The cellular triumvirate: fibroblasts entangled in the crosstalk between cancer cells and immune cells.

This review article will focus on subpopulations of fibroblasts that get reprogrammed by tumor cells into cancer-associated fibroblasts. Throughout this article, we will discuss the intricate interactions between fibroblasts, immune cells, and tumor cells. Unravelling complex intercellular crosstalk will pave the way for new insights into cellular mechanisms underlying the reprogramming of the local tumor immune microenvironment and propose novel immunotherapy strategies that might have potential in harnessing and modulating immune system responses.

Bruton Tyrosine Kinase-Dependent Immune Cell Cross-talk Drives Pancreas Cancer.

UNLABELLED: Pancreas ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here, we report that targeting Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, restores T cell-dependent antitumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy. We report that PDAC tumor growth depends on cross-talk between B cells and FcRγ(+) tumor-associated macrophages, resulting in T(H)2-type macrophage programming via BTK activation in a PI3Kγ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a T(H)1 phenotype that fostered CD8(+) T-cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacologic inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. SIGNIFICANCE: We report that BTK regulates B-cell and macrophage-mediated T-cell suppression in pancreas adenocarcinomas. Inhibition of BTK with the FDA-approved inhibitor ibrutinib restores T cell-dependent antitumor immune responses to inhibit PDAC growth and improves responsiveness to chemotherapy, presenting a new therapeutic modality for pancreas cancer.

Ibrutinib exerts potent antifibrotic and antitumor activities in mouse models of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal fibroinflammatory reaction that is a major obstacle to effective therapy. The desmoplastic stroma comprises many inflammatory cells, in particular mast cells as key components of the PDAC microenvironment, and such infiltration correlates with poor patient outcome. Indeed, it has been hypothesized that stromal ablation is critical to improve clinical response in patients with PDAC. Ibrutinib is a clinically approved Bruton's tyrosine kinase inhibitor that inhibits mast cells and tumor progression in a mouse model of ß-cell tumorigenesis. Here, we show that ibrutinib is highly effective at limiting the growth of PDAC in both transgenic mouse and patient-derived xenograft models of the disease. In these various experimental settings, ibrutinib effectively diminished fibrosis, extended survival, and improved the response to clinical standard-of-care therapy. Our results offer a preclinical rationale to immediately evaluate the clinical efficacy of ibrutinib in patients with PDAC.

B cells regulate macrophage phenotype and response to chemotherapy in squamous carcinomas.

B cells foster squamous cell carcinoma (SCC) development through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγ receptor-dependent activation of myeloid cells. Because human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced support for SCC growth. Although ineffective as a single agent, treatment of mice bearing preexisting SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and Taxol-based chemotherapy. Improved chemoresponsiveness was dependent on altered chemokine expression by macrophages that promoted tumor infiltration of activated CD8(+) lymphocytes via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anticancer therapy in select tumors.

Cathepsin C is a tissue-specific regulator of squamous carcinogenesis.

Serine and cysteine cathepsin (Cts) proteases are an important class of intracellular and pericellular enzymes mediating multiple aspects of tumor development. Emblematic of these is CtsB, reported to play functionally significant roles during pancreatic islet and mammary carcinogenesis. CtsC, on the other hand, while up-regulated during pancreatic islet carcinogenesis, lacks functional significance in mediating neoplastic progression in that organ. Given that protein expression and enzymatic activity of both CtsB and CtsC are increased in numerous tumors, we sought to understand how tissue specificity might factor into their functional significance. Thus, whereas others have reported that CtsB regulates metastasis of mammary carcinomas, we found that development of squamous carcinomas occurs independently of CtsB. In contrast to these findings, our studies found no significant role for CtsC during mammary carcinogenesis but revealed squamous carcinogenesis to be functionally dependent on CtsC. In this context, dermal/stromal fibroblasts and bone marrow-derived cells expressed increased levels of enzymatically active CtsC that regulated the complexity of infiltrating immune cells in neoplastic skin, development of angiogenic vasculature, and overt squamous cell carcinoma growth. These studies highlight the important contribution of tissue/microenvironment context to solid tumor development and indicate that tissue specificity defines functional significance for these two members of the cysteine protease family.

Differential macrophage programming in the tumor microenvironment.

Of the multiple unique stromal cell types common to solid tumors, tumor-associated macrophages (TAMs) are significant for fostering tumor progression. The protumor properties of TAMs derive from regulation of angiogenic programming, production of soluble mediators that support proliferation, survival and invasion of malignant cells, and direct and indirect suppression of cytotoxic T cell activity. These varied activities are dependent on the polarization state of TAMs that is regulated in part by local concentrations of cytokines and chemokines, as well as varied interactions of TAMs with normal and degraded components of the extracellular matrix. Targeting molecular pathways regulating TAM polarization holds great promise for anticancer therapy.

Stromal regulation of vessel stability by MMP14 and TGFbeta.

Innate regulatory networks within organs maintain tissue homeostasis and facilitate rapid responses to damage. We identified a novel pathway regulating vessel stability in tissues that involves matrix metalloproteinase 14 (MMP14) and transforming growth factor beta 1 (TGFbeta(1)). Whereas plasma proteins rapidly extravasate out of vasculature in wild-type mice following acute damage, short-term treatment of mice in vivo with a broad-spectrum metalloproteinase inhibitor, neutralizing antibodies to TGFbeta(1), or an activin-like kinase 5 (ALK5) inhibitor significantly enhanced vessel leakage. By contrast, in a mouse model of age-related dermal fibrosis, where MMP14 activity and TGFbeta bioavailability are chronically elevated, or in mice that ectopically express TGFbeta in the epidermis, cutaneous vessels are resistant to acute leakage. Characteristic responses to tissue damage are reinstated if the fibrotic mice are pretreated with metalloproteinase inhibitors or TGFbeta signaling antagonists. Neoplastic tissues, however, are in a constant state of tissue damage and exhibit altered hemodynamics owing to hyperleaky angiogenic vasculature. In two distinct transgenic mouse tumor models, inhibition of ALK5 further enhanced vascular leakage into the interstitium and facilitated increased delivery of high molecular weight compounds into premalignant tissue and tumors. Taken together, these data define a central pathway involving MMP14 and TGFbeta that mediates vessel stability and vascular response to tissue injury. Antagonists of this pathway could be therapeutically exploited to improve the delivery of therapeutics or molecular contrast agents into tissues where chronic damage or neoplastic disease limits their efficient delivery.

FcRgamma activation regulates inflammation-associated squamous carcinogenesis.

Chronically activated leukocytes recruited to premalignant tissues functionally contribute to cancer development; however, mechanisms underlying pro- versus anti-tumor programming of neoplastic tissues by immune cells remain obscure. Using the K14-HPV16 mouse model of squamous carcinogenesis, we report that B cells and humoral immunity foster cancer development by activating Fcgamma receptors (FcgammaRs) on resident and recruited myeloid cells. Stromal accumulation of autoantibodies in premalignant skin, through their interaction with activating FcgammaRs, regulate recruitment, composition, and bioeffector functions of leukocytes in neoplastic tissue, which in turn promote neoplastic progression and subsequent carcinoma development. These findings support a model in which B cells, humoral immunity, and activating FcgammaRs are required for establishing chronic inflammatory programs that promote de novo carcinogenesis.

Lymphocytes in cancer development: polarization towards pro-tumor immunity.

The classic view that the role of immune cells in cancer is primarily one of tumor rejection has been supplanted by a more complex view of leukocytes having both pro- and anti-tumor properties. This shift is due to the now well recognized capabilities of several myeloid cell types that foster pro-tumor programming of premalignant tissue, as well as the discovery that subsets of leukocytes also suppress development and effector functions of lymphocytes important for mediating anti-tumor immunity. In this review, we focus on the underappreciated role that T lymphocytes play in promoting tumor development. This includes, in addition to the role of T regulatory cells, a role for natural killer T cells and CD4(+) T helper cells in suppressing anti-tumor immunity and promoting cancer growth and metastasis.

Delineating protease functions during cancer development.

Much progress has been made in understanding how matrix remodeling proteases, including metalloproteinases, serine proteases, and cysteine cathepsins, functionally contribute to cancer development. In addition to modulating extracellular matrix metabolism, proteases provide a significant protumor advantage to developing neoplasms through their ability to modulate bioavailability of growth and proangiogenic factors, regulation of bioactive chemokines and cytokines, and processing of cell-cell and cell-matrix adhesion molecules. Although some proteases directly regulate these events, it is now evident that some proteases indirectly contribute to cancer development by regulating posttranslational activation of latent zymogens that then directly impart regulatory information. Thus, many proteases act in a cascade-like manner and exert their functionality as part of a proteolytic pathway rather than simply functioning individually. Delineating the cascade of enzymatic activities contributing to overall proteolysis during carcinogenesis may identify rate-limiting steps or pathways that can be targeted with anti-cancer therapeutics. This chapter highlights recent insights into the complexity of roles played by pericellular and intracellular proteases by examining mechanistic studies as well as the roles of individual protease gene functions in various organ-specific mouse models of cancer development, with an emphasis on intersecting proteolytic activities that amplify programming of tissues to foster neoplastic development.

IKKalpha at the crossroads of inflammation and metastasis.

The development of solid tumors is regulated by dynamic interactions between evolving neoplastic cells and their microenvironment. Luo et al. (2007) recently demonstrated that tumor-infiltrating immune cells expressing RANKL induce activation and nuclear localization of IKKalpha in prostatic epithelial tumor cells. This leads to repression of maspin, a critical suppressor of metastasis, and thus commits malignant prostatic epithelial cells to a metastatic fate.

Activation of Akt and mTOR in CD34+/K15+ keratinocyte stem cells and skin tumors during multi-stage mouse skin carcinogenesis.

BACKGROUND: The goal of the present studies was to localize two proteins known to be involved in regulation of cell proliferation and survival in specific cell populations in normal SENCAR mouse skin and during multi-stage skin carcinogenesis. The proteins evaluated included activated Akt, as defined by phosphorylation of Akt at Serine-473 (pAkt) and mammalian target of rapamycin (pmTOR), defined by phosphorylation of mTOR at Serine-2448 (pmTOR). The cell populations examined included mouse keratinocyte stem cells (KSCs) within hair follicles and preneoplastic papilloma cells. MATERIALS AND METHODS: Immunochemical staining analysis as well as triple color immunofluorescence in combination with confocal microscopy were used to evaluate the presence of activated Akt and mammalian target of rapamycin (mTOR) in KSCs within the bulge niche of hair follicles, as identified by expression of the specific markers of mouse KSCs, CD34 and cytokeratin 15 (K15). Western blot analysis was used to examine CD34 and K15 protein levels in dorsal skin isolated from SENCAR mice during multi-stage skin carcinogenesis. RESULTS: CD34+/K15+ KSCs were located only in the outer root sheath (ORS) of a specific niche within hair follicles defined as "the bulge". The location of CD34+/K15+ KSCs remained restricted to the bulge region throughout the 22-week time-period examined during which pre-malignant papillomas developed and rapidly expanded. There was a significant decrease in K15 protein levels at 24 h and 15 weeks in dorsal skin treated with DMBA/TPA compared to CD34 protein levels. CD34+ cells within the numerous hair follicles in hyperplastic skin were found to undergo proliferation during the process of multi-stage skin carcinogenesis based on their staining with antibodies directed against proliferating cell nuclear antigen (PCNA). While pAkt was present within the bulge region of hair follicles, pmTOR was present in cells in the ORS of the bulge region as well as the upper infundibulum of hair follicles in dorsal skin treated with acetone. Within papillomas tissues isolated at 15 weeks following DMBA/TPA treatment, pAkt was localized to suprabasal cells with nominal staining of pAkt in the basal cell layer. There were fewer cells within the basal cell layer that contained pmTOR, in addition to the presence of pmTOR in suprabasal cells within papillomas. CONCLUSION: These results provide first time evidence for pAkt and pmTOR in CD34+/K15+ KSCs localized to the outer root sheath niche of the bulge region of mouse hair follicles. Taken together, the present observations suggest that pAkt and pmTOR may allow this cell population to evade terminal differentiation and to persist for long periods of time in their specific niche. Strategies that target pAkt and pmTOR may deplete both cells within the CD34+/K]5+ KSCs compartment, as well as impacting the survival of nonproliferating suprabasal cells within pre-malignant papillomas.

Activated Akt-1 in specific cell populations during multi-stage skin carcinogenesis.

The goal of the present study was to identify specific populations of cells that contain activated Akt-1, as determined by the presence ofphosphorylated Akt at serine 473 (p Akt), during development of skin tumors using a murine multi-stage carcinogenesis model. Nucleated papillomas cells as well as both epidermal and follicular keratinocytes in hyperplastic skin contained increased pAkt compared to skin treated only with acetone or 7, 12 dimethylbenz[a]anthracene (DMBA). Although the numbers of both mast cells and neutrophils were significantly increased in the stroma of papillomas (p<0.0005; p<0.0001, respectively), only mast cells contained pAkt. The amount of total Akt protein was similar regardless of time or treatment group examined. The present results suggest that activation of Akt-1 may provide specific populations of epidermal keratinocytes that develop into skin tumors with the ability to resist terminal differentiation and have enhanced proliferation during multi-stage skin carcinogenesis. In addition, mast cells which contain activated Akt-1 may persist within the stroma of papillomas during skin tumor development and progression through this signaling pathway, thereby contributing to a pro-oxidant and proangiogenic microenvironment.

Vascular endothelial growth factor as a survival factor in tumor-associated angiogenesis.

This review focuses on vascular endothelial growth factors (VEGF), a group of structurally-related proteins that serve as key growth factors for tumor-associated angiogenesis. Pathways induced by VEGF proteins that regulate biological functions of key cell types involved in tumor angiogenesis, including vascular endothelial cells, pericytes and tumor cells, are discussed. Strategies that are currently being developed and tested based on the emerging definitions of the roles of the multiple cell types involved in tumor vessel development, their selective production of VEGF-related proteins and other pro-angiogenic growth factors, their expression of associated receptors as well as identification of signal transduction pathways involved in VEGF-induced tumor survival and tumor-associated angiogenesis will be reviewed.