costimulate antigen-specific naïve T cells.90 Presentation of tumor-associated antigens by dendritic cells and their recognition by CTLs play an important role in the eradication of tumor cells.91 Based on the importance of dendritic cells in tumor immunity, a variety of strategies have been used to exploit this cell type in cancer immunotherapy.92–94 Advances in the isolation and in vitro propagation of dendritic cells, combined with identification of specific tumor antigens, have facilitated the start of clinical trials to evaluate dendritic cell-based vaccines,92–94 and dendritic cell transfer has since been demonstrated to be a safe approach for clinical evaluation.95–100 Strategies involving the use of dendritic cells in immunotherapy have included pulsing isolated dendritic cells with tumor antigen peptides, apoptotic tumor cells, or tumor lysates ex vivo.101–103 Dendritic cells have also been genetically modified with genes encoding tumor antigens or immunomodulatory proteins.104–106 There is evidence that dendritic cells transduced with adenoviral vectors (AdV) have prolonged survival and resistance to spontaneous and Fas-mediated cell death, suggesting their utility in delivering immunotherapy more efficiently and robustly.107 AdV transduction itself can also augment the capacity of dendritic cells to induce protective antitumor immunity.108 In addition, enhanced local and systemic antitumor effects have been demonstrated when AdV-transduced dendritic cells expressing cytokine genes have been injected intratumorally.109 AdVs are often used to transduce dendritic cells, because they efficiently induce strong heterologous gene expression in these cells.108,109 C-C motif chemokine ligand 21 (CCL21) is a cysteine-cysteine motif (CC) chemokine that belongs to a family of proteins involved in leukocyte chemotaxis and activation. Expressed in high endothelial venules and T cell zones of the spleen and lymph nodes, CCL21 exerts potent attraction of naïve T cells and mature dendritic cells, promoting their colocalization in secondary lymphoid organs and promoting cognate T cell activation.110 Potent antitumor properties of CCL21 in murine cancer models have been reported.111–113 CCL21 has also shown antiangiogenic activities in mice, thus strengthening its immunotherapeutic potential in cancer.114,115 Based on the so-called ectopic lymph node concept posited by Dieu-Nosjean et al.46 and the body of dendritic cell (DC)-CCL21 preclinical data available at the time,

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our group initiated a phase I clinical trial at the University of California, Los Angeles in patients with advanced stage NSCLC. The trial consisted of intratumoral administration of autologous DCs transduced with a replication deficient adenoviral vector to express the CCL21.116 In situ vaccination with DC-CCL21 was well tolerated and induced systemic tumor antigen-specific immune responses and enhanced CD8+ T cell infiltration of the primary tumor. This study is one clinically relevant approach by which to harness the cellular component of the TME and manipulate the soluble component of the TME to the advantage of patients. 

PROTOTYPICAL CELL-SECRETED PRODUCTS COMPRISING THE SOLUBLE COMPONENT OF THE DEVELOPING LUNG TUMOR MICROENVIRONMENT Chronic or dysregulated inflammation in the pulmonary microenvironment characterizes pulmonary diseases associated with the greatest risk for the development of lung cancer, such as emphysema, chronic obstructive pulmonary disease, and pulmonary fibrosis.117–119 Here, we will discuss the induction, targeting, and potential/pitfalls associated with manipulating the following prototypical inflammatory mediators found in the developing lung TME: IL-2, IL-6, and transforming growth factor-beta (TGF-β).120

Interleukin-2 IL-2, produced by T cells during an immune response,121 is necessary for the growth, proliferation, and differentiation of naïve T cells into effector T cells. The use of IL-2 is approved by the US Food and Drug Administration (FDA) for cancer immunotherapy, and it is currently in clinical trials for the treatment of chronic viral infection.122 Combination treatment with IL-2 and anti-IL-2 monoclonal antibodies protects against tumor metastases in the lung,123 and although pulmonary edema was a side effect, high-dose IL-2 led to an antitumor response against pulmonary tumor nodules.124 IL-2 with a D20T mutation retains the antimetastatic activity of IL-2 via its interaction with the highaffinity IL-2 receptor, but it has a lower toxicity profile.125 Of interest is a recent study demonstrating that acupoint stimulation elicited a pronounced immunomodulatory effect among patients with lung cancer, as shown by increased production of IL-2.126 Collectively, these studies support the potential of harnessing IL-2 production for the benefit of patients. 

Interleukin-6 IL-6 is a multifunctional cytokine that can act as both a proinflammatory and an anti-inflammatory mediator. It is secreted by T cells and macrophages to stimulate immune responses, and increased levels of IL-6 have been associated with trauma, infection, and elevated cancer risk. IL-6 function is mediated primarily through the Janus kinase-signal transducer and activator of transcription-zinc finger protein 1–2 signaling pathway, and an elevated level of IL-6 has been shown to increase the production of collagen and alpha-actin, which together induce interstitial lung disease. High levels of IL-6 are also responsible for enhanced neoangiogenesis, inhibition of cancer cell apoptosis, and dysregulation of other control mechanisms in the TME.127 IL-6 has also been implicated in acquired resistance to EGFR inhibitors in patients with lung cancer. Furthermore, IL-6 is associated with poor prognosis and many of the debilitating symptoms that often affect patients with late-stage lung cancer, such as fatigue, thromboembolism, cachexia, and anemia. Consequently, a monoclonal antibody targeting IL-6 (ALD518) was recently developed to treat these

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IL-6-dependent morbidities. In preclinical, phase I, and phase II trials in advanced stage NSCLC, ALD518 appears to be well tolerated and to effectively ameliorate anemia and cachexia.128 

Transforming Growth Factor-β TGF-β is a cytokine that controls proliferation, cellular differentiation, and other functions in most cells. Secreted by many cell types, including macrophages, it plays a role in immunity and carcinogenesis. When a cell is transformed into a cancer cell, parts of the TGF-β signaling pathway are mutated, resulting in proliferation of the cancer cells and surrounding stromal cells (fibroblasts). Additionally, both cell types increase their production of TGF-β, which then acts on the surrounding stromal, immune, endothelial, and smooth-muscle cells to induce immunosuppression and angiogenesis and to make the cancer more invasive.129 TME-derived TGF-β induces malignant phenotypes, such as epithelial mesenchymal transition (EMT) and aberrant cell motility, in lung cancer. TGF-β-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes.130 Many studies have indicated that high levels of TGF-β characterize most tumor tissues, primarily released from tumor cells to maintain their metastatic potential and the protumorigenic TME.131 A TME enriched in TGF-β is broadly immunosuppressive, in part, due to its inhibition of natural killer cell function. Several studies have shown that miR-183-dependent repression of DNA polymerase III subunit tau (DNAX) activating protein 12 kDa (DAP12) transcription and translation in NSCLC is mediated by TGF-β.132,133 TGF-β also converts effector T cells into T regs. Of interest, IL-6 enhances epithelial cell EMT and stimulates tumor progression by enhancing TGF-β signaling. Thus, IL-6 and TGF-β may play a contributing role in the maintenance of a paracrine loop between fibroblasts and NSCLC cells that facilitates tumor progression.134 Like IL-6, TGF-β is a pleiotropic inflammatory mediator that interacts with premalignant lesions and the developing tumor in ways that are malleable and potentially manipulatable for the advantage of patients. 

RECENT ATTEMPTS TO MOLECULARLY DEFINE THE FIELD COMPONENT OF THE LUNG TUMOR MICROENVIRONMENT In our review of the recent translational research, several studies highlight the field’s renewed appreciation for the urgent need to better define the key events driving lung carcinogenesis, if we are to ever achieve effective targeted lung cancer prevention. In the first of these studies, Ooi et al.135 identified molecular alterations that characterize premalignant lesions and carcinogenesis in lung squamous cell carcinoma using a novel approach. In this first report of a gene expression profiling study of airway premalignant lesions and patient-matched normal tissue and squamous cell carcinoma samples, the authors discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of squamous cell carcinoma within individual patients. Additionally, their analysis identified coordinate changes in the activity of upstream regulators and the expression of downstream genes within the same patient during early- and late-stage carcinogenesis, enhancing our understanding of the stepwise carcinogenesis of squamous cell carcinoma. In another study, by Perdomo et al.,136 next-generation sequencing of small RNA from human bronchial airway epithelium identified miR-4423 as a regulator of airway epithelium differentiation and a repressor of lung carcinogenesis. Expression of miR-4423 is downregulated in the cytologically normal bronchial airway epithelium of smokers with lung cancer, which suggests that expression of miR-4423 and/or other miRNAs may be influenced by a field cancerization

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effect and could be useful for the early detection of lung cancer in the relatively accessible proximal airway. Inflammationinduced upregulation of the zinc-finger transcription factor Snail has also been demonstrated to contribute to diverse aspects of lung carcinogenesis and progression, including EMT and angiogenesis.137,138 Snail was previously shown to be upregulated in human NSCLC tissues, to be associated with poor prognosis in patients, and to have promoted cancer cell growth and progression in vivo.138 More recently, we discovered that one mechanism by which Snail acts is via upregulation of secreted protein, acidic and rich in cysteine (SPARC), which drives SPARC-dependent invasion in a model of human lung premalignancy.139 The literature now suggests that the adjacent histologically normal-appearing epithelium is a participant in the dynamic process of lung tumor initiation and carcinogenesis. Work to define the interconnectedness of the field of cancerization to the other components of the TME and the developing or established primary tumor may be a rich source for biomarkers of initiation, progression, and targets for prevention and therapy. Development of more accurate in vitro and in vivo models of human premalignancy and lung carcinogenesis will further advance these efforts. 

RECENT ATTEMPTS TO MANIPULATE THE CELLULAR (IMMUNITY) AND SOLUBLE (INFLAMMATION) COMPONENTS OF THE TUMOR MICROENVIRONMENT FOR LUNG CANCER CHEMOPREVENTION AND THERAPY One of the consequences of the inflammatory TME is suppression of antitumor immunity, thus recent strategies have been designed to specifically target the immune system. As mentioned briefly, one approach to enhance immune responses is DC-based vaccines, in which DCs are used as a vehicle to intratumorally deliver chemokines and subsequently redirect the trafficking of immune cells into the tumor and enhance their activation.82,116 Using two murine models of lung cancer, we demonstrated for the first time that intratumoral administration of recombinant CCL21 could lead to potent immune-dependent antitumor responses and, consequently, reduce tumor growth.140 Importantly, CCL21mediated antitumor responses were lymphocyte-dependent. Therapy did not alter tumor growth in severe combined immunodeficiency mice, whereas intratumoral injection of CCL21 led to a significant increase in CD4+ and CD8+ T lymphocytes and DCs infiltrating both the tumor and draining lymph nodes in immunocompetent mice. Further studies in CD4 and CD8 gene knockout mice determined that both CD4+ and CD8+ T cell subsets accounted for the CCL21-mediated tumor regression.140 Intratumoral administration of CCL21 gene-modified DCs was also shown to generate systemic antitumor responses and confer tumor immunity via recruitment and activation of T effector cells in a transplantable and a spontaneous bronchoalveolar cell carcinoma model of lung cancer.141,142 These studies additionally demonstrated that elaboration of CCL21 in the tumors by DCs promotes the CXCR3/CXCR3 ligand efferent arm of the immune response for the modulation of antitumor activity; i.e., neutralization of the CXCR3 ligands CXCL9 or CXCL10 inhibited the antitumor responses.82,141 As the number of circulating competent DCs is decreased in patients with lung cancer,143 injecting DCs within the lung tumor site may be a particularly effective approach. In fact, there is a relationship between tumor-infiltrating DC aggregation and apoptosis in situ in human NSCLC.144 To this end, intratumoral administration of clinical grade CCL21-transduced DCs was evaluated in a phase I clinical trial for late-stage NSCLC.116 Patients with stage IIIB/IV NSCLC with a tumor accessible by computed tomography–guided or bronchoscopic intervention and disease refractory to standard therapy were selected. The objectives of the trial were to (1) determine the safety and

maximum tolerated dose of CCL21 gene-modified DCs (AdCCL21-DC) when administered into the primary lung cancer of patients with advanced NSCLC and (2) determine the local and systemic biologic activity of AD-CCL21 DC. Intratumoral vaccination with Ad-CCL21-DC was well tolerated and resulted in (1) induction of systemic tumor antigen-specific immune responses and (2) enhanced tumor CD8+ T cell infiltration accompanied by increased PD-L1 expression.82,116 Thus, DCs are a cellular component of the TME that can be utilized to redistribute soluble components of the TME (e.g., CCL21), ultimately redirecting trafficking of the immune cells into the tumor and enhancing specific immune activation. DC-CCL21 in situ vaccination will next be evaluated in combination with checkpoint inhibitor therapy. Intratumoral immunization represents another avenue for reversing cancer-induced immunotolerance, allowing an antitumor response to occur.145–147 This strategy has recently been supported by the positive results of clinical trials in metastatic melanoma, renal cell carcinoma, and NSCLC, cancers with low sensitivity to conventional cytotoxic therapies.146 Two families of drugs that are currently directed at the immune system and in clinical development include pattern recognition receptor agonists (PRRago) and immunostimulatory monoclonal antibodies (immune checkpoint inhibitors). In contrast to conventional anticancer drugs, these immunostimulatory drugs can be directly delivered into the tumor and generate a systemic antitumor immune response. Furthermore, intratumoral delivery can potentially trigger more potent antitumor immune responses while causing less autoimmune toxicity. PRRs constitute a growing family of receptors that recognize pathogen-associated molecular patterns, such as viral DNA or bacterial cell wall molecules, and damage-associated molecular patterns (DAMP) that are released upon cell death, stress, or tissue injury. PRRs are typically known for their role in the activation of immune responses against infectious pathogens, and evidence now suggests that activation of PRRs, such as toll-like receptors (TLRs) expressed by immune cells, also plays a role in immune responses against tumor cells.146 In this regard, it has been demonstrated that TLR stimulation of antigen-presenting cells within mice and in the human TME modifies their phenotype from tolerogenic to immunogenic, with an upregulation of class II major histocompatibility complex, CD80, and CD86.148,149 TLRs can also be expressed by tumor cells, and the direct activation of these TLRs can result in the death of the targeted tumor cell and/or upregulate antigenpresentation molecules.150,151 Furthermore, with chemotherapy or tumor-targeted therapy, tumor cells can release DAMPs, which can then stimulate the immune cells surrounding the tumor cells. This is exemplified by high mobility group protein B1, an intracellular protein released in the TME upon tumor cell death that is subsequently recognized by TLR-4 expressed on tumor-infiltrating immune cells. Although the mechanism of the therapeutic effect of intratumoral PRRago is multifactorial, depending on the tumor cell type, the TME, and the PRRago used, a common feature is stimulation of tumor-infiltrating antigen-presenting cells, including B cells, DCs, tumor-associated macrophages, and other myeloid-derived suppressor cells. It should be noted, however, that although activation of tumor-infiltrating antigen-presenting cells is a prerequisite for mounting an efficient adaptive antitumor immune response against tumor-associated antigens, it does not address immunosuppressive tumor-infiltrating T regs and exhausted tumor-infiltrating CTLs. Immunostimulatory monoclonal antibodies are designed to reverse tumor immunotolerance and stimulate antitumor immune responses by targeting checkpoints for T cell activation. Of the checkpoint inhibitors in clinical development, the anti-CTL antigen-4 (CTLA-4) monoclonal antibody ipilimumab has already been approved for metastatic melanoma.146,147 CTLA-4 is a cell surface receptor constitutively expressed by FOXP3+ CD4+ T regs, and it is a critical negative immune checkpoint that limits the induction of potent CTL responses. In two randomized phase III clinical trials,

CHAPTER 14  Microenvironment and Lung Cancer

systemic intravenous therapy with ipilimumab generated long-lasting tumor responses in up to 20% of patients with refractory/relapsing melanoma.152,153 However, this therapy was associated with major autoimmune toxicities requiring high-dose corticosteroids in about 60% of patients treated. The efficacy of anti-CTLA-4 has thus far been attributed to its ability to block the inhibitory interaction of CTLA-4 expressed on effector T cells with CD80/86 expressed by tolerogenic tumor antigen-presenting cells and, more recently, to intratumoral depletion of T regs rather than an interaction with CD4+ effector T cells.146,147,152,153 Intratumoral tumor-specific T regs express high levels of CTLA-4, which can be depleted by therapy with anti-CTLA-4 via FcγR+ tumor-infiltrating cells.146 Although no biomarkers exist to definitively predict which patients will benefit from anti-CTLA-4 therapy, there is a pattern in which a pretreatment gene signature demonstrating CD8 T cell infiltrates and CD8-attracting chemokines is, at least to some degree, positively correlated with benefit.154 Current use of anti-CTLA-4 agents in NSCLC is still limited to phase I–III trials. Based on the positive results of anti-CTLA-4 monoclonal antibodies, a second negative immune checkpoint mediated through interactions of PD-1 with its ligands PD-L1 and PD-L2 has been investigated as a target for cancer immunotherapy.145–147 Monoclonal antibodies targeting the PD-1/PD-L1 axis have demonstrated strong and encouraging clinical activity in patients with metastatic melanoma, renal cell carcinoma, and NSCLC.155,156 Late-phase clinical trials of these anti-PD-1 agents in patients with advanced lung cancers translated into improved clinical outcomes compared with standard-of-care chemotherapy.157–161 Thus, two of the agents, nivolumab and pembrolizumab, are now FDA-approved for NSCLC in the second-line setting.157,158 FDA approval for these agents as first-line therapy for NSCLC is anticipated. Importantly, preclinical models have demonstrated that the efficacy of immunostimulatory monoclonal antibodies may be potentiated when used in combination. Indeed, in murine models of melanoma, the combination of anti-PD-1 and anti-CTLA-4 monoclonal antibodies may be more effective than either agent alone, due to the complementary functional roles of these two negative immune checkpoints. Intratumoral injection of immunostimulatory agents is also postulated to have a potentiating effect. Local delivery, rather than systemic, allows concentration of the agent in the TME, limiting the toxicity of the monoclonal antibodies and increasing the efficacy of PRRago. This strategy relies on accessibility of the tumor site for injection, however, which can be an issue if repeated injections are needed. As with anti-CTLA-4 therapy, no definitive predictive biomarkers exist for monoclonal antibodies targeting the PD-1/ PD-L1 axis. However, transcriptomic profiling and whole-exome sequencing of melanoma from patients treated with anti-PD-1, a subset of whom had received prior mitogen-activated protein kinase inhibitor treatment, has given us insight into the relevance of transcriptomic changes and tumor mutations to therapeutic responsiveness.162 Description of an innate anti-PD-1 resistance signature (IPRES) consisting of a set of coenriched genes in nonresponders is an important first step toward the identification of better biomarkers of response. With the approval of nivolumab and pembrolizumab for NSCLC, similar advances may soon be brought to bear against lung cancer as well. In addition to their report of a melanoma IPRES, Hugo et al.162 also described a correlation between tumor mutational load and improved patient survival, but no statistically significant association between high mutational load and response to anti-PD-1 therapy was observed. Conversely, a number of other groups have reported a positive correlation between overall mutational load and both antiCTLA-4 and anti-PD-1 treatment responsiveness.162–166 There are still other preclinical reports suggesting that it is not mutational load in general that predicts response, but rather key driver mutations specifically upregulate PD-L1 for the purpose of immune evasion, thereby linking those specific mutations to antiPD-1 treatment responsiveness.145,167,168 For example, Akbay

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et al.145 suggest that EGFR-driven tumors may be characterized by host T cell exhaustion via upregulation of the PD-1/PD-L1 axis. Using a mouse model of EGFR-driven lung cancer, the authors demonstrated that administration of anti-PD-1 monoclonal antibodies reduced tumor growth and improved survival by enhancing T cell effector function and reducing the levels of tumor-promoting cytokines. Preclinical investigations of KRAS and MYC driver mutations also identify upregulation of PD-L1 by these oncogenic drivers, along with a concomitant increase in other key tumorigenic phenotypes.167,168 Perhaps in alignment with these preclinical observations, Rizvi et al.158,169 found that mutations in KRAS were evident in 7 of 14 tumors from NSCLC patients with partial or stable response >6 months compared with 1 of 17 in those that had no durable benefit from pembrolizumab. However, this finding may be explained by the association between KRAS mutations in NSCLC with smoking, given that smokers often harbor a substantially greater mutational load with each mutation serving as a potential source of neoantigens.169,170 

CONCLUSION Although the epithelial compartment remains central, investigators now understand that lung carcinogenesis proceeds in complicity with each of the four main components of the TME—the field, cellular, soluble, and stromal components. The epithelial and field compartments are definitively interconnected, but a more complete understanding of the molecular pathogenesis of lung cancer is required for the development of biomarker signatures, noninvasively obtained from the field, that are useful for risk assessment, diagnosis, disease monitoring, and predicting adjuvant therapy efficacy following surgery. Numerous cell types and cell-secreted products comprise the developing lung TME, and there are both advantages and disadvantages associated with attempting to harness each for the benefit of patients. Our review of the most recent translational and clinical literature highlights the field’s evolving approach to the manipulation of these two particular TME components, including the rise of immunotherapeutics targeting the tumor-TME interface. On the whole, targeting the interplay between the epithelial compartment and the developing lung TME as a lung cancer prevention and therapy strategy has clear clinical potential that finally appears to be approaching fruition.

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