George F. Murphy

Identification of cells initiating human melanomas.

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

BRAF Inhibition Is Associated with Enhanced Melanoma Antigen Expression and a More Favorable Tumor Microenvironment in Patients with Metastatic Melanoma

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. Results: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

Interleukin-22 Protects Intestinal Stem Cells from Immune-Mediated Tissue Damage and Regulates Sensitivity to Graft versus Host Disease

Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

Effects of α-hydroxy acids on photoaged skin: Apilot clinical, histologic, and ultrastructural study

Background : α-Hydroxy acids (AHAs) have been reported to improve aging skin. The mechanisms of action of AHAs on epidermal and dermal compartments need clarification. Objective : Our purpose was to determine the effects of AHAs on photoaged human skin by clinical and microanalytic means. Methods : Patients applied a lotion containing 25% glycolic, lactic, or citric acid to one forearmand a placebo lotion to the opposite forearm for an average of 6 months. Thickness of forearm skin was measured throughout the study. Biopsy specimens from both forearms were processed for analysis at the end of the study. Results : Treatment with AHAs caused an approximate 25% increase in skin thickness. The epidermis was thicker and papillary dermal changes included increased thickness, increased acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen. No inflammation was evident. Conclusion : Treatment with AHAs produced significant reversal of epidermal and dermalmarkers of photoaging.

Bevacizumab plus Ipilimumab in Patients with Metastatic Melanoma

Hodi and colleagues report the safety and efficacy of targeting angiogenesis and CTLA-4 in a phase I trial of 46 patients with metastatic melanoma, which revealed the influence of VEGF-A blockade on inflammation, lymphocyte trafficking, and immune regulation, and their synergistic therapeutic effects. Ipilimumab improves survival in advanced melanoma and can induce immune-mediated tumor vasculopathy. Besides promoting angiogenesis, vascular endothelial growth factor (VEGF) suppresses dendritic cell maturation and modulates lymphocyte endothelial trafficking. This study investigated the combination of CTLA4 blockade with ipilimumab and VEGF inhibition with bevacizumab. Patients with metastatic melanoma were treated in four dosing cohorts of ipilimumab (3 or 10 mg/kg) with four doses at 3-week intervals and then every 12 weeks, and bevacizumab (7.5 or 15 mg/kg) every 3 weeks. Forty-six patients were treated. Inflammatory events included giant cell arteritis (n = 1), hepatitis (n = 2), and uveitis (n = 2). On-treatment tumor biopsies revealed activated vessel endothelium with extensive CD8+ and macrophage cell infiltration. Peripheral blood analyses demonstrated increases in CCR7+/−/CD45RO+ cells and anti-galectin antibodies. Best overall response included 8 partial responses, 22 instances of stable disease, and a disease-control rate of 67.4%. Median survival was 25.1 months. Bevacizumab influences changes in tumor vasculature and immune responses with ipilimumab administration. The combination of bevacizumab and ipilimumab can be safely administered and reveals VEGF-A blockade influences on inflammation, lymphocyte trafficking, and immune regulation. These findings provide a basis for further investigating the dual roles of angiogenic factors in blood vessel formation and immune regulation, as well as future combinations of antiangiogenesis agents and immune checkpoint blockade. Cancer Immunol Res; 2(7); 632–42. ©2014 AACR.

Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen

BACKGROUND Filaggrin is important for skin barrier function and is mutated in 15% to 20% of patients with atopic dermatitis. OBJECTIVE To examine whether filaggrin deficiency predisposes to skin inflammation and epicutaneous sensitization with protein antigen. METHODS Skin histology in filaggrin-deficient flaky tail (ft)/ft mice and wild-type controls was assessed by Hematoxylin and Eosin (H&E) staining and immunohistochemistry. Cytokine mRNA expression was examined by quantitative RT-PCR. Serum antibody levels and splenocyte secretion of cytokines were measured by ELISA. RESULTS The ft/ft mice developed eczematous skin lesions after age 28 weeks and a progressive increase in serum IgE and IgG(1) levels. Normal-appearing skin from 8-week-old ft/ft mice had epidermal thickening and increased dermal infiltration with CD4(+) cells and expression of mRNA for IL-17, IL-6, and IL-23, but not IL-4, IL-13, or IFN-gamma. Lesional skin of 32-week-old ft/ft mice exhibited qualitatively similar, but more pronounced, changes, and elevated IL-4 mRNA levels. Epicutaneous application of ovalbumin to shaved skin of 8-week-old ft/ft mice, but not WT mice, resulted in increased epidermal thickening, dermal infiltration by CD4(+) cells but not eosinophils, and expression of IL-17, IL-6, IL-23, IL-4, and IFN-gamma, but not IL-5 or IL-13, mRNA. Splenocytes from epicutaneously sensitized ft/ft mice, but not controls, secreted cytokines in response to ovalbumin stimulation, and their sera, but not those of controls, contained ovalbumin-specific IgE and IgG(1) antibodies. CONCLUSION Filaggrin-deficient mice exhibit T(H)17-dominated skin inflammation and eczematous changes with age, and are permissive to epicutaneous sensitization with protein antigen.

Gamma-interferon inhibits collagen synthesis in vivo in the mouse.

Subcutaneous implantation of osmotic pumps into CAF1 mice resulted in the formation of thick fibrous capsules around the pumps. When pumps were loaded with recombinant murine gamma-interferon (rMuIFN-gamma) to deliver 2 X 10(3) U/h for 14 d, there was a marked decrease in thickness and collagen content of the capsules from rMuIFN-gamma-treated animals compared with capsules from animals receiving diluent alone. The collagen content of the capsules was estimated by hydroxyproline analysis of the tissue and by quantitative electron microscopy of collagen bundles. Heat-inactivated rMuIFN-gamma failed to reduce the fibrotic response in this assay. These results provide compelling evidence that gamma-interferon can down-regulate collagen synthesis in vivo and suggest the possibility that this lymphokine may be useful in the treatment of disease states characterized by excessive fibrosis.

Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells

Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particularly numerous in patients on T cell immunosuppression. We found that blood vessels in SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen (CLA)+ T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induction of E-selectin on tumor vessels, recruitment of CLA+ CD8+ T cells, and histological evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular E-selectin. Approximately 50% of the T cells infiltrating untreated SCCs were FOXP3+ regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth factor (TGF)-β. Treatment of T reg cells in vitro with imiquimod inhibited their suppressive activity and reduced FOXP3, CD39, CD73, IL-10, and TGF-β by indirect mechanisms. In vivo and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In summary, we find that SCCs evade the immune response at least in part by down-regulating vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these strategies, supporting their use in SCCs and other tumors with similar immune defects.

T cell subsets and Langerhans cells in lichen planus: in situ characterization using monoclonal antibodies.

Skin biopsies from four patients with lichen planus were studied using monoclonal antibodies directed against T lymphocytes. Anti‐T1 and anti‐T3 antibodies, which react with all peripheral T cells, stained most cells in the dermal infiltrates. The majority of infiltrating cells also stained with anti‐T4 and anti‐T4b antibodies, which react with helper/inducer cells, whereas a minority of cells stained with anti‐T8 antibody, which reacts with cytotoxic/suppres‐sor cells. Surface IgM was not identified on any infiltrating cells, providing evidence against B cell participation. Intraepidermal and dermal cells with long cytoplasmic extensions stained with anti‐T6 antibody in all cases, defining them as Langerhans cells or their precursors. T6‐positive cells were seen in greater number than in normal control epidermis and dermis. The results indicate that well‐developed lesions of lichen planus are characterized by an influx of helper/inducer T lymphocytes and increased numbers of Langerhans cells. These observations support the contention that cellular immunity is important in the pathogenesis of this disorder.