Immune Response In Melanoma Research Articles

A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Targeting Dectin-1 and or VISTA enhances anti-tumor immunity in melanoma but not colorectal cancer model.

Acquired resistance to immune checkpoint blockers (ICBs) is a major barrier in cancer treatment, emphasizing the need for innovative strategies. Dectin-1 (gene Clec7a) is a C-type lectin receptor best known for its ability to recognize β-glucan-rich structures in fungal cell walls. While Dectin-1 is expressed in myeloid cells and tumor cells, its significance in cancer remains the subject of controversy. Using Celc7a-/- mice and curdlan administration to stimulate Dectin-1 signaling, we explored its impact. VISTA KO mice were employed to assess VISTA's role, and bulk RNAseq analyzed curdlan effects on neutrophils. Our findings reveal myeloid cells as primary Dectin-1 expressing cells in the tumor microenvironment (TME), displaying an activated phenotype. Strong Dectin-1 co-expression/co-localization with VISTA and PD-L1 in TME myeloid cells was observed. While Dectin-1 deletion lacked protective effects, curdlan stimulation significantly curtailed B16-F10 tumor progression. RNAseq and pathway analyses supported curdlan's role in triggering a cascade of events leading to increased production of pro-inflammatory mediators, potentially resulting in the recruitment and activation of immune cells. Moreover, we identified a heterogeneous subset of Dectin-1+ effector T cells in the TME. Similar to mice, human myeloid cells are the prominent cells expressing Dectin-1 in cancer patients. Our study proposes Dectin-1 as a potential adjunctive target with ICBs, orchestrating a comprehensive engagement of innate and adaptive immune responses in melanoma. This innovative approach holds promise for overcoming acquired resistance to ICBs in cancer treatment, offering avenues for further exploration and development.

Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

CRABP2 regulates infiltration of cancer-associated fibroblasts and immune response in melanoma.

Finding biomarkers for immunotherapy is an urgent issue in cancer treatment. Cellular retinoic acid-binding protein 2 (CRABP2) is a controversial factor in the occurrence and development of human tumors. However, there is limited research on the relationship between CRABP2 and immunotherapy response. This study found that negative correlations of CRABP2 and immune checkpoint markers (PD-1, PD-L1, and CTLA-4) were observed in breast invasive carcinoma (BRCA), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD) and testicular germ cell tumors (TGCT). In particular, in SKCM patients who were treated with PD-1 inhibitors, high levels of CRABP2 predicted poor prognosis. Additionally, CRABP2 expression was elevated in cancer-associated fibroblasts (CAFs) at the single-cell level. The expression of CRABP2 was positively correlated with markers of CAFs, such as MFAP5, PDPN, ITGA11, PDGFRα/β and THY1 in SKCM. To validate the tumor-promoting effect of CRABP2 in vivo, SKCM xenograft mice models with CRABP2 overexpression have been constructed. These models showed an increase in tumor weight and volume. Enrichment analysis indicated that CRABP2 may be involved in immune-related pathways of SKCM, such as extracellular matrix (ECM) receptor interaction and epithelial-mesenchymal transition (EMT). The study suggests that CRABP2 may regulate immunotherapy in SKCM patients by influencing infiltration of CAFs. In conclusion, this study provides new insights into the role of CRABP2 in immunotherapy response. The findings suggest that CRABP2 may be a promising biomarker for PD-1 inhibitors in SKCM patients. Further research is needed to confirm these findings and to explore the clinical implications of CRABP2 in immunotherapy.

Multifunctional hybrid oncolytic virus-mimicking nanoparticles for targeted induce of tumor-specific pyroptosis and enhanced anti-tumor immune response in melanoma

Pyroptosis, a newly discovered form of programmed cell death, has emerged as a promising antitumor approach by releasing pro-inflammatory cytokines and immunogenic substances upon cell rupture. However, non-specific pyroptosis potentially causes systemic damage to healthy tissues, and the heterogeneity of tumors limits the anti-tumor effect of pyroptosis. To address these challenges, we propose a simple yet powerful method for creating multifunctional hybrid oncolytic virus-mimicking nanovesicles (HVMNVs) that possess tumor-targeting capabilities, adjuvant activity, and the ability to evade immune clearance induced by virus modification. Based on this multifunctional nanovesicle, we developed a functional nanoparticle (HVMNVs@Fe-C) that significantly enhances the tumor targetability and cell uptake efficiency of the extremely small iron oxide nanoparticles (ESIONPs) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), leading to rapid tumor pyroptosis. More importantly, HVMNVs@Fe-C ingeniously achieves a synergistic effect by incorporating the adjuvantic and tumor-targeting properties of oncolytic viruses with the cell lytic and pro-inflammatory functions of pyroptosis in a single nanoparticle. This leads to a cascade of specific anti-tumor immune responses and reprogramming of the immunosuppressive tumor microenvironment, resulting in excellent tumor inhibition efficacy against primary melanoma growth and pulmonary metastasis. Overall, our novel strategy offers considerable promise for precisive targeted delivery and cancer immunotherapy.

B cell profiles, antibody repertoire and reactivity reveal dysregulated responses with autoimmune features in melanoma

B cells are known to contribute to the anti-tumor immune response, especially in immunogenic tumors such as melanoma, yet humoral immunity has not been characterized in these cancers to detail. Here we show comprehensive phenotyping in samples of circulating and tumor-resident B cells as well as serum antibodies in melanoma patients. Memory B cells are enriched in tumors compared to blood in paired samples and feature distinct antibody repertoires, linked to specific isotypes. Tumor-associated B cells undergo clonal expansion, class switch recombination, somatic hypermutation and receptor revision. Compared with blood, tumor-associated B cells produce antibodies with proportionally higher levels of unproductive sequences and distinct complementarity determining region 3 properties. The observed features are signs of affinity maturation and polyreactivity and suggest an active and aberrant autoimmune-like reaction in the tumor microenvironment. Consistent with this, tumor-derived antibodies are polyreactive and characterized by autoantigen recognition. Serum antibodies show reactivity to antigens attributed to autoimmune diseases and cancer, and their levels are higher in patients with active disease compared to post-resection state. Our findings thus reveal B cell lineage dysregulation with distinct antibody repertoire and specificity, alongside clonally-expanded tumor-infiltrating B cells with autoimmune-like features, shaping the humoral immune response in melanoma.

Siglec-7/9 are novel immune checkpoints for prostate cancer

Abstract Immune checkpoint-based therapy has led to striking improvements in survival in some types of cancer, but it is ineffective for others, including prostate cancer. Changes in glycosylation, particularly hypersialylation, have been found in diverse malignancies. Glycosylation can result in immune evasion through direct interactions of sialylated glycoproteins on cancer cell surfaces with Siglecs on immune cells including T cells, NK cells, and dendritic cells. Interactions between Siglec-7/9 and sialic acid have been implicated in inhibiting immune response in melanoma, leukemia, and lung cancer. Here, we found that Siglec-7/9 was highly expressed in immune cells including T cells and myeloid cells in the prostate cancer TCGA dataset. Immunohistochemistry demonstrated that Siglec-7/9 ligands were highly expressed in surgically resected prostate cancer tumor tissues but showed no or low expression in adjacent normal tissues. Siglec-7/9 ligands were also found in the prostate cancer cell lines. The disruption of Siglec-7/9 and their ligands by sialidase, which cleaves the sialic acid, or by anti-Siglec-7/9 blocking antibodies, promoted T cell-mediated cytotoxic killing of prostate cancer cells. Furthermore, anti-Siglec-7/9 treatment suppressed prostate cancer tumor growth in a humanized mouse model. Immunohistochemistry demonstrated that prostate cancers in mice treated with anti-Siglec 7/9 antibodies exhibited less Ki67 and CD31, and more cleaved caspase 3 and CD8 compared with the isotype control. This study provides insight into Siglec-7/9-sialic acid targeting strategies for prostate cancer and lays the groundwork for developing a novel class of immunotherapy-based drugs. Supported by grants from DoD (W81XWH2110195) and NIH (#LINK#U01CA226051#ENDLINK#)

Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma

BackgroundLoss of Ambra1 (autophagy and beclin 1 regulator 1), a multifunctional scaffold protein, promotes the formation of nevi and contributes to several phases of melanoma development. The suppressive functions of...

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy.

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed.

Ezh2Y641F mutations co-operate with Stat3 to regulate MHC class I antigen processing and alter the tumor immune response in melanoma.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2, a chromatin modifying complex, which mediates methylation of lysine 27 on histone 3 (H3K27me3), a repressive chromatin mark. Genetic alterations in EZH2 in melanoma include amplifications and activating point mutations at tyrosine 641 (Y641) whose underlying oncogenic mechanisms remain largely unknown. Here, we found that expression of Ezh2Y641F causes upregulation of a subset of interferon-regulated genes in melanoma cells. Upregulation of these genes was not a direct effect of changes in H3K27me3, but via a non-canonical interaction between Ezh2 and Signal Transducer and Activator of Transcription 3 (Stat3). Ezh2 and Stat3 together function as transcriptional activators to mediate gene activation of numerous genes, including MHC Class 1b antigen processing genes. Furthermore, expression of Stat3 is required to maintain an anti-tumor immune response in Ezh2Y641F melanomas and to prevent melanoma progression and recurrence.

Spatial biology analysis reveals B cell follicles in secondary lymphoid structures may regulate anti-tumor responses at initial melanoma diagnosis.

IntroductionB cells are key regulators of immune responses in melanoma. We aimed to explore differences in the histologic location and activation status of B cell follicles in sentinel lymph nodes (SLN) of melanoma patients.MethodsFlow cytometry was performed on fresh tumor draining lymph nodes (LN). Paraffin slides from a separate cohort underwent NanoString Digital Spatial Profiling (DSP)®. After staining with fluorescent markers for CD20 (B cells), CD3 (T cells), CD11c (antigen presenting cells) and a nuclear marker (tumor) was performed, regions of interest (ROI) were selected based on the location of B cell regions (B cell follicles). A panel of 68 proteins was then analyzed from the ROIs.ResultsB cell percentage trended higher in patients with tumor in LN (n=3) compared to patients with nSLN (n=10) by flow cytometry. B cell regions from a separate cohort of patients with tumor in the (pSLN) (n=8) vs. no tumor (nSLN) (n=16) were examined with DSP. Within B cell regions of the SLN, patients with pSLN had significantly higher expression of multiple activation markers including Ki-67 compared to nSLN patients. Among 4 patients with pSLN, we noted variability in arrangement of B cell follicles which were either surrounding the tumor deposit or appeared to be infiltrating the tumor. The B cell follicle infiltrative pattern was associated with prolonged recurrence free survival.ConclusionThese data suggest a role for B cell follicles in coordinating effective adaptive immune responses in melanoma when low volume metastatic disease is present in tumor draining LN.

Ferroptosis Associates With Diagnosis and Prognosis by Promoting Antitumor Immune Response in Melanoma.

Immunotherapy has greatly improved the clinical benefits of cancer treatment, especially in melanoma. Ferroptosis is a novel mechanism of cell death which relates to immunity. This study aimed at understanding the potential link between ferroptosis and cancer immunocompetent in melanoma using multiple bioinformatics analyses. By the WGCNA assay, we first constructed a key module–gene of ferroptosis, which was strongly correlated with the diagnosis, prognosis, and infiltration of immune cells in melanoma. The elevated module–gene could effectively distinguish melanoma from normal tissues and acted as a good prognostic marker. The module–gene of ferroptosis was positively correlated with the infiltration of immune cells. In particular, the module was positively correlated with the expression of PD-L1 and sensitively increased after effective anti-PD-1 treatment. Furthermore, the differential expression of the module–gene between normal and tumor tissues was observed in pan-cancer. The similarity correlations of the module–gene with infiltration of immune cells and the expressions of PD-L1 were confirmed in the pan-cancer level. Our study demonstrated that the key module–gene of ferroptosis was closely related with diagnosis, prognosis, and anti-immune response in melanoma, as well as in pan-cancer.

MiRNA as a Modulator of Immunotherapy and Immune Response in Melanoma.

Immune checkpoint inhibitors are a promising therapy for the treatment of cancers, including melanoma, that improved benefit clinical outcomes. However, a subset of melanoma patients do not respond or acquire resistance to immunotherapy, which limits their clinical applicability. Recent studies have explored the reasons related to the resistance of melanoma to immune checkpoint inhibitors. Of note, miRNAs are the regulators of not only cancer progression but also of the response between cancer cells and immune cells. Investigation of miRNA functions within the tumor microenvironment have suggested that miRNAs could be considered as key partners in immunotherapy. Here, we reviewed the known mechanism by which melanoma induces resistance to immunotherapy and the role of miRNAs in immune responses and the microenvironment.

Prognostic significance of platelet lymphocyte ratio in patients with melanoma: A meta-analysis.

The aim of this study was to systematically evaluate the prognostic role of platelet lymphocyte ratio (PLR) in patients with melanoma through performing a meta-analysis. PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure were searched for potential studies. The basic characteristics and relevant data were extracted. Hazard ratios with 95% confidence intervals (CIs) were pooled to evaluate the prognostic role of PLR in patients with melanoma. Ten studies enrolling 2422 patients were included. The pooled hazard ratios of higher PLR for overall survival and progression-free survival in melanoma were 1.70 (95% CI, 1.22-2.37) and 1.65 (95% CI, 1.10-2.47), respectively. Sensitivity analysis and subgroup analyses were also performed. No significant publication bias was observed. Our results showed that higher PLR was associated with poorer overall survival and progression-free survival in patients with melanoma. These findings may help to determine the prognosis and explore future novel therapies based on modulating inflammation and immune responses in melanoma.

Stapled Wasp Venom-Derived Oncolytic Peptides with Side Chains Induce Rapid Membrane Lysis and Prolonged Immune Responses in Melanoma.

Peptide stapling chemistry represents an attractive strategy to promote the clinical translation of protein epitope mimetics, but its use has not been applied to natural cytotoxic peptides (NCPs) to produce new oncolytic peptides. Based on a wasp venom peptide, a series of stapled anoplin peptides (StAnos) were prepared. The optimized stapled Ano-3/3s were shown to be protease-resistant and exerted superior tumor cell-selective cytotoxicity by rapid membrane disruption. In addition, Ano-3/3s induced tumor ablation in mice through the direct oncolytic effect and subsequent stimulation of immunogenic cell death. This synergistic oncolytic-immunotherapy effect is more remarkable on melanoma than on triple-negative breast cancer in vivo. The efficacies exerted by Ano-3/3s on melanoma were further characterized by CD8+ T cell infiltration, and the addition of anti-CD8 antibodies diminished the long-term antitumor effects. In summary, these results support stapled peptide chemistry as an advantageous method to enhance the NCP potency for oncolytic therapy.

Proof of Concept: Protein S As an Immune Modulatory Agent to Control Pancreatic Cancer

Background: Pancreatic cancer (PC) remains one of the most difficult tumors to treat. PC is nearly always diagnosed late with 80% of PC patients having Stage 4 disease and an average life expectancy of a mere 4-6 months. It affects men and women equally. In spite of our understanding of KRAS as the driver mutation in the overwhelming majority of PC patients, drugs to target KRAS remain elusive so far. Chemotherapy and radiation remain the standard of care for PC patients, many of whom are unresectable at diagnosis. This bleak prognosis encourages scientists and drug developers to propose bold, creative treatment strategies to better manage PC. Protein S (PS) is a 75 KDa vitamin K-dependent potent natural anticoagulant. PC patients have much higher risk of thrombosis than the general population. In 2018, we have shown that HIF1α, commonly upregulated in hypoxic cancer tissues, suppresses PS expression. PS has coagulation-independent, receptor-mediated effects through the TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK). We recently published (in press) data that indicate that PS suppresses the growth of PC cells in vitro and that PROS1 (PS) gene expression is associated with better survival in PC. Survival analysis based on public domain RNASeq data shows that PROS1 mRNA expression in PC is significantly correlated with better overall survival (OS) up to 18 months (Figure 1). Additionally, very recent data indicate that PS receptor MERTK is a late costimulatory signal in CD8 T-cells, which is required for the establishment of immunological memory and anti-tumor immune responses in melanoma. Our proof of concept (POC) shown in the schematic is PS directly suppresses PC cell growth and promotes CD8-mediated tumor immunity against PC. Results &amp; Discussion: PS and its properties as a signaling molecule in the context of cancer progression and cellular aggressiveness have not been studied in-depth. The expression levels of the individual TAM receptors showed less than 1-fold variation among the cell lines tested; whereas, PS and GAS6 levels were significantly different. PS/GAS6 ratio directly correlates with the aggressiveness of PC cell lines. We found that the lower the ratio, the higher was the aggressiveness. We used three cell lines, MIA PaCa-2, PANC-1, and BXPC-3 with population doubling times of 40 hours, 52 hours and 72 hours, respectively (per information from ATCC). The basal levels of the three TAM receptors were relatively uniform among the three cell lines, i.e., less than 5% difference in expression level. Interestingly, although PANC-1 cells, with an intermediate degree of aggressiveness, had a PS/GAS6 ratio of 1, MIA-PaCa-2 cells had a significantly lower PS/GAS6 ratio indicating that more aggressive cell line has more GAS6. The least aggressive cell line BXPC-3 cells had a much higher PS/GAS6 ratio. PS overexpression reduced survival and proliferation of PANC-1 and MIA PaCa-2 cells. To further confirm that PS overexpression accelerated apoptosis of PC cells, we generated flow data using Annexin V/7AAD (7-Aminoactinomycin D) staining. Notably, PS gene knockdown by SiRNA or GAS6 overexpression resulted in increase in aggressiveness of those cell lines tested in our published data (In press). Conversely, GAS6 gene knockdown attenuated the aggressive phenotypes of these cell lines tested. Conclusion: We propose to test PS infusion in PC to downregulate HIF1α and modulate the tumor microenvironment (TME) to promote sustained tumor immunity. Our preliminary data and publicly available gene expression profiles suggest that PS may have therapeutic value in PC. Disclosures No relevant conflicts of interest to declare.

Abstract IA07: UVB-induced tumor heterogeneity directs immune response in melanoma

Abstract Little is known regarding the relationship between intra-tumor heterogeneity (ITH) and immune response in melanoma. Here, we explored the role of ITH in tumor rejection by establishing a melanoma mouse model and inducing UVB-derived mutations that increase both ITH and mutational load. This induction gives rise to highly aggressive tumors and decreased cytotoxic activity of tumor infiltrating lymphocytes (TILs). Conversely, single cell-derived melanoma clones with reduced ITH are swiftly rejected. Tumor rejection is accompanied by increased TIL reactivity and increased infiltration into the tumor core. Using phylogenetic tree analyses and mixing experiments of 20 single cell clones that lie along the phylogenetic tee we show that tumor rejection is strongly affected by number of injected clones and genetic diversity. We have, thus set up a novel, highly controlled system that enables us to study the interphase between the immune system and different layers of intra-tumor heterogeneity. Finally, the analysis of melanoma patient data identifies parallel observations, supporting the importance of ITH in determining patient survival and response to checkpoint blockade. Citation Format: Osnat Bartok, Sushant Patkar, Sapir Cohen, Kevin Litchfield, Hiren Karathia, Joo Sang Lee, Alejandro Jiménez-Sánchez, Chi-Ping Day, Lea Eisenbach, Martin Miller, Glenn Merlino, Eli Pikarsky, Arie Admon, Charles Swanton, Eytan Ruppin, Yardena Samuels, Yochai Wolf. UVB-induced tumor heterogeneity directs immune response in melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA07.

Melanoma Stem Cell-Like Phenotype and Significant Suppression of Immune Response within a Tumor Are Regulated by TRIM28 Protein.

Simple SummaryA growing body of evidence indicates that stem cell-associated molecular features, collectively known as stemness, are biologically important in cancer development and progression, and negatively associate with anticancer immunity. The aim of our study was to investigate the association between TRIM28 level and melanoma stemness accompanied by low antitumor immune response. Furthermore, we aimed to evaluate potential value for TRIM28 in predicting stem-like melanoma phenotype. Our results indicate that TRIM28 might facilitate the “stemness high/immune low” melanoma phenotype by attenuating interferon signaling leading to a worse prognosis for melanoma patients. TRIM28 emerged as a regulator Interferon Regulatory Factor family of transcription factors’ expression, mediating epigenetic repression of IRF family members in “stemness high/immune low” melanomas.TRIM28 emerged as a guard of the intrinsic “state of cell differentiation”, facilitating self-renewal of pluripotent stem cells. Recent reports imply TRIM28 engagement in cancer stem cell (CSC) maintenance, although the exact mechanism remains unresolved. TRIM28 high expression is associated with worse melanoma patient outcomes. Here, we investigated the association between TRIM28 level and melanoma stemness, and aligned it with the antitumor immune response to find the mechanism of “stemness high/immune low” melanoma phenotype acquisition. Based on the SKCM TCGA data, the TRIM28 expression profile, clinicopathological features, expression of correlated genes, and the level of stemness and immune scores were analyzed in patient samples. The biological function for differentially expressed genes was annotated with GSEA. Results were validated with additional datasets from R2: Genomics Analysis and Visualization Platform and in vitro with a panel of seven melanoma cell lines. All statistical analyses were accomplished using GraphPad Prism 8. TRIM28HIGH-expressing melanoma patients are characterized by worse outcomes and significantly different gene expression profiles than the TRIM28NORM cohort. TRIM28 high level related to higher melanoma stemness as measured with several distinct scores and TRIM28HIGH-expressing melanoma cell lines possess the greater potential of melanosphere formation. Moreover, TRIM28HIGH melanoma tumors were significantly depleted with infiltrating immune cells, especially cytotoxic T cells, helper T cells, and B cells. Furthermore, TRIM28 emerged as a good predictor of “stemness high/immune low” melanoma phenotype. Our data indicate that TRIM28 might facilitate this phenotype by direct repression of interferon signaling. TRIM28 emerged as a direct link between stem cell-like phenotype and attenuated antitumor immune response in melanoma, although further studies are needed to evaluate the direct mechanism of TRIM28-mediated stem-like phenotype acquisition.

UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma.

SummaryAlthough clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

831 Bortezomib-induced immunogenic cell death enhances immune response in melanoma

831 Bortezomib-induced immunogenic cell death enhances immune response in melanoma