AbstractBackgroundThe regulatory functions of microRNAs (miRNAs) in anti‐tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell—target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell‐mediated cytotoxicity.MethodsLuciferase expressing B16F10 melanoma (B16F10 Luci+) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma‐associated antigen tyrosinase‐related protein 2. miRNAs with the most pronounced effects on T cell‐mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)‐mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA‐seq) data from miRNA‐overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)‐derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients’ survival.ResultsThe miRNA screen resulted in the selection of seven miRNAs enhancing CTL‐mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa‐miR‐320a‐3p, mmu‐miR‐7037‐5p and mmu‐miR‐666‐3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA‐mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA‐seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA‐mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined.ConclusionsFor the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell‐mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.

Radiotherapy can act as an in situ vaccine, activating preventive tumor-specific immune responses in patients. Although carbon ion radiotherapy has superior biophysical properties over conventional photon irradiation, the immunological effects induced by this radiation type are poorly understood. Multiple strategies combining radiotherapy with immune checkpoint inhibition (radioimmunotherapy) to enhance antitumor immunity have been described; however, immune cell composition in tumors following radioimmunotherapy with carbon ions remains poorly explored. We developed a bilateral tumor model based on time-shifted subcutaneous injection of murine Her2+ EO771 tumor cells into immune-competent mice followed by selective irradiation of the primary tumor. αCTLA4-, but not αPD-L1-based radioimmunotherapy, induced complete tumor rejection and mediated the eradication of even non-irradiated, distant tumors. Cured mice were protected against the EO771 rechallenge, indicating long-lasting, tumor-specific immunological memory. Single-cell RNA sequencing and flow cytometric analyses of irradiated tumors revealed activation of NK cells and distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes. Distant tumors in the irradiated mice showed higher frequencies of naïve T cells activated upon the combination with CTLA4 blockade. Thus, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data suggest combining radiotherapy approaches with CTLA4 blockade to achieve durable antitumor immunity. Evaluation of future radioimmunotherapy approaches should not be restricted to immunological impact at the irradiation site but should also consider systemic immunological effects on non-irradiated tumors.

The immunosuppressive tumor microenvironment (TME) established by tumor cells, stromal cells and inhibitory immune cells counteracts the function of tumor reactive T cells. Tumor associated macrophages (TAMs) showing functional plasticity contribute to this process as so called M2-like macrophages can suppress the function of effector T cells and promote their differentiation into regulatory T cells (Tregs). Furthermore, tumor antigen specific CD4+ T effector cells can essentially sustain anti-tumoral immune responses as shown for various tumor entities, thus suggesting that cognate interaction between tumor antigen-specific CD4+ Th1 cells and TAMs might shift the intra-tumoral M1/M2 ratio toward M1. This study demonstrates repolarization of M2-like PECs upon MHC II-restricted interaction with tumor specific CD4+ Th1 cells in vitro as shown by extensive gene and protein expression analyses. Moreover, adoptive transfer of OVA-specific OT-II cells into C57BL/6 mice bearing OVA expressing IAb-/- tumors resulted in increased accumulation of M1-like TAMs with enhanced M1 associated gene and protein expression profiles. Thus, this paper highlights a so far underestimated function of the CD4+ Th1/TAM axis in re-conditioning the immunosuppressive tumor microenvironment.

[This corrects the article DOI: 10.1371/journal.pone.0174077.].

Distribution, patterns and prognostic impact of spontaneous antibody responses against different tumor-associated antigens (TAAs) in malignant melanoma patients are unknown so far and were investigated in this study for the first time in a large cohort at different stages of the disease, identifying new prognostic biomarkers for malignant melanoma. Serum samples from 365 melanoma patients (97 Stage I melanoma patients, 87 Stage II, 92 Stage III and 89 Stage IV) and 100 age and gender matched healthy control donors were analyzed. Samples were drawn at the time of diagnosis (Stages I-III) or at time of diagnosis of distant metastasis (Stage IV). Applying a novel multiplex assay, humoral immune responses against 29 TAAs were determined and the association between response and patient survival was investigated. Antibody responses were mainly found in melanoma patients and all tested antigens elicited immune responses in all disease stages. Antibody responses against single antigens were either associated with poor prognosis and/or shorter progression-free survival (PFS) or had no influence. While in Stages I-III significant associations were observed between an antibody response and overall survival or PFS, among Stage IV patients, no significant association was found. Multivariate analyses identified specific humoral immune responses as prognostic factors independently of age, chemotherapy and immunotherapy. Antibody responses against specific TAA in Stage I-III melanoma patients correlate with poor prognosis and/or shorter PFS. These results may help to design clinical studies in order to evaluate the potential of these responses as prognostic serological biomarkers.

The microRNA miR-101 has been reported to be a tumor suppressor. Here we show that low expression of miR-101 is associated with poor survival in stage IV melanoma patients. We identified microphthalmia-associated transcription factor (MITF) as a direct target of miR-101. In melanoma cells, overexpression of miR-101 downregulated protein levels of MITF and a previously reported target protein, enhancer of zeste homolog 2 (EZH2). Functional assays showed that miR-101 suppressed invasion and proliferation – an outcome that could be phenocopied by siRNA knockdown of MITF and EZH2. Our data suggest that miR-101 might have a beneficial role in melanoma.

MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmia-associated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that nniR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF. Journal of Investigative Dermatology (2013) 133, 768-775; doi:10.1038/jid.2012.357; published online 15 November 2012

Background: Interleukin-10 is an immunosuppressive cytokine produced by a variety of cells such as monocytes, macrophages, B-and T-lymphocytes.It was proved that IL-10 is also secreted by different cancer types including ovarian cancer.In patients with advanced ovarian cancer IL-10 is overexpressed in sera and ascites.The role of intracellular expression of IL-10 in ovarian cancer microenvironment remains unclear.The aim of study was to compare the percentage of peripheral blood (PB), peritoneal fluid (PF) and cancer tissue CD4+ and CD8+ T cells producing IL-10 in ovarian cancer patients (OVC).Material and Methods: Three-color flow cytometry was used to evaluate the percentage of CD4+ and CD8+ T cells producing IL-10 from peripheral blood (n = 32), peritoneal fluid (n = 24) and tissue (n = 15) of ovarian cancer patients.Peripheral blood, peritoneal fluid and cancer tissue mononuclear cells were obtained by density gradient centrifugation using gradisol and washed twice with PBS.For CD4+ and CD8+ T cells producing IL-10 detection the cells were stimulated for 4 hours ex vivo with phorbol myristate acetate (PMA) and ionomycin.Cancer tissue was previously fragmented with a surgical blade and digested with an enzyme mixture.Results: The percentage of CD4+ T cells producing IL-10 was significantly higher in the cancer tissue (1.91%) than in the peripheral blood (0.24%).Additionally, we found that the percentage of CD4+/IL-10+ T cells was significantly higher in the peritoneal fluid (0.94%) compared with the peripheral blood (0.24%).There was no significant difference between the percentage of CD4+/IL-10+ T cells between the peritoneal fluid (0.94%) and ovarian cancer tissue (1.91%).The percentage of CD8+ T cells producing IL-10 was also significantly higher in the cancer tissue (0.44%) than in the peripheral blood (0.08%).There were no significant differences between the percentage of CD8+/IL-10+ T cells between the peripheral blood (0.08%) and the peritoneal fluid (0.24%) and between the peritoneal fluid (0.24%) and cancer tissue (0.44%).Conclusions: We demonstrated the presence of IL-10+ T cells in both CD4+ and CD8+ T lymphocytes in ovarian cancer patients.The highest percentage of CD4+/IL-10+ and CD8+/IL-10+ in ovarian cancer tissue suggest that these cells are accumulated in tumor tissue or that they may migrate from the peripheral blood to the tumor tissue.

Single nucleotide polymorphisms, besides influencing susceptibility can potentially alter progression and survival in melanoma patients. In this study we evaluated the association of polymorphisms in the base-excision repair genes XRCC1 and APEX1 with overall survival (OS), metastasis-free survival (MFS) and survival following the first metastasis (SFM) in patients with cutaneous melanoma. We genotyped the D148E APEX1, -77 T > C XRCC1, R280H XRCC1, and R399Q XRCC1 polymorphisms in 400 German melanoma patients (Tx, N0, M0) using an allelic discrimination method. The results were correlated with the patient follow-up parameters. The significant association detected between the R399Q XRCC1 polymorphism and MFS was also evaluated in 529 Spanish melanoma patients. In a Kaplan–Meier survival model the AA genotype of the polymorphism showed a median OS of 24.4 years compared to 11.5 years for two other genotypes. Similarly patients with the AA genotype showed median MFS of 20.9 years compared to 5.3 years for two other genotypes. In a multivariate Cox proportional hazard model, the AA genotype was associated with a hazard ratio (HR) of 0.40, 95% (CI 0.21–0.78, p = 0.007) for MFS and 0.32 (95% CI 0.11–0.90, p = 0.03) for OS in 400 German melanoma patients. The decreased risk of metastasis was confirmed by adding 529 Spanish melanoma patients with a combined HR of 0.40 (95% CI 0.24–0.68, p = 0.0006). A significant association with SFM was also found for -77 T > C XRCC1 (HR 1.73, 95% CI 1.02–2.94, p = 0.04). Our results show that non-synonymous variants or those located in potential regulatory regions of DNA repair genes probably influence the disease outcome in melanoma patients and have potentially significant implications for patient surveillance and tailored treatment.