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Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

Background VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically. Methods The expression of ten TMEMs ( RTP3 , SLC35G2 , TMEM30B , TMEM45A , TMEM45B , TMEM61 , TMEM72 , TMEM116 , TMEM207 and TMEM213 ) was measured by qPCR. T -test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools. Results We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER. Conclusions The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.

Understanding the biological factors that are characteristic of metastasis in melanoma remains a key approach to improving treatment. In this study, we seek to identify a gene signature of metastatic melanoma. We configured a new network-based computational pipeline, combined with a machine learning method, to mine publicly available transcriptomic data from melanoma patient samples. Our method is unbiased and scans a genome-wide protein-protein interaction network using a novel formulation for network scoring. Using this, we identify the most influential, differentially expressed nodes in metastatic as compared to primary melanoma. We evaluated the shortlisted genes by a machine learning method to rank them by their discriminatory capacities. From this, we identified a panel of 6 genes, ALDH1A1 , HSP90AB1 , KIT , KRT16 , SPRR3 and TMEM45B whose expression values discriminated metastatic from primary melanoma (87% classification accuracy). In an independent transcriptomic data set derived from 703 primary melanomas, we showed that all six genes were significant in predicting melanoma specific survival (MSS) in a univariate analysis, which was also consistent with AJCC staging. Further, 3 of these genes, HSP90AB1 , SPRR3 and KRT16 remained significant predictors of MSS in a joint analysis (HR = 2.3, P = 0.03) although, HSP90AB1 (HR = 1.9, P = 2 × 10 −4 ) alone remained predictive after adjusting for clinical predictors.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune‐excluded tumor phenotype. However, the molecular mechanisms that sustain TEC‐mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T‐cells, thereby restricting melanoma growth. In melanoma‐bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF‐kB and STING signaling. In line, single‐cell transcriptomic datasets from melanoma patients disclose an enriched Inflammatory High /Autophagy Low TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8 + T‐cells. Mechanistically, STING‐dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF‐kB‐driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti‐inflammatory mechanism dampening melanoma antitumor immunity. Synopsis Tumor endothelial cell (EC)‐autophagy was identified as a major barrier to anti‐tumor immune responses against melanoma. Endothelial cell‐specific knockout of autophagy promoted the concomitant activation of the STING‐Type I Interferon and NF‐κB signaling. An “inflamed TEC” signature, identified in EC‐specific autophagy‐deficient mice, correlated with low vascular autophagy levels across several human cancer types. An Inflamed High /Autophagy Low status in TECs of melanoma patients was associated with better response to anti‐PD1 therapy. Graphical Abstract Tumor endothelial cell (EC)‐autophagy was identified as a major barrier to anti‐tumor immune responses against melanoma.

BackgroundImmune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy.MethodsWe used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs.ResultsHere, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry).ConclusionAs HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.

Systematic tumour profiling is essential for biomarker research and clinically for assessing response to therapy. Solving the challenge of delivering informative copy number (CN) profiles from formalin-fixed paraffin embedded (FFPE) material, the only likely readily available biospecimen for most cancers, involves successful processing of small quantities of degraded DNA. To investigate the potential for analysis of such lesions, whole-genome CNVseq was applied to 300 FFPE primary tumour samples, obtained from a large-scale epidemiological study of melanoma. The quality and the discriminatory power of CNVseq was assessed. Libraries were successfully generated for 93% of blocks, with input DNA quantity being the only predictor of success (success rate dropped to 65% if <20 ng available); 3% of libraries were dropped because of low sequence alignment rates. Technical replicates showed high reproducibility. Comparison with targeted CN assessment showed consistency with the Next Generation Sequencing (NGS) analysis. We were able to detect and distinguish CN changes with a resolution of ≤10 kb. To demonstrate performance, we report the spectrum of genomic CN alterations (CNAs) detected at 9p21, the major site of CN change in melanoma. This successful analysis of CN in FFPE material using NGS provides proof of principle for intensive examination of population-based samples.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2 −/− mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22 , and Foxp3 . Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2 −/− Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2 −/− mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy. The ubiquitin ligase Siah2 has been implicated in immune responses. Here, the authors show that Siah2 null immune cells have an increased inflammatory response to inoculated melanoma cells, along with a reduced number of infiltrating immunosuppressive regulatory T cells, resulting in inhibition of tumour growth.

Most clinically applied cancer immunotherapies rely on the ability of CD8 + cytolytic T cells to directly recognize and kill tumour cells 1 – 3 . These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment 4 – 6 . The ability of CD4 + effector cells to contribute to antitumour immunity independently of CD8 + T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified 7 – 10 . Here, we describe a mechanism whereby a small number of CD4 + T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8 + T cell targeting. The CD4 + effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II + CD11c + antigen-presenting cells. We show that T helper type 1 cell-directed CD4 + T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4 + T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4 + T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8 + T cells and natural killer cells and advance cancer immunotherapies. This article describes a mechanism through which CD4 + T cells can eradicate MHC-deficient tumours that escape direct CD8 + T cell targeting and thereby complement the activity of CD8 + T cells and natural killer cells to advance cancer immunotherapies.