1315PD - Unsorted single-cell RNA sequencing profiles of metastatic melanoma patients reveal the heterogeneity of melanoma-associated fibroblasts

Abstract

BackgroundSingle cell transcriptomic analyses of the tumor microenvironment provides unprecedented opportunities to guide precision immune therapy of melanoma patients. Previous single-cell RNA sequencing studies in metastatic melanoma patients have shown that the TME composition can be deciphered with a FACS-based single cell assay. Here, we applied droplet-based single-cell RNA sequencing for the decomposition of the tumor microenvironment of metastatic melanoma patients before PD1-based immune therapies and, when possible, during or after PD1 therapy. MethodsFresh biopsies and surgical excisions from melanoma patients destined for standard-of-care immune therapy were collected and processed within 2hours. To capture the global cell composition, cells were not FACS sorted. We performed single-cell RNA sequencing using the 10x Chromium platform. We used both 3’ and 5’ RNA sequencing. Libraries were sequenced at the Campus Biotech platform in Geneva. Standard analytical pipelines were applied (Cell Ranger 3.0, Seurat 2.0). ResultsOur current dataset comprises a total of 26 tumors from 18 patients with a total number of 66’923 cells. The mean number of cells analyzed per patient was 2573 (Range: 160-12’676). Immune TME cell types could be readily identified, but with a significant patient-to-patient heterogeneity. Importantly, melanoma-associated fibroblasts (MAFs) were observed in most tumors (23/26), with an abundance of>1% in 19 out of 26 tumors. The MAF population, which in total comprised 4722 cells (median 2.1%/tumor, range: 0-59.8%) was heterogeneous and was composed of at least two different sub-clusters. The MAF subtypes were correlated with outcomes from PD1 therapy, with an immune evasion gene signature, and with bulk TCGA melanoma data, as will be presented. ConclusionsUnsorted single-cell RNA sequencing of fresh human melanoma samples demonstrably provides an insightful snapshot of heterotypic cell compositions. In contrast to previous FACS-based scRNAseq assays, both immune and non-immune components of the tumor microenvironment are efficiently captured. MAFs form a sizeable and heterogenous component of melanoma tumors, heretofore underappreciated in melanoma. Legal entity responsible for the studyCHUV. FundingSystemsX. DisclosureAll authors have declared no conflicts of interest.