Human Melanoma Patients Research Articles

Abstract 3733: Gut microbiota-dependent bile acidtaurodeoxycholic acid, TDCAfacilitates anti-tumor immune responses by modulating cytotoxic CD8 T cell immunity

Abstract Gut microbiota are critical for effective immune checkpoint blockade therapy (ICT) for cancer. However, the underlying mechanisms by which gut microbiota and/or microbiota-derived metabolites augment antitumor response are largely unknown. Here, we utilized a preclinical melanoma (B16-F10) model and treated groups of mice (Jackson, C57BL/6J, female, 6-8 wks, n=14-26 per group) with seven distinct antibiotic treatments to induce unique gut microbiomes and metabolomes. Mice were then treated with ICT (anti-PD-1 and anti-CTLA-4), resulting in markedly heterogeneous tumor phenotypes (tumor volume and weight) as dictated by the specific antibiotic treatment. Using machine learning (e.g. random forest classifier), we identified metabolite candidates most strongly associated with ICT response (i.e. tumor volume and weight).One metabolite candidate, the bile acid taurodeoxycholic acid, TDCA, had previously been identified by my group as associated with anti-PD-1/anti-CTLA-4 response in human melanoma patients (PMID 28923537). Administration of TDCA alone is sufficient to overcome antibiotic-induced ICT hyporesponsiveness in mice. Utilizing both in vitro/ex vivo immune cell assays and in vivo preclinical models, we have validated that TDCA treatment directly boosted the antitumor cytotoxic CD8+ T cell responses (i.e. IFN-gamma production, anti-tumor cytotoxicity, tumor growth, etc.) without affecting innate immune responses. Bulk RNAseq of CD8 T cell analysis indicated that TDCA modulate CD8 T cell function through IFN pathway. Together, our findings suggest that the gut microbial metabolite TDCA could promote immunotherapy efficacy through regulation of CD8+ T cell immunity and has the potential to be utilized as a novel therapeutic agent to enhance ICT efficacy. Citation Format: Wenng li, Hesuiyuan Wang, Laura Coughlin, Nicole Poulides, Priscilla Del Valle, Jake Lichterman, Xiaowei Zhan, Chen Liao, Suzette N. Palmer, Xin Li, Andrew Y. Koh. Gut microbiota-dependent bile acidtaurodeoxycholic acid, TDCAfacilitates anti-tumor immune responses by modulating cytotoxic CD8 T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3733.

Abstract 1547: Melanotransferrin (MELTF, CD228) as a determinant of melanomagenesis: MELTF expression attenuates melanoma progression in the A375-luc2 murine metastasis model and human patients

Abstract The cell-surface glycoprotein melanotransferrin (MELTF, CD228) is a member of the iron-binding transferrin superfamily expressed in human melanocytes and other cell types. Dysregulated MELTF expression in malignant melanoma has been reported before but its specific role in tumorigenesis has remained elusive. Following our interest in the role of the transferrin receptor (TFRC) in melanomagenesis, our initial TCGA-analysis suggested that high MELTF expression is associated with increased melanoma patient survival. In order to explore the mechanistic role of MELTF in melanoma we performed CRISPR/Cas9-based MELTF deletion in A375-luc2 human malignant melanoma cells. ICP/MS-analysis indicated no difference in iron-content between MELTF_WT and MELTF_KO cells. NanoString nCounterTM ('PanCancer-Progression-Panel'; 770 genes) comparative transcriptomic profiling (MELTF_KO versus MELTF_WT) demonstrated an upregulation of EMT-related expression networks in MELTF_KO cells, an observation consistent with phenotypic detection of enhanced matrigel invasiveness; likewise, inclusion of recombinant glycosylated MELTF (Gly20-Gly711 His-tag; 92 kDa; 10-100 nM) attenuated invasiveness of MELTF_KO cells. In SCID mice, subcutaneous growth of MELTF_KO exceeded that of MELTF_WT xenografts, and intracardial injection followed by non-invasive bioluminescent monitoring revealed an increase in lung metastatic burden associated with decreased survival of the MELTF_KO group. These data support a heretofore unrecognized tumor-suppressive function of MELTF expression in melanoma progression, a mechanistic role consistent with a potential survival benefit associated with high MELTF expression observed in human melanoma patients. Citation Format: Jana Jandova, Georg T. Wondrak. Melanotransferrin (MELTF, CD228) as a determinant of melanomagenesis: MELTF expression attenuates melanoma progression in the A375-luc2 murine metastasis model and human patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1547.

Tumor stroma-derived ANGPTL2 potentiates immune checkpoint inhibitor efficacy.

Use of immune checkpoint inhibitors (ICIs) as cancer immunotherapy has advanced rapidly in the clinic. We recently reported that tumor stroma-derived angiopoietin-like protein 2 (ANGPTL2) has tumor suppressive activity by enhancing dendritic cell-mediated CD8+ T cell anti-tumor immune responses. However, a direct impact of ANGPTL2 on ICI anti-tumor effect remains unclear. Here, we use a murine syngeneic model to show that host ANGPTL2 facilitates CD8+ T cell cross-priming and contributes to anti-tumor responses to ICIs in this context. Importantly, our analysis of public datasets indicated that ANGPTL2 expression is associated with positive responses to ICI therapy by human melanoma patients. We conclude that ANGPTL2-mediated stromal cell crosstalk facilitates anti-tumor immunity and ICI responsiveness. These findings overall provide novel insight into ANGPTL2 anti-tumor function and regulation of ICI-induced anti-tumor immunity.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNβ and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Lipid droplets are a metabolic vulnerability in melanoma

Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the melanocytic cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved across human melanoma cell lines and patient tumors. This melanocytic state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon fatty acid oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt cell cycle progression and slow melanoma growth in vivo. Because the melanocytic cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.

Fluorescent Detection of Peroxynitrite Produced by Myeloid-Derived Suppressor Cells in Cancer and Inhibition by Dasatinib.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that expand dramatically in many cancer patients. This expansion contributes to immunosuppression in cancer and reduces the efficacy of immune-based cancer therapies. One mechanism of immunosuppression mediated by MDSCs involves production of the reactive nitrogen species peroxynitrite (PNT), where this strong oxidant inactivates immune effector cells through destructive nitration of tyrosine residues in immune signal transduction pathways. As an alternative to analysis of nitrotyrosines indirectly generated by PNT, we used an endoplasmic reticulum (ER)-targeted fluorescent sensor termed PS3 that allows direct detection of PNT produced by MDSCs. When the MDSC-like cell line MSC2 and primary MDSCs from mice and humans were treated with PS3 and antibody-opsonized TentaGel microspheres, phagocytosis of these beads led to production of PNT and generation of a highly fluorescent product. Using this method, we show that splenocytes from a EMT6 mouse model of cancer, but not normal control mice, produce high levels of PNT due to elevated numbers of granulocytic (PMN) MDSCs. Similarly, peripheral blood mononuclear cells (PBMCs) isolated from blood of human melanoma patients produced substantially higher levels of PNT than healthy human volunteers, coincident with higher peripheral MDSC levels. The kinase inhibitor dasatinib was found to potently block the production of PNT both by inhibiting phagocytosis in vitro and by reducing the number of granulocytic MDSCs in mice in vivo, providing a chemical tool to modulate the production of this reactive nitrogen species (RNS) in the tumor microenvironment.

Splicing of ultraconserved poison exon elements in RNA binding proteins reprograms the T effector immune response

Abstract Our current understanding of pathways governing the strength and longevity of T cell mediated immune responses primarily focuses on transcriptional and epigenetic changes. However, our prior work demonstrated that optimal T cell effector function is also dependent on RNA binding proteins (RBPs) that carry out post-transcriptional RNA splicing and polyadenylation. Here, we find that a specific family of RBPs, serine- and arginine-rich (SR) RBPs, are significantly upregulated by T cell activation and required for CD8 +T cell mediated responses. SR transcripts contain ultraconserved poison exon (PE) elements that are alternatively spliced and introduce premature stop codons that trigger nonsense-mediated decay. We show that differential splicing of specific RBP PE regions is both induced by TCR stimulation and required for CD8 +T cell activation in both human and mouse. Further, we demonstrate that PEs are cell-state associated by using single-cell RNA sequencing analysis of splicing changes across mouse thymic T cell subsets, in CD8 +T cells over the course of an acute Listeria infection, as well as in human lymphoblastic leukemia, atherosclerosis, and melanoma patient T cells. CRISPR-Cas9 mediated deletion of the PE element of SR-like RBP Tra2β in mouse T effector cells during influenza infection resulted in a significant increase in proliferating and surviving T cells and altered the splicing pattern of critical cell survival, proliferation, and adhesion genes. In total, our work demonstrates a fundamental role for alternatively spliced ultraconserved elements in defining T cell states, controlling T effector-mediated immunity, and altering the T cell response in pathologic conditions. This work was supported in part by NIH grants AI136955 and R21AI139891 awarded to A.T.V, institutional support, and Boehringer Ingelheim endowed Chair in Immunology.

Abstract 3755: Dlx4os: a lncRNA associated with malignant melanocyte transformation

Abstract It is estimated that more than 90% of the human genome is transcribed into non-coding RNAs (ncRNA). It has been demonstrated that ncRNAs have an important role in biological processes such as proliferation, differentiation, and cell migration. Changes in their expression have been associated with several diseases, including cancer. Melanoma is one of the most aggressive types of cancer, with high probability of metastasis and an unfavorable response to therapies. Our lab has developed an in vitro melanoma progression model, which was established by exposing nontumorigenic melanocytes (melan-a) to sustained stress conditions (cycles of adhesion blockage), sequentially generating different cell lines (i.e., 4C, premalignant melanocytes; 4C11-, non-metastatic melanoma cells; and 4C11+, metastatic melanoma cells), in which each cell line represents a distinct stage of tumor progression. Previous RNA sequencing data comparing these 4 cell lines identified a total of 3404 long non-coding transcripts which displayed differential expression (DE) levels when comparing two or more cell lines. We have clustered 531 of them into three distinct signatures: malignancy, epithelial-to-mesenchymal transition (EMT), or metastasis, according to their expression profiles. We also analyzed the neighboring genes of those lncRNAs, as it is known that lncRNAs might regulate gene expression in cis. We identified the lncRNA Dlx4os, highly expressed in undifferentiated and mesenchymal-like 4C and 4C11- cells, and 6 neighboring genes whose expression correlate to the EMT signature and overall survival in human melanoma patients. Among these genes we found Dlx4, which shares the same promoter site with Dlx4os and whose expression correlates to a poor prognosis. We silenced Dlx4os in the 4C11- cells through interference RNA technology. The downregulation of Dlx4os led to expression changes in EMT-related genes, such as Snail1, Sox10 and Mitf, indicating that it might play a role in EMT. Dlx4os downregulation also inhibited cell migration in vitro and tumorigenesis in vivo. These results overall indicate a role of Dlx4os in melanocyte malignant transformation. Citation Format: Ana Luísa P. Ayub, Hatylas Azevedo, Diogo Pessoa, Leinal Sejour, Ioannis Vlachos, Eduardo M. Reis, Frank J. Slack, Miriam G. Jasiulionis. Dlx4os: a lncRNA associated with malignant melanocyte transformation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3755.

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...

A Sampling of Highlights from the Literature.

Knockout of RASA2 enhances T-cell antitumor function (from Public Domain Vectors)Several immunosuppressive mechanisms limit the efficacy of adoptive T-cell therapies. Carnevale et al. performed six genome-wide CRISPR–Cas9 knock-out screens for regulators of T-cell resistance to immunosuppression by inhibitors of calcium and calcineurin, TGFβ, and regulatory T cells, identifying a role for RAS p21 protein activator 2 (RASA2) in each case. Knocking out RASA2A in human T cells, including T-cell receptor (TCR) and chimeric antigen receptor (CAR)–engineered T cells, enhances their antitumor function in vitro and in xenograft models of melanoma, osteosarcoma, and acute lymphoblastic leukemia. The data suggest ablation of RASA2 may enable the generation of more efficacious adoptive T-cell therapies.Carnevale J, …, Marson A. Nature 2022 August 24;609:174–82.CD62L+ TPEX cells mediate responses to anti–PD-1 (from NIAID via NIH Flickr)Precursors of exhausted T (TPEX) cells, characterized by expression of TCF1, mediate responsiveness to anti–PD-1. Following single-cell RNA sequencing of TPEX cell–enriched CD8+ T cells from mice chronically infected with lymphocytic choriomeningitis virus (LCMV), Tsui et al. stratify TPEX cells by expression of Sell, which encodes CD62L. The transcription factor MYB is required for the development of CD62L+ TPEX cells, which show enhanced capacity for self-renewal, multipotency, and long-term proliferation compared with CD62L– TPEX cells. CD62L+ TPEX cells are pivotal for responsiveness to anti–PD-1 in mice chronically infected with LCMV. Thus, it is vital that we better understand their role in T cell–mediated cancer immunotherapy.Tsui C, …, Kallies A. Nature 2022 August 17;609:354–60.iNKT cells outperform other cell therapies (from Todd van Hoosear via Flickr)The immune suppressive tumor microenvironment (TME) can curb responses to an array of cell-based therapies. Delfanti et al. demonstrate the utility of T-cell receptor (TCR)–engineered invariant natural killer T (iNKT) cells to overcome immune-suppressive barriers in the TME. The engineered iNKT cells act as bispecific effectors, bridging CD1d to MHC-restricted antigens on tumor cells, while also maintaining their known TME-modulating functions. Treatment with these cells outperforms nontransduced iNKT cells and CD8+ T cells expressing the same TCR, and efficacy can be boosted with addition of an iNKT-cell agonist. These data show that TCR-engineered iNKT cells could be a potential cell-based therapy for patients with cancer.Delfanti G, …, Casorati G. Sci Immunol 2022 August 19;7:sciimmunol.abn65.Targeting COX2+ fibroblasts can improve antitumor responses (from go digital via Flickr).How the premetastatic niche develops and what cell types in the metastasis-supporting organ are involved has not been fully elucidated. Gong et al. identify a subset of cyclooxygenase 2–positive (COX-2+) lung fibroblasts that modulate the function of multiple myeloid cell subsets, rendering them dysfunctional or immune suppressive. This process is enhanced by neutrophil-derived IL1β produced because of tumor-associated inflammation. In breast cancer models, targeting the COX2 pathway in these fibroblasts improves immunotherapy efficacy and reduces lung metastasis, highlighting how understanding the microenvironment of the premetastatic niche can facilitate identification of therapeutic targets to boost antitumor responses.Gong Z, …, Ren G. Immunity 2022 July 30;55:1483–1500.E9.In situ generation of CAR-macrophages effectively prevent GBM recurrence (from Christaras A via Wikimedia Commons)Recurrence of glioblastoma multiforme (GBM) after surgical resection is driven by residual glioma stem cells (GSC). Chen et al. developed a cavity-injectable hydrogel embedded with macrophage-targeted nanoporter particles that they used to deliver plasmids encoding a CD133-directed chimeric antigen receptor (CAR) to macrophages in situ following tumor resection in preclinical models. The CAR+ macrophages target GSCs and show antitumor activity in vitro and in vivo, including preventing recurrence of surgically resected GBM in an orthotopic patient-derived xenograft model. These findings suggest that the injectable CAR-macrophage platform may be an effective therapeutic strategy to improve long-term survival of GBM patients.Chen C, …, Jiang X. Sci Trans Med 2022 Aug 3;14:scitranslmed.abn1128.Tumor-reactive T cells exhibit signatures of activation indicative of their TCR avidity (from Takuma-sa via Wikimedia Commons)The impact of T-cell receptor (TCR) avidity on T-cell antitumor activity remains unclear. Through adoptive transfer of ultra-low (<1000) numbers of T cells with OVA-specific TCRs of varying avidity into mice bearing OVA-expressing tumors, Purcarea et al. found that tumor-reactive T cells in tissues outside of tumors express PD-1 proportional to the avidity of their TCR and to their protective capacity. Tumor-infiltrating and peripheral T cells isolated from human melanoma patients exhibit gene expression and phenotypic signatures of ex vivo TCR functionality and cytotoxicity, altogether presenting molecular markers of highly active tumor-specific T cells.Purcarea A, …, Schober K. Sci Immunol 2022 August 12;7:sciimmunol.abm207.

Pilot Study: Assessing the Expression of Serum Lactate Dehydrogenase and Peripheral Leukocyte Ratios in Canine Oral Malignant Melanoma.

Simple SummaryThis study aimed to assess the expression of novel blood biomarkers in dogs with oral malignant melanoma, an aggressive and common oral cancer in dogs. The results of this pilot study suggest that the concentrations of lactate dehydrogenase, which can be easily measured with blood samples, are significantly higher in dogs with oral malignant melanoma. These blood biomarkers have been shown to be of prognostic value in human melanoma patients, meaning that they can act to predict the potential behaviour of the tumour. Therefore, research into our canine patients should be viewed as being potentially very valuable, as the discovery of easily measurable prognostic biomarkers could significantly further not only our understanding of the underlying physiology of melanoma itself, but also change the way veterinary surgeons investigate and treat the disease in the future. This study lays the foundations for further, more extensive investigation, into the topic.Measurement of blood biomarkers such as lactate dehydrogenase (LDH) and peripheral leukocyte ratios have been shown to be of prognostic value in human melanoma patients. Previous veterinary studies have demonstrated that changes in these values are detectable in multiple canine cancer patients. However, to the authors’ knowledge, no studies have yet demonstrated an increase in LDH in canine oral malignant melanoma patients, nor has the effect of metastasis on LDH levels been explored. This retrospective pilot study included 18 dogs, of which 10 were healthy controls, 5 OMM patients with metastasis and 3 without metastasis. Serum LDH was measured and pre-treatment peripheral leucocyte ratios were calculated. LDH was measurable within all patient groups and a statistically significant difference in LDH levels was detected between patients with OMM and healthy controls (p < 0.05); however, no significant difference was detected between patients with or without metastatic disease. This study suggests that serum LDH levels are significantly increased in dogs with OMM compared to healthy controls, paving the way for further research to investigate the prognostic value of this biomarker.

Lipid Nanoparticle Delivery of Fas Plasmid Restores Fas Expression to Suppress Melanoma Growth In Vivo.

Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-Tcell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to Tcell cytotoxicity. DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas+ tumor cells and suppresses established melanoma growth in immune-competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein levels on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted Tcells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data demonstrate that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.

Vasculogenic mimicry structures in melanoma support the recruitment of monocytes

ABSTRACT The progression of cancer is facilitated by infiltrating leukocytes which can either actively kill cancer cells or promote their survival. Our current understanding of leukocyte recruitment into tumors is largely limited to the adhesion molecules and chemokines expressed by conventional blood vessels that are lined by endothelial cells (ECs). However, cancer cells themselves can form their own vascular structures (a process known as vasculogenic mimicry (VM)); but whether they actively participate in the recruitment of leukocytes remains to be elucidated. Herein, we demonstrate that VM-competent human melanoma cell lines express multiple adhesion molecules (e.g. CD44, intercellular adhesion molecule (ICAM)-1 and junction adhesion molecules (JAMs)) and chemokines (e.g. CXCL8 and CXCL12) relevant for leukocyte recruitment. Microfluidic-based adhesion assays revealed that similar to ECs, VM-competent melanoma cells facilitate the rolling and adhesion of leukocytes, particularly monocytes, under conditions of shear flow. Moreover, we identified ICAM-1 to be a key participant in this process. Transwell assays showed that, similar to ECs, VM-competent melanoma cells facilitate monocyte transmigration toward a chemotactic gradient. Gene expression profiling of human melanoma patient samples confirmed the expression of numerous leukocyte capture adhesion molecules and chemokines. Finally, immunostaining of patient tissue microarrays revealed that tumors with high VM content also contained higher numbers of leukocytes (including macrophages). Taken together, this study suggests an underappreciated role of VM vessels in solid tumors via their active participation in leukocyte recruitment and begins to identify key adhesion molecules and chemokines that underpin this process.

UBIAD1 and CoQ10 protect melanoma cells from lipid peroxidation-mediated cell death

Cutaneous melanoma is the deadliest type of skin cancer, although it accounts for a minority of all skin cancers. Oxidative stress is involved in all stages of melanomagenesis and cutaneous melanoma can sustain a much higher load of Reactive Oxygen Species (ROS) than normal tissues. Melanoma cells exploit specific antioxidant machinery to support redox homeostasis. The enzyme UBIA prenyltransferase domain-containing protein 1 (UBIAD1) is responsible for the biosynthesis of non-mitochondrial CoQ10 and plays an important role as antioxidant enzyme. Whether UBIAD1 is involved in melanoma progression has not been addressed, yet. Here, we provide evidence that UBIAD1 expression is associated with poor overall survival (OS) in human melanoma patients. Furthermore, UBIAD1 and CoQ10 levels are upregulated in melanoma cells with respect to melanocytes. We show that UBIAD1 and plasma membrane CoQ10 sustain melanoma cell survival and proliferation by preventing lipid peroxidation and cell death. Additionally, we show that the NAD(P)H Quinone Dehydrogenase 1 (NQO1), responsible for the 2-electron reduction of CoQ10 on plasma membranes, acts downstream of UBIAD1 to support melanoma survival. By showing that the CoQ10-producing enzyme UBIAD1 counteracts oxidative stress and lipid peroxidation events in cutaneous melanoma, this work may open to new therapeutic investigations based on UBIAD1/CoQ10 loss to cure melanoma.

Canine Melanoma Immunology and Immunotherapy: Relevance of Translational Research.

In veterinary oncology, canine melanoma is still a fatal disease for which innovative and long-lasting curative treatments are urgently required. Considering the similarities between canine and human melanoma and the clinical revolution that immunotherapy has instigated in the treatment of human melanoma patients, special attention must be paid to advancements in tumor immunology research in the veterinary field. Herein, we aim to discuss the most relevant knowledge on the immune landscape of canine melanoma and the most promising immunotherapeutic approaches under investigation. Particular attention will be dedicated to anti-cancer vaccination, and, especially, to the encouraging clinical results that we have obtained with DNA vaccines directed against chondroitin sulfate proteoglycan 4 (CSPG4), which is an appealing tumor-associated antigen with a key oncogenic role in both canine and human melanoma. In parallel with advances in therapeutic options, progress in the identification of easily accessible biomarkers to improve the diagnosis and the prognosis of melanoma should be sought, with circulating small extracellular vesicles emerging as strategically relevant players. Translational advances in melanoma management, whether achieved in the human or veterinary fields, may drive improvements with mutual clinical benefits for both human and canine patients; this is where the strength of comparative oncology lies.

Circulating CD8+ mucosal-associated invariant T cells correlate with improved treatment responses and overall survival in anti-PD-1-treated melanoma patients.

ObjectivesWhile much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide‐specific T cells, the role of unconventional T cells, such as mucosal‐associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate‐like T cells expressing a semi‐invariant T‐cell receptor restricted to the MHC class I‐like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes.MethodsIn this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor‐infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non‐responders as well as healthy donors.ResultsWe identified tumor‐infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival.ConclusionOur results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell‐directed therapies in combination with current and next‐generation ICIs.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

A Deep Learning-Based Model That Reduces Speed of Sound Aberrations for Improved In Vivo Photoacoustic Imaging.

Photoacoustic imaging (PAI) has attracted great attention as a medical imaging method. Typically, photoacoustic (PA) images are reconstructed via beamforming, but many factors still hinder the beamforming techniques in reconstructing optimal images in terms of image resolution, imaging depth, or processing speed. Here, we demonstrate a novel deep learning PAI that uses multiple speed of sound (SoS) inputs. With this novel method, we achieved SoS aberration mitigation, streak artifact removal, and temporal resolution improvement all at once in structural and functional in vivo PA images of healthy human limbs and melanoma patients. The presented method produces high-contrast PA images in vivo with reduced distortion, even in adverse conditions where the medium is heterogeneous and/or the data sampling is sparse. Thus, we believe that this new method can achieve high image quality with fast data acquisition and can contribute to the advance of clinical PAI.