Melanoma Tumor Microenvironment Research Articles

The role of FoxP3+ regulatory T cells and IDO+ immune and tumor cells in malignant melanoma \u2013 an immunohistochemical study

BackgroundFoxP3+ Regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) participate in the formation of an immunosuppressive tumor microenvironment (TME) in malignant cutaneous melanoma (CM). Recent studies have reported that IDO expression correlates with poor prognosis and greater Breslow’s depth, but results concerning the role of FoxP3+ Tregs in CM have been controversial. Furthermore, the correlation between IDO and Tregs has not been substantially studied in CM, although IDO is known to be an important regulator of Tregs activity.MethodsWe investigated the associations of FoxP3+ Tregs, IDO+ tumor cells and IDO+ stromal immune cells with tumor stage, prognostic factors and survival in CM. FoxP3 and IDO were immunohistochemically stained from 29 benign and 29 dysplastic nevi, 18 in situ -melanomas, 48 superficial and 62 deep melanomas and 67 lymph node metastases (LNMs) of CM. The number of FoxP3+ Tregs and IDO+ stromal immune cells, and the coverage and intensity of IDO+ tumor cells were analysed.ResultsThe number of FoxP3+ Tregs and IDO+ stromal immune cells were significantly higher in malignant melanomas compared with benign lesions. The increased expression of IDO in melanoma cells was associated with poor prognostic factors, such as recurrence, nodular growth pattern and increased mitotic count. Furthermore, the expression of IDO in melanoma cells was associated with reduced recurrence˗free survival. We further showed that there was a positive correlation between IDO+ tumor cells and FoxP3+ Tregs.ConclusionsThese results indicate that IDO is strongly involved in melanoma progression. FoxP3+ Tregs also seems to contribute to the immunosuppressive TME in CM, but their significance in melanoma progression remains unclear. The positive association of FoxP3+ Tregs with IDO+ melanoma cells, but not with IDO+ stromal immune cells, indicates a complex interaction between IDO and Tregs in CM, which demands further studies.

Identification of Genes Related to Immune Infiltration in the Tumor Microenvironment of Cutaneous Melanoma

Cutaneous melanoma (CM) is the leading cause of skin cancer deaths and is typically diagnosed at an advanced stage, resulting in a poor prognosis. The tumor microenvironment (TME) plays a significant role in tumorigenesis and CM progression, but the dynamic regulation of immune and stromal components is not yet fully understood. In the present study, we quantified the ratio between immune and stromal components and the proportion of tumor-infiltrating immune cells (TICs), based on the ESTIMATE and CIBERSORT computational methods, in 471 cases of skin CM (SKCM) obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were analyzed by univariate Cox regression analysis, least absolute shrinkage, and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis to identify prognosis-related genes. The developed prognosis model contains ten genes, which are all vital for patient prognosis. The areas under the curve (AUC) values for the developed prognostic model at 1, 3, 5, and 10 years were 0.832, 0.831, 0.880, and 0.857 in the training dataset, respectively. The GSE54467 dataset was used as a validation set to determine the predictive ability of the prognostic signature. Protein–protein interaction (PPI) analysis and weighted gene co-expression network analysis (WGCNA) were used to verify “real” hub genes closely related to the TME. These hub genes were verified for differential expression by immunohistochemistry (IHC) analyses. In conclusion, this study might provide potential diagnostic and prognostic biomarkers for CM.

Tumor Immune Microenvironment Characterization Identifies Prognosis and Immunotherapy-Related Gene Signatures in Melanoma.

BackgroundThe tumor microenvironment (TME) involves infiltration of multiple immune cell subsets, which could influence the prognosis and clinical characteristics. The increasing evidence on the role of tumor-infiltrating lymphocytes (TILs) in primary and metastatic melanomas supports that the immune system is involved in the progression and outcomes of melanoma. However, the immune infiltration landscape in melanoma has not been systematically elucidated.MethodsIn this study, we used CIBERSORT and ESTIMATE algorithms to analyze immune infiltration pattern of 993 melanoma samples. Then we screened differential expression genes (DEGs) related to immune subtypes and survival. The immune cell infiltration (ICI) score was constructed by using principal-component analysis (PCA) based on immune signature genes from DGEs. Gene set enrichment analysis (GSEA) was applied to explore high and low ICI score related pathways. Finally, the predictive ability of ICI score was evaluated in survival prognosis and immunotherapy benefit.ResultWe identified three ICI clusters and three gene clusters associated with different immune subtypes and survival outcomes. Then the ICI score was constructed, and we found that high ICI score exhibited activated immune characteristics and better prognosis. High ICI score was significantly enriched in immune pathways and highly expressed immune signature genes. More importantly, we confirmed that melanoma patients with high ICI score had longer overall survival and rate of response to immunotherapy.ConclusionWe presented a comprehensive immune infiltration landscape in melanoma. Our results will facilitate understanding of the melanoma tumor microenvironment and provide a new immune therapy strategy.

In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment.

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

Clinical Significance of Tumor Microenvironment in Acral Melanoma: A Large Single-Institution Study of Caucasians.

Background: The presence of tumor-infiltrating lymphocytes (TILs) in many studies is associated with a better prognosis in melanoma patients. Programmed death-ligand 1 (PD-L1) expression has a significant value in predicting several cancers, but its role in melanoma remains ambiguous. The study aims to report a comprehensive analysis of TILs characteristics and their impact on survival in primary acral melanoma (AM). Methods: Clinical and pathological features and survival outcomes were investigated in 70 patients with AM. Immunohistochemical quantitative analysis of TILs, including expression of CD4, CD8, FOXP3, PD-1, and PD-L1, on melanoma cells was performed. Results: Kaplan-Meier analysis showed significant differences in overall survival (OS) for CD4+ (p = 0.021), CD8+ (p = 0.037), FOXP3+ (p = 0.007), and TILs density (p = 0.043). In univariate analysis of immunohistochemical features, FOXP3, CD4, CD8, PD-1, and Melanoma Institute of Australia (MIA) grading TILs (grade, density, and distribution) were correlated with survival. The higher density of FOXP3-positive cells was an independent factor associated with better survival. Conclusions: High TILs content (classed as brisk Clark scale and marked/diffuse TILs MIA grade) regardless of its immunophenotype was associated with better survival outcomes in AM. PD-L1 expression on tumor cells did not influence OS and was independent of clinical and pathological characteristics. We demonstrated that TILs are significant biomarkers in sentinel lymph node status prediction.

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

Extracellular Microparticles Encapsulated with Diallyl Trisulfide Interfere with the Inflammatory Tumor Microenvironment and Lung Metastasis of Invasive Melanoma.

Lung metastasis is a fatal and late-stage event for many solid tumors. Multiple lines of evidence have demonstrated that diallyl trisulfide (DATS), an active ingredient of garlic, possesses striking antimetastatic effects. However, the lack of highly efficient organ-compatible carriers restricts its application. In the present study, we showed that extracellular microparticles encapsulated with DATS (DATS-MPs) were capable of interfering with the prometastatic inflammatory microenvironment in local tissues. DATS-MPs were successfully prepared and exhibited typical characteristics of B16BL6-derived extracellular vesicles. The DATS-MPs preferentially fused with cancer cells and endogenous cells (mouse lung epithelial MLE-12 cells) from the metastatic organs in vitro. More interestingly, the systemically administered MPs predominantly accumulated in the lung tissue that serves as their main metastatic organ. The drug-loaded MPs exerted higher antimetastatic effects than DATS alone in both the spontaneous and the experimental metastasis models in mice (*p < 0.05). Additionally, we found that DATS-MPs inhibited tumor cell migration and interfered with the prometastatic inflammatory microenvironment via decreasing the release of S100A8/A9, serum amyloid A (SAA), and interleukin-6 (IL-6) and inhibiting the expression of fibronectin, MRP8, myeloperoxidase (MPO), and the toll-like receptor 4 (TLR4)-Myd88 in the lung tissues. Collectively, DATS-MPs appeared to enhance the antimetastatic efficiency of DATS in animal models under study.

Neem leaf glycoprotein salvages T cell functions from Myeloid-derived suppressor cells-suppression by altering IL-10/STAT3 axis in melanoma tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune functions. We have observed that an immunomodulator, neem leaf glycoprotein (NLGP), inhibits tumor-resident MDSCs and enhances antitumor CD8+ T cell immunity. NLGP inhibits the number as well as functions of tumor-resident MDSCs (Gr1±CD11b±) and enhances antitumor CD8± T cell immunity by downregulating arginase 1 and inducible nitric oxide synthase production in MDSCs. Accordingly, decreased T cell anergy and helper to regulatory T cell conversion have been observed in the presence of NLGP, which ultimately augments T cell functions. Mechanistically, NLGP-mediated rectification of T cell suppressive functions of MDSCs was primarily associated with downregulation of the interleukin (IL)-10/signal transducer and activator of transcription 3 (STAT3) signaling axis within the tumor microenvironment, as confirmed by knockdown of STAT3 (by STAT3-siRNA) and using IL-10-/- mice. Thus, NLGP-mediated suppression of MDSC functions in tumor hosts is appeared to be another associated effective mechanism for the eradication of murine melanoma by NLGP.

Single-cell melanoma transcriptomes depicting functional versatility and clinical implications of STIM1 in the tumor microenvironment.

Rationale: Previous studies have implicated the functions of stromal interaction molecule 1 (STIM1) in immunity and malignancy, however, the specificity and effects of STIM1 expression in malignant and non-malignant cells in the tumor microenvironment are unclear.Methods: In the current study, we posed two central questions: (1) does STIM1 expression elicit different cellular programs in cell types within the melanoma tumor microenvironment (2) whether the expression of STIM1 and STIM1-coexpressed genes (SCGs) serve as prognostic indicators of patient's outcomes? To answer these questions, we dissected cell-specific STIM1-associated cellular programs in diverse cell types within the melanoma tumor microenvironment by measuring cell-type specificity of STIM1 expression and SCGs.Results: A distinct set of SCGs was highly affected in malignant melanoma cells, but not in the other cell types, suggesting the existence of malignant-cell-specific cellular programs reflected by STIM1 expression. In contrast to malignant cells, STIM1 expression appeared to trigger universal and non-specific biological functions in non-malignant cell types, as exemplified by the transcriptomes of macrophages and CD4+ T regulatory cells. Results from bioinformatic analyses indicated that SCGs in malignant cells may alter cell-cell interactions through cytokine/chemokine signaling and/or orchestrate immune infiltration into the tumor. Moreover, a prognostic association between SCGs in CD4+ T regulatory cells and patient's outcomes was identified. However, we didn't find any correlation between SCGs and responsiveness of immunotherapy.Conclusions: Overall, our results provide an integrated biological framework for understanding the functional and clinical consequences of cell-specific STIM1 expression in melanoma.

Immune landscape of inflammatory breast cancer suggests vulnerability to immune checkpoint inhibitors

ABSTRACT Background. Anti-PD1/PDL1 immune checkpoint inhibitors (ICIs) showed promising results in breast cancer, and exploration of additional actionable immune checkpoints is ongoing. Inflammatory breast cancer (IBC) is an aggressive form of disease, the immune tumor microenvironment (TME) of which is poorly known. We aimed at providing the first comprehensive immune portrait of IBCs. Methods. From the gene expression profiles of 137 IBC and 252 non-IBC clinical samples, we measured the fractions of 22 immune cell types, expression of signatures associated with tertiary lymphoid structures (TLS) and with the response to ICIs (T cell-inflamed signature: TIS) and of 18 genes coding for major actionable immune checkpoints. The IBC/non-IBC comparison was adjusted upon the clinicopathological variables. Results. The immune profiles of IBCs were heterogeneous. CIBERSORT analysis showed profiles rich in macrophages, CD8+ and CD4 + T-cells, with remarkable similarity with melanoma TME. The comparison with non-IBCs showed significant enrichment in M1 macrophages, γδ T-cells, and memory B-cells. IBCs showed higher expression of TLS and TIS signatures. The TIS signature displayed values in IBCs close to those observed in other cancers sensitive to ICIs. Two-thirds of actionable immune genes (HAVCR2/TIM3, CD27, CD70, CTLA4, ICOS, IDO1, LAG3, PDCD1, TNFRSF9, PVRIG, CD274/PDL1, and TIGIT) were overexpressed in IBCs as compared to normal breast and two-thirds were overexpressed in IBCs versus non-IBCs, with very frequent co-overexpression. For most of them, the overexpression was associated with better pathological response to chemotherapy. Conclusion. Our results suggest the potential higher vulnerability of IBC to ICIs. Clinical trials.

Immune marker signature helps to predict survival in uveal melanoma

The detailed molecular function of tumor microenvironment (TEM) in uveal melanoma (UVM) remains unclear. This study generated the immune index and the stromal index scores by ESTIMATE algorithm based on RNA-sequencing data with 80 UVM patients. There was no correlation between the immune stromal index and clinical parameters. The differentially expressed genes related to the immune stromal index were calculated and were described by functional annotations and protein-protein interaction network diagrams. After univariate and multivariate Cox regression analyses, there were four genes (HLA-J, MMP12, HES6, and ADAMDEC1) with significant prognostic significance. The prognostic model was constructed using these four characteristic genes, and the KM curve and tROC curve were described to show that the model had a better ability to predict survival outcomes and prognosis. The verification results in GSE62075 showed that HLA-J and HES6 were expressed differently in the cancer group than in the non-cancer group. This study indicates that the risk signature based on the immune index can be used as an indicator to evaluate the prognosis of patients with UVM.

Quantitative analysis of CMTM6 expression in tumor microenvironment in metastatic melanoma and association with outcome on immunotherapy

ABSTRACT Chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 6 (CMTM6) modulates degradation of a number of proteins, including programmed death ligand-1 (PD-L1) by protecting it from ubiquitin-mediated degradation. In this role, it could modulate the effectiveness of immunotherapy. Here, for the first time, we characterize CMTM6 expression in melanoma and evaluate its association with response to immune checkpoint inhibitors (ICI). We evaluated the expression of CMTM6, PD-L1 and other immune-related proteins in 60 pretreatment biopsies from metastatic melanoma patients who received immunotherapy, in a tissue microarray (TMA) using quantitative immunofluorescence (QIF). Expression of mRNA from control patients obtained from The Cancer Genome Atlas (TCGA) database was also compared. CMTM6 expression was positively correlated with PD-L1, CD3, CD20, and CD68 markers, at protein (Pearson’s r = 0.53–0.81, all P < .0001) and mRNA (Spearman’s r = 0.15–0.44, all P < .002, except for CD68 where P = .26) levels. CMTM6 protein was associated with longer survival after immunotherapy when measured in the stromal (P = .007) and all the immune compartments tested (T cells, B cells, and macrophages). Multivariable analyses also revealed significant CMTM6 survival associations when measured in stromal (Hazard Ratio (HR) = 0.12, P = .001) and CD68-positive (HR = 0.30, P = .043) compartments. Additionally, PD-L1 but not CMTM6 showed prognostic value in control patients. Finally, high CMTM6 and PD-L1 co-expression in the stromal compartment was significantly associated with longer survival in treated patients (P = .028). Consequently, CMTM6 expression shows potential as a predictive factor for ICI treatments.

Evaluation of galectins 1 and 3 expression in canine melanoma

ABSTRACT: Canine melanoma is a frequently-occuring neoplasm in dogs and presents as malignant and highly metastatic in this context, studies that contribute to the understanding of the tumor microenvironment in melanoma include the role of galectins. Galectins are proteins of the family of animal lectins that display carbohydrate recognition domains. Galectin-1 and galectin-3 are associated with neoplastic transformation, neoplastic cell survival, angiogenesis, immune system evasion, and metastasis. The goal of this study was to establish a correlation between expression patterns of galectin-1 and galectin-3 and the different degrees of aggressiveness of canine melanoma, as well as to determine serum concentration of galectin-3 in dogs with melanoma. Galectin-1 and galectin-3 expression was analyzed by immunohistochemistry in 30 canine melanomas, six melanocytomas and nine metastatic lymph nodes from patients whose primary tumors were also processed and analyzed. Serum samples from 30 dogs were collected and galectin-3 concentration was determined by ELISA and compared to the samples of 10 healthy dogs. Canine melanoma samples expressed galectin-1 in the cytoplasm and presented a variable pattern of galectin-3 staining depending on melanoma aggressiveness. We observed a decrease in the percentage of cells with cytoplasmic galectin-3 immunolabeling simultaneous to the increased nuclear staining intensity, while there was also a decrease in the percent frequency of nuclear galectin-3 immunolabeled cells according to progression of melanoma, comparing the least to the most aggressive cases. Dogs with melanoma had increased serum levels of galectin-3 when compared to healthy animals, suggesting its potential biomarker of patients with melanoma.

Data mining of immune-related prognostic genes in metastatic melanoma microenvironment.

Skin cutaneous melanoma (SKCM) is one of the most deadly malignancies. Although immunotherapies showed the potential to improve the prognosis for metastatic melanoma patients, only a small group of patients can benefit from it. Therefore, it is urgent to investigate the tumor microenvironment in melanoma as well as to identify efficient biomarkers in the diagnosis and treatments of SKCM patients. A comprehensive analysis was performed based on metastatic melanoma samples from the Cancer Genome Atlas (TCGA) database and ESTIMATE algorithm, including gene expression, immune and stromal scores, prognostic immune‐related genes, infiltrating immune cells analysis and immune subtype identification. Then, the differentially expressed genes (DEGs) were obtained based on the immune and stromal scores, and a list of prognostic immune‐related genes was identified. Functional analysis and the protein–protein interaction network revealed that these genes enriched in multiple immune-related biological processes. Furthermore, prognostic genes were verified in the Gene Expression Omnibus (GEO) databases and used to predict immune infiltrating cells component. Our study revealed seven immune subtypes with different risk values and identified T cells as the most abundant cells in the immune microenvironment and closely associated with prognostic outcomes. In conclusion, the present study thoroughly analyzed the tumor microenvironment and identified prognostic immune‐related biomarkers for metastatic melanoma.

Divergent Resistance Mechanisms to Immunotherapy Explain Responses in Different Skin Cancers.

Simple SummaryDespite the successes of immune checkpoint therapy in treating metastatic skin cancers, most patients either fail to respond or become unresponsive to immunotherapy. We found that the cell–cell communication of two different immune cell types, macrophages and memory B cells, correlate very strongly with the response to immunotherapy. We built a mathematical model based on these results that predict different skin cancers would have different response rates and predict that a high ratio of memory B cells to macrophages is optimal for a response to immunotherapy.The advent of immune checkpoint therapy for metastatic skin cancer has greatly improved patient survival. However, most skin cancer patients are refractory to checkpoint therapy, and furthermore, the intra-immune cell signaling driving response to checkpoint therapy remains uncharacterized. When comparing the immune transcriptome in the tumor microenvironment of melanoma and basal cell carcinoma (BCC), we found that the presence of memory B cells and macrophages negatively correlate in both cancers when stratifying patients by their response, with memory B cells more present in responders. Moreover, inhibitory immune signaling mostly decreases in melanoma responders and increases in BCC responders. We further explored the relationships between macrophages, B cells and response to checkpoint therapy by developing a stochastic differential equation model which qualitatively agrees with the data analysis. Our model predicts BCC to be more refractory to checkpoint therapy than melanoma and predicts the best qualitative ratio of memory B cells and macrophages for successful treatment.

Transcriptional characterization of conjunctival melanoma identifies the cellular tumor microenvironment and prognostic gene signatures

This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.

Abstract A27: Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets

Abstract Resistance to targeted therapies remains an obstacle in curing cancers. A reason for this is testing of therapeutic agents in 2D culture conditions that do not recapitulate growth conditions of tumors in vivo. A critical component for defining the cell survival in vivo is the extracellular matrix (ECM). The ECM not only modulates cell-matrix and cell-cell interactions, but also determines drug accessibility to the tumor tissue. Many labs thus evaluate drug perturbations in 3D cell culture platforms using Matrigel and collagen scaffolds. A caveat with these models is the simplistic use of only basement membrane proteins or individual ECM proteins that, again, do not efficiently mimic the tumor microenvironment (TME). Although in vivo mouse models can be a better alternative, the time to conduct experiments, the associated cost, and inability to recapitulate patient TME remain a crucial challenge. Thus, we aim to regenerate an in vitro melanoma TME by engineering a melanoma extracellular matrix. We aim to do this by first identifying the patient melanoma ECM. On conducting proteomics for ECM proteins on patient tissues we identified collagen VI to be the most dominant ECM protein. However, prior work from other labs reports collagen I as the most abundant protein. This information was used to construct a platform through which we can recapitulate the melanoma ECM. We did this by generating cell-derived matrices (CDM) using human foreskin fibroblasts. On comparing the CDM from HFF1 to patient ECM, we could detect ~80 of ~120 core ECM proteins. Upon performing proteomic studies on CDMs, we again identified collagen VI as the dominant ECM protein. We also tested CDM deposition with cancer-associated fibroblasts, yielding similar results. This brought us to the question of whether collagen VI plays a role in drug resistance in melanoma. To address the role of cell-derived matrices and collagen VI, we created collagen VI KO fibroblasts through CRISPR/Cas9, which were then used to generate these matrices and will be used to evaluate its effect on drug treatments. While building this in vitro melanoma TME, we understood the need for a 3D assay to have a robust readout on effect of drugs in 3D compared to 2D. Although many viability and proliferation assays are used by various labs, these assays do not look at the effect of treatments at a single-cell resolution and neither distinguishes if the drug is cytostatic or cytotoxic. Towards that goal, we established a 3D assay by culturing melanoma cells in collagen and tested for Raf-Mek combination therapies. We made comparisons of cells either cultured in 3D as single cells or spheroids. Imaging of these platforms was performed on epifluorescence and light-sheet microscopes. We tested the platform for both viability and proliferation and the images were analyzed through individual Z planes of the 3D image. While we observed differences in viability and proliferation in 2D vs. 3D upon drug treatments, there was no difference in 3D single cells vs. spheroids. Citation Format: Vasanth Siruvallur Murali, Justin Cillay, Erik Welf, Gaudenz Danuser, Murat Can Cobanoglu. Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A27.

Abstract CT241: Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma

Abstract Background: Anti-PD-1 and anti-CTLA4 immune-based therapeutics have vastly improved melanoma survival rates, but many patients still do not benefit from these therapies. IFx-Hu2.0 is a plasmid DNA encoding the streptococcal membrane protein, Emm55, contained within a cationic polymer. Preliminary correlative laboratory data from the first in human IFx-Hu2.0 phase I feasibility study for use as an intralesional melanoma therapy is reported. No clinical data will be presented. The melanoma tumor microenvironment, pre- and post-treatment, was analyzed on FFPE tissues collected from the first three patients with a panel consisting of CD3, CD8, FOXP3, PD1, PDL1, SOX10 and DAPI. The numbers of SOX10+ melanoma cells tended to decrease with treatment (Patient 1: 16.7% SOX10+ cells pre-treatment, 0.3% SOX10+ cells post treatment; Patient 2: 47.1% pre/6.0% post; Patient 3: 49.0% pre/52.7% post). Multi-parameter analysis revealed that when the numbers of SOX10+ cells decreased, there were an increased number of T cells in the tumor environment. This observation also held true for the whole section for patient 2. In this patient, there were 0.3% CD3+CD8+ T cells prior to treatment but this number increased to 15.2% after treatment. Interestingly, the numbers of FOXP3 regulatory T cells also increased in this patient suggesting the formation of an immune response. Taken together, IFx-Hu2.0 is proposed to create an immune response in melanoma patients and is being tested in a first in human trial. Methods: Up to six patients (&amp;gt;18 years) with unresectable stage III or IV cutaneous melanoma will have cutaneous melanoma lesions injected with IFx-Hu2.0 to test the safety, tolerability, and feasibility of intralesional IFx-Hu2.0 injection. The subjects must have at least one lesion available for injection and another biopsiable lesion. This trial is unique as a cutaneous lesion as small as 3 mm may be injected. To be eligible, subjects should have been considered for anti-PD-1 therapy, TVEC and/or BRAF inhibition as indicated in the FDA labels for those agents. Small brain metastases (1 cm) are permitted assuming the subjects receive concurrent radiation at a site distant from intralesional injection with a greater than 3-month life expectancy. These patients may not be receiving any other systemic therapies, may not have uncontrolled hepatitis/HIV infection, and may not have a history of immunosuppression as is seen in patients who have undergone organ transplantation. For correlative studies, 4 tubes of peripheral blood will be collected at the beginning of the study and at the dose limiting toxicity (DLT) assessment visit 28 days later. Trial subjects will have the option of continued dosing every 3 weeks if they have a response to therapy. Success of the study is defined as being able to treat 5/6 subjects without DLT at 28 days. Three of the six planned trial subjects have been enrolled as of January 2020 (Clinical trial registry number: NCT03655756). Citation Format: Joseph Markowitz, Andrew Brohl, Amod A. Sarnaik, Zeynep Eroglu, Deanryan B. De Aquino, Nikhil Khushalani, Ahmad Tarhini, Vernon K. Sondak, Shari Pilon-Thomas. Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT241.

Abstract 2656: Myeloid derived Wnt5a influences the immune-dynamics of melanoma tumor microenvironment

Abstract Despite breakthroughs in targeted therapies and immunotherapies, over 75% of patients either do not respond to therapy, or relapse. This may be due in part to changes in their tumor microenvironment (TME). Wnt5A is a glycoprotein that has been shown to influence melanoma phenotype, promoting a slow-cycling, but more invasive cancer. Wnt5A was thought to be produced from tumor cells and fibroblasts in the TME. However, our data now suggest that unique pathologically induced myeloid derived suppressor cells (MDSCs) produce a major proportion of Wnt5A in the TME which is taken up by melanoma cells to increase invasiveness. This finding highlights the diverse roles that MDSCs play in melanoma, going beyond immunosuppression. We created novel transgenic animals with MDSC specific knockdown of Wnt5A and demonstrate a clear decrease in the Wnt5A expression within the TME. Wnt5A deficient MDSCs caused an increase in CD8+ T-cells in peripheral blood and decreased tumoral MDSCs and Tregs. Tumor infiltrating lymphocytes also had significantly decreased PD-1 expression suggesting a less exhausted phenotype, and increased ratios of effector T-cells to immunosuppressive cells resulting in a more immunogenic TME. Wnt5A promotes a switch from proliferative to an invasive melanoma cell state. Myeloid specific Wnt5A knockdown caused decreased lung metastasis and significantly increased tumor growth in vivo, with the lack of MDSC infiltration negatively correlating to tumor volume. Tumor infiltrating MDSCs from control animals had a strong positive correlation with Tregs, whereas, this was completely ablated in animals with Wnt5A-negative MDSCs. Overall, our data suggests that while MDSCs contribute to an immunosuppressive and less immunogenic environment, they may have an additional function as the major source of Wnt5A in the tumor microenvironment. These two functions likely synergize to drive a highly metastatic and therapy-resistant phenotype altering immunogenicity. Citation Format: Stephen Douglass, Mitchell Fane, Emilio Sanseviero, Dmitry Gabrilovich, Ashani Weeraratna. Myeloid derived Wnt5a influences the immune-dynamics of melanoma tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2656.

Abstract 2832: Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-cells

Abstract Tumor-infiltrating lymphocytes (TIL) hold strong prognostic value in determining patient survival as well as response to immune checkpoint blockade across many tumor types, especially metastatic melanoma. Tumors often evolve mechanisms to prevent TIL access and/or function to allow for continued growth. One such mechanism is the recruitment and reprogramming of tumor-associated macrophages (TAM) which suppress TILs through a variety of pathways. The interactions between TAMs and TILs that result in a suppressive tumor microenvironment (TME) have been studied in vitro and with spatially-agnostic sequencing, leaving the location-specific contributions underexplored. Interrogating the TAM-TIL interactions in an intact TME will uncover novel mechanisms responsible for TAM function and may be key to reversing TIL immunosuppression, improving anti-tumor immune responses, survival, and potentially therapy response. We use histocytometry, a multiplex quantitative tissue imaging method capable of capturing whole-slide tumor sections, to explore the human metastatic melanoma TME in situ and resolve the spatial contributions of TAM-TIL interactions. We demonstrate a T-cell preference for physical contact with phagocytic TAMs in the tumor but non-phagocytic TAMs in the stroma. We further model immune synapses from histocytometry to probe the function underlying the TAM-TIL preference based on the directionality of signaling components. The model suggests T-cells in contact with macrophages in the tumor are communicating more frequently with the phagocytic TAMs as compared to their non-phagocytic counterparts, indicating that this interaction extends beyond a simple proximity preference. In addition, proliferating T-cells communicate more frequently with macrophages than non-proliferating T-cells but this effect limited to the intratumor space. Together we demonstrate the functional consequences of physical TAM-TIL interactions with unprecedented spatial resolution. This work unravels important TAM interactions that shape the TME which have not been previously appreciated, providing novel insight into the forces modifying T-cell function in the TME and revealing new TAM biology to explore further. Citation Format: Victor G. Wang, Jan Martinek, Ananya Gulati, Hannah Boruchov, Kelly Ray, Karolina Palucka, Jeffrey H. Chuang. Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2832.