Tumor cell-intrinsic PD-1 promotes Merkel cell carcinoma growth by activating downstream mTOR-mitochondrial ROS signaling.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. Inhibitors targeting the programmed cell death 1 (PD-1) immune checkpoint have improved MCC patient outcomes by boosting antitumor T cell immunity. Here, we identify PD-1 as a growth-promoting receptor intrinsic to MCC cells. In human MCC lines and clinical tumors, RT-PCR-based sequencing, immunoblotting, flow cytometry, and immunofluorescence analyses demonstrated PD-1 gene and protein expression by MCC cells. MCC-PD-1 ligation enhanced, and its inhibition or silencing suppressed, in vitro proliferation and in vivo tumor xenograft growth. Consistently, MCC-PD-1 binding to PD-L1 or PD-L2 induced, while antibody-mediated PD-1 blockade inhibited, protumorigenic mTOR signaling, mitochondrial (mt) respiration, and ROS generation. Last, pharmacologic inhibition of mTOR or mtROS reversed MCC-PD-1:PD-L1-dependent proliferation and synergized with PD-1 checkpoint blockade in suppressing tumorigenesis. Our results identify an MCC-PD-1-mTOR-mtROS axis as a tumor growth-accelerating mechanism, the blockade of which might contribute to clinical response in patients with MCC.

Inhibition of Melanoma Cell-Intrinsic Tim-3 Stimulates MAPK-Dependent Tumorigenesis.

T-cell immunoglobulin mucin family member 3 (Tim-3) is an immune checkpoint receptor that dampens effector functions and causes terminal exhaustion of cytotoxic T cells. Tim-3 inhibitors are under investigation in immuno-oncology (IO) trials, because blockade of T-cell-Tim-3 enhances antitumor immunity. Here, we identify an additional role for Tim-3 as a growth-suppressive receptor intrinsic to melanoma cells. Inhibition of melanoma cell-Tim-3 promoted tumor growth in both immunocompetent and immunocompromised mice, while melanoma-specific Tim-3 overexpression attenuated tumorigenesis. Ab-mediated Tim-3 blockade inhibited growth of immunogenic murine melanomas in T-cell-competent hosts, consistent with established antitumor effects of T-cell-Tim-3 inhibition. In contrast, Tim-3 Ab administration stimulated tumorigenesis of both highly and lesser immunogenic murine and human melanomas in T-cell-deficient mice, confirming growth-promoting effects of melanoma-Tim-3 antagonism. Melanoma-Tim-3 activation suppressed, while its blockade enhanced, phosphorylation of pro-proliferative downstream MAPK signaling mediators. Finally, pharmacologic MAPK inhibition reversed unwanted Tim-3 Ab-mediated tumorigenesis in T-cell-deficient mice and enhanced desired antitumor activity of Tim-3 interference in T-cell-competent hosts. These results identify melanoma-Tim-3 blockade as a mechanism that antagonizes T-cell-Tim-3-directed IO therapeutic efficacy. They further reveal MAPK targeting as a combination strategy for circumventing adverse consequences of unintended melanoma-Tim-3 inhibition. SIGNIFICANCE: Tim-3 is a growth-suppressive receptor intrinsic to melanoma cells, the blockade of which promotes MAPK-dependent tumorigenesis and thus counteracts antitumor activity of T-cell-directed Tim-3 inhibition.

Distinct antibody clones detect PD-1 checkpoint expression and block PD-L1 interactions on live murine melanoma cells.

Monoclonal antibodies (abs) targeting the programmed cell death 1 (PD-1) immune checkpoint pathway have revolutionized tumor therapy. Because T-cell-directed PD-1 blockade boosts tumor immunity, anti-PD-1 abs have been developed for examining T-cell-PD-1 functions. More recently, PD-1 expression has also been reported directly on cancer cells of various etiology, including in melanoma. Nevertheless, there is a paucity of studies validating anti-PD-1 ab clone utility in specific assay types for characterizing tumor cell-intrinsic PD-1. Here, we demonstrate reactivity of several anti-murine PD-1 ab clones and recombinant PD-L1 with live B16-F10 melanoma cells and YUMM lines using multiple independent methodologies, positive and negative PD-1-specific controls, including PD-1-overexpressing and PD-1 knockout cells. Flow cytometric analyses with two separate anti-PD-1 ab clones, 29F.1A12 and RMP1-30, revealed PD-1 surface protein expression on live murine melanoma cells, which was corroborated by marked enrichment in PD-1 gene (Pdcd1) expression. Immunoblotting, immunoprecipitation, and mass spectrometric sequencing confirmed PD-1 protein expression by B16-F10 cells. Recombinant PD-L1 also recognized melanoma cell-expressed PD-1, the blockade of which by 29F.1A12 fully abrogated PD-1:PD-L1 binding. Together, our data provides multiple lines of evidence establishing PD-1 expression by live murine melanoma cells and validates ab clones and assay systems for tumor cell-directed PD-1 pathway investigations.

Comparative efficacy analysis identifies immune checkpoint blockade as a new survival benchmark in advanced cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma is a common type of skin cancer that may progress to locally advanced or metastatic disease. Both disease stages are managed by a variety of treatment options, including immune checkpoint blockade (ICB), targeted therapy to epidermal growth factor, chemotherapy or treatment combinations. However, the comparative efficacy of such treatments is unclear. METHODS: We performed a systematic literature search of Medline, Embase and Central to identify eligible studies reporting Kaplan-Meier curves or individual patient data for overall survival (OS) or progression-free survival (PFS). Kaplan-Meier curves were digitised using the "'WebPlotDigitizer" program. Individual patient data was subsequently remodelled and pooled for distinct treatment groups. RESULTS: Overall, 22 independent studies were included of which n = 927 patients were evaluable for PFS and n = 1054 for OS. ICB showed the highest median PFS (mPFS 9.9 months (95% CI: 8.1-19.9)) and median OS (mOS not reached (95% CI: 31.5 months-not reached)) compared to chemotherapy (mPFS 3.0 months (95% CI: 2.2-4.8), mOS 12.6 months (95% CI: 9.6-15.8)), targeted therapy to epidermal growth factor (mPFS 4.9 months (95% CI: 4.4-5.6), mOS 12.7 months (95% CI: 11.9-14.9)) and combination therapies without ICB (mPFS 9.1 months (95% CI: 8.0-12.1), mOS 18.1 months (95% CI: 16.3-22.8)). The survival benchmark with ICB after 26 months for metastatic squamous cell carcinoma was 70.8% (95% CI: 61.5%-81.5%) versus 37.9% (95% CI: 29.5%-48.8%) for the combination group and 17.1% (95% CI: 9.5%-30.8%) for chemotherapy. CONCLUSION: ICB is superior to other systemic treatments and sets a novel survival benchmark for advanced cutaneous squamous cell carcinoma.

Targeting antigen-presenting cells by anti-PD-1 nanoparticles augments antitumor immunity.

Recent studies in cancer research have focused intensely on the antineoplastic effects of immune checkpoint inhibitors. While the development of these inhibitors has progressed successfully, strategies to further improve their efficacy and reduce their toxicity are still needed. We hypothesized that the delivery of anti-PD-1 antibody encapsulated in PLGA nanoparticles (anti-PD-1 NPs) to the spleen would improve the antitumor effect of this agent. Unexpectedly, we found that mice treated with a high dose of anti-PD-1 NPs exhibited significantly higher mortality compared with those treated with free anti-PD-1 antibody, due to the overactivation of T cells. Administration of anti-PD-1 NPs to splenectomized LT-α-/- mice, which lack both lymph nodes and spleen, resulted in a complete reversal of this increased mortality and revealed the importance of secondary lymphoid tissues in mediating anti-PD-1-associated toxicity. Attenuation of the anti-PD-1 NPs dosage prevented toxicity and significantly improved its antitumor effect in the B16-F10 murine melanoma model. Furthermore, we found that anti-PD-1 NPs undergo internalization by DCs in the spleen, leading to their maturation and the subsequent activation of T cells. Our findings provide important clues that can lead to the development of strategies to enhance the efficacy of immune checkpoint inhibitors.

PD-L1 expression is an independent predictor of favorable outcome in patients with localized esophageal adenocarcinoma.

Background. The outcome of patients with adenocarcinoma of the esophagogastric junction (AEG) remains poor. The programmed cell-death-protein-1 (PD-1), a co-inhibitory receptor primarily expressed by T-cells, represents a potential new therapeutic target. PD-1, PD-1 ligand 1 (PD-L1), and PD-L2 expression have all been described as prognostic factors in a variety of cancers. Their expression patterns in AEG, however, are poorly understood. We analyzed PD-L1, PD-L2 and PD-1 expression by tumor-infiltrating lymphocytes (TILs) and cancer-cells in tumor-biospecimens in AEG-patients. Methods. 168 patients who underwent esophagectomy because of AEG between 1992-2011 were included in this study. PD-L1, PD-L2 and PD-1 expression were evaluated by immunohistochemistry and correlated with various clinicopathological parameters, disease-free survival (DFS) and long-term overall survival (OS). Results. PD-L1 expression by cancer-cells (cancer-cell-PD-L1+) was found in 43.5% of patients whereas PD-L1 expression by TILs (TILs-PD-L1+) was observed in 69%. PD-L2 expression by cancer-cells and TILs was only found in 3.5% and 1.8%, respectively. Additionally, 77.4% of tumors contained PD-1+-cancer-cells and 81% PD-1+-TILs. Patients with increased expression of PD-1 by cancer-cells and TILs showed significantly reduced OS and DFS, as determined by univariate, but not multivariate analysis. Expression of PD-L1 by cancer-cells was found to be an independent predictor for improved DFS (p = 0.038) and OS (p = 0.042) in multivariate analysis. Conclusions. Cancer cells and TILs displayed PD-L1 expression in around 50% and PD-1 expression in around 80% of tumor-biospecimens obtained from AEG patients. Expression of PD-L1 is an independent predictor of favorable outcome in AEG, whereas PD-1 expression is associated with worse outcome and advanced tumor stage.

Expression of Programmed Cell Death Protein 1 by Tumor-Infiltrating Lymphocytes and Tumor Cells is Associated with Advanced Tumor Stage in Patients with Esophageal Adenocarcinoma.

BACKGROUND: Despite recent advances in the therapy for adenocarcinoma of the esophagogastric junction (AEG), overall prognosis remains poor. Programmed cell death protein 1 (PD1) is a co-inhibitory receptor primarily expressed by T-cells. Tumor cells can escape anticancer immune responses by triggering the PD1 pathway. Moreover, PD1 receptor engagement on cancer cells may trigger tumor-intrinsic growth signals. This study aimed to evaluate the potential clinical relevance of PD1 expression by tumor-infiltrating lymphocytes (TILs) and cancer cells in the AEG. METHODS: Patients with AEG who underwent esophagectomy from 1992 to 2011 were included in the study. Expression of PD1was evaluated by immunohistochemistry and correlated with long-term overall survival (OS), disease-free survival (DFS), and various clinicopathologic parameters. RESULTS: Tumor biospecimens from 168 patients were analyzed. In the analysis, 81% of the patients showed high tumoral frequencies (>5%) of PD1-expressing TILs (TIL-PD1+), and 77% of patient tumors harbored high levels (>5%) of PD1+ cancer cells (cancer-PD1+). Expression of PD1 by TILs and cancer cells correlated significantly (p < 0.05) with patients' tumor stage and lymph node involvement. Compared with the patients who had low tumoral frequencies of PD1+ TILs or cancer cells, the TIL-PD1+ and cancer-PD1+ patients demonstrated significantly reduced DFS in the univariate analysis (5-year DFS: 73.3 vs. 41.9%, log-rank 0.008 and 71.3 vs. 41.6%, p = 0.008, respectively). Additionally, the cancer-PD1+ patients showed significantly decreased OS in the univariate analysis compared with the cancer-PD1- patients (5-year OS: 68.8 vs. 43.5%; p = 0.047). However, these correlations did not reach significance in the multivariate analysis. CONCLUSIONS: The PD1 receptor is expressed by both TILs and cancer cells in AEG. High expression of PD1 is associated with advanced tumor stage and lymph node involvement, but not with survival.

T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy.

Advances in cancer immunotherapy have offered new hope for patients with metastatic disease. This unfolding success story has been exemplified by a growing arsenal of novel immunotherapeutics, including blocking antibodies targeting immune checkpoint pathways, cancer vaccines, and adoptive cell therapy (ACT). Nonetheless, clinical benefit remains highly variable and patient-specific, in part, because all immunotherapeutic regimens vitally hinge on the capacity of endogenous and/or adoptively transferred T-effector (Teff) cells, including chimeric antigen receptor (CAR) T cells, to home efficiently into tumor target tissue. Thus, defects intrinsic to the multi-step T-cell homing cascade have become an obvious, though significantly underappreciated contributor to immunotherapy resistance. Conspicuous have been low intralesional frequencies of tumor-infiltrating T-lymphocytes (TILs) below clinically beneficial threshold levels, and peripheral rather than deep lesional TIL infiltration. Therefore, a Teff cell 'homing deficit' may arguably represent a dominant factor responsible for ineffective immunotherapeutic outcomes, as tumors resistant to immune-targeted killing thrive in such permissive, immune-vacuous microenvironments. Fortunately, emerging data is shedding light into the diverse mechanisms of immune escape by which tumors restrict Teff cell trafficking and lesional penetrance. In this review, we scrutinize evolving knowledge on the molecular determinants of Teff cell navigation into tumors. By integrating recently described, though sporadic information of pivotal adhesive and chemokine homing signatures within the tumor microenvironment with better established paradigms of T-cell trafficking under homeostatic or infectious disease scenarios, we seek to refine currently incomplete models of Teff cell entry into tumor tissue. We further summarize how cancers thwart homing to escape immune-mediated destruction and raise awareness of the potential impact of immune checkpoint blockers on Teff cell homing. Finally, we speculate on innovative therapeutic opportunities for augmenting Teff cell homing capabilities to improve immunotherapy-based tumor eradication in cancer patients, with special focus on malignant melanoma.

Homing in on the Sweet Side of Immune Checkpoint Biology.

P-selectin glycoprotein ligand-1 (PSGL-1) and its glycostructural determinants facilitate responses to infection and cancer by promoting immune effector-cell trafficking into inflamed tissue. In this issue of Immunity, Tinoco et al. (2016) report homing-independent functions of PSGL-1 in immune checkpoint regulation and T cell effector activity, in models of chronic viral infection and melanoma.

Tumour-infiltrating lymphocytes in melanoma prognosis and cancer immunotherapy.

The field of systemic cancer therapy for metastatic disease has entered an exciting era with the advent of novel immunomodulatory strategies targeting immune checkpoints. At the heart of these promising efforts are the tumour-infiltrating lymphocytes (TILs). As the reports demonstrating efficacy of modulating TIL effector function in patients with advanced stage cancer continue to accrue, it has become essential to better understand TIL immunobiology in order to further improve clinical outcome. In addition to providing an overview of the current immunotherapies available for metastatic melanoma, this review will briefly introduce the history and classification of TILs. Moreover, we will dissect the multifaceted roles of TILs in tumour-specific immunity and melanoma immune escape. The significance of TILs in melanoma prognosis and cancer immunotherapy will also be discussed, with a particular focus on their potential utility as biomarkers of patient response. The goal of personalised medicine appears to be in realistic sight, as new immunomodulatory techniques and technological innovations continue to advance the field of cancer immunotherapy. In light of recent studies highlighting the possible utility of TILs in determining therapeutic outcome, further characterisation of TIL phenotype and function has the potential to help translate individualised care into current medical practice.

ABCB5-Targeted Chemoresistance Reversal Inhibits Merkel Cell Carcinoma Growth.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with profound but poorly understood resistance to chemotherapy, which poses a significant barrier to clinical MCC treatment. Here we show that ATP-binding cassette member B5 (ABCB5) confers resistance to standard-of-care MCC chemotherapeutic agents and provide proof-of-principle that ABCB5 blockade can inhibit human MCC tumor growth through sensitization to drug-induced cell cytotoxicity. ABCB5 expression was detected in both established MCC lines and clinical MCC specimens at levels significantly higher than those in normal skin. Carboplatin- and etoposide-resistant MCC cell lines exhibited increased expression of ABCB5, along with enhanced ABCB1 and ABCC3 transcript expression. ABCB5-expressing MCC cells in heterogeneous cancers preferentially survived treatment with carboplatin and etoposide in vitro and in human MCC xenograft-bearing mice in vivo. Moreover, patients with MCC also exhibited enhanced ABCB5 positivity after carboplatin- and etoposide-based chemotherapy, pointing to clinical significance of this chemoresistance mechanism. Importantly, ABCB5 blockade reversed MCC drug resistance and impaired tumor growth in xenotransplantation models in vivo. Our results establish ABCB5 as a chemoresistance mechanism in MCC and suggest utility of this molecular target for improved MCC therapy.

Effects of Malignant Melanoma Initiating Cells on T-Cell Activation.

Although human malignant melanoma is a highly immunogenic cancer, both the endogenous antitumor immune response and melanoma immunotherapy often fail to control neoplastic progression. Accordingly, characterizing melanoma cell subsets capable of evading antitumor immunity could unravel optimized treatment strategies that might reduce morbidity and mortality from melanoma. By virtue of their preferential capacity to modulate antitumor immune responses and drive inexorable tumor growth and progression, malignant melanoma-initiating cells (MMICs) warrant closer investigation to further elucidate the cellular and molecular mechanisms underlying melanoma immune evasion and immunotherapy resistance. Here we describe methodologies that enable the characterization of immunoregulatory effects of purified MMICs versus melanoma bulk populations in coculture with syngeneic or allogeneic lymphocytes, using [3H]thymidine incorporation, enzyme-linked immunosorbent spot (ELISPOT), or ELISA assays. These assays were traditionally developed to analyze alloimmune processes and we successfully adapted them for the study of tumor-mediated immunomodulatory functions.

ABCB5 Identifies Immunoregulatory Dermal Cells.

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth.

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

Melanoma Cell Galectin-1 Ligands Functionally Correlate with Malignant Potential.

Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening antitumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity, and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely dysplastic nevi, as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAM(KD)) or ST6GalNAc2-overexpressing (ST6(O/E)) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAM(KD) or ST6(O/E) melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1/melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy.

Interleukin-10+ regulatory B cells arise within antigen-experienced CD40+ B cells to maintain tolerance to islet autoantigens.

Impaired regulatory B cell (Breg) responses are associated with several autoimmune diseases in humans; however, the role of Bregs in type 1 diabetes (T1D) remains unclear. We hypothesized that naturally occurring, interleukin-10 (IL-10)-producing Bregs maintain tolerance to islet autoantigens, and that hyperglycemic nonobese diabetic (NOD) mice and T1D patients lack these potent negative regulators. IgVH transcriptome analysis revealed that islet-infiltrating B cells in long-term normoglycemic (Lnglc) NOD, which are naturally protected from diabetes, are more antigen-experienced and possess more diverse B-cell receptor repertoires compared to those of hyperglycemic (Hglc) mice. Importantly, increased levels of Breg-promoting CD40(+) B cells and IL-10-producing B cells were found within islets of Lnglc compared to Hglc NOD. Likewise, healthy individuals showed increased frequencies of both CD40(+) and IL-10(+) B cells compared to T1D patients. Rituximab-mediated B-cell depletion followed by adoptive transfer of B cells from Hglc mice induced hyperglycemia in Lnglc human CD20 transgenic NOD mouse models. Importantly, both murine and human IL-10(+) B cells significantly abrogated T-cell-mediated responses to self- or islet-specific peptides ex vivo. Together, our data suggest that antigen-matured Bregs may maintain tolerance to islet autoantigens by selectively suppressing autoreactive T-cell responses, and that Hglc mice and individuals with T1D lack this population of Bregs.

Nestin depletion induces melanoma matrix metalloproteinases and invasion.

Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated the expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. The development of three-dimensional melanospheres that in vitro partially recapitulate noninvasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase and increased melanoma cell responsiveness to transforming growth factor-beta, both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

Merkel cell carcinoma expresses vasculogenic mimicry: demonstration in patients and experimental manipulation in xenografts.

Merkel cell carcinoma (MCC) is a highly virulent cutaneous neoplasm that, like melanoma, is a frequent cause of patient morbidity and mortality. The cellular mechanisms responsible for the aggressive behavior of MCC remain unknown. Vasculogenic mimicry (VM) is a phenomenon associated with cancer virulence, including in melanoma, whereby anastomosing laminin networks form in association with tumor cells that express certain endothelial genes. To determine whether VM is a factor in MCC, we employed a relevant xenograft model using two independent human MCC lines. Experimentally induced tumors were remarkably similar histologically to patient MCC, and both contained laminin networks associated with vascular endothelial-cadherin (CD144) and vascular endothelial growth factor receptor 1, as well as Nodal expression typical of VM in melanoma. Moreover, two established chemotherapeutic agents utilized for human MCC, etoposide and carboplatin, induced necrosis in xenografts on systemic administration while enriching for laminin networks in apparently resistant viable tumor regions that persisted. These findings for the first time establish VM-like laminin networks as a biomarker in MCC, demonstrate the experimental utility of the MCC xenograft model, and suggest that VM-rich regions of MCC may be refractory to conventional chemotherapeutic agents.

ABCB5 maintains melanoma-initiating cells through a proinflammatory cytokine signaling circuit.

The drug efflux transporter ABCB5 identifies cancer stem-like cells (CSC) in diverse human malignancies, where its expression is associated with clinical disease progression and tumor recurrence. ABCB5 confers therapeutic resistance, but other functions in tumorigenesis independent of drug efflux have not been described that might help explain why it is so broadly overexpressed in human cancer. Here we show that in melanoma-initiating cells, ABCB5 controls IL1ß secretion, which serves to maintain slow cycling, chemoresistant cells through an IL1ß/IL8/CXCR1 cytokine signaling circuit. This CSC maintenance circuit involved reciprocal paracrine interactions with ABCB5-negative cancer cell populations. ABCB5 blockade induced cellular differentiation, reversed resistance to multiple chemotherapeutic agents, and impaired tumor growth in vivo. Together, our results defined a novel function for ABCB5 in CSC maintenance and tumor growth.