Unraveling the testicular seminoma tumor microenvironment by single-cell RNA-sequencing.

Abstract

522 Background: Seminomas is a malignant germ cell tumor (GCT) affecting male adults and is characterized by low somatic mutation burden, histological homogeneity, and high survival rate. It is currently unknown what drives tumorigenesis of this curable testicular cancer. Tumor/immune microenvironment (TME) has a vital role in cancer survival and progression with implications in cell death resistance and immune escape. Herein, we aimed to unravel the transcriptomic TME landscape of seminomas and better understand seminoma pathogenesis. Methods: We performed single cell RNA-sequencing (scRNA-seq; 10X-Illumina platform) on surgically resected pure seminomas from four patients. A total of ~24,000 single cells were analyzed using Seurat/Bioconductor R packages and standard means for data visualization. Key findings were validated by in situ hybridization RNA analysis (RNAscope) on histological sections from these samples. Results: T-cells and Natural-Killer cell populations were mostly predominantly enriched in this seminoma cohort, where tumor cells consisted of a distinct subpopulation with limited presence (1%). Other distinct cell populations were also identified for additional immune cell types e.g., B-cells, macrophages/monocytes, endothelial and smooth muscle cells. ScRNA-seq revealed a unique set of signature marker genes (e.g., POU5F1, KHDC3L, DPPA5, NANOS3) highly expressed in seminoma cells which are associated with pivotal cellular processes e.g., developmental pluripotency and germ cell development. Notably, this gene set was also highly differentially expressed (DE) in The Cancer Genome Atlas (TGCA) seminomas (n=73) compared to TCGA non-seminomas (n=38) (FDR<0.01). Amongst top marker genes, KHDC3L, also higher expressed in seminomas compared to normal testis, was validated by RNA-scope. KHDC3L demonstrated the strongest positive correlation with DPPA5 (Spearman/Pearson; R2=0.8, p<0.0001), top #3 ranked DE gene as well as with other top 20 DE genes identified by scRNA-seq. Remarkably, similar strong correlations among other top DE genes were also found in the testicular TGCA cohort. Top DE genes including master transcription factors (e.g., POU5F1, NANOG, SOX4) are involved in the same protein pathway/networks likely suggesting synergic effect/involvement in the same regulatory circuitry. Gene Set Enrichment (GSEA) analyses demonstrated enrichment in immune signatures and pivotal pathways e.g., Wnt/beta catenin signaling and MYC targets. Conclusions: Our analyses revealed novel marker genes and immune signatures, uniquely expressed in seminomas which consist of a homogeneous cell population across all 4 tumors analyzed, clearly distinct from other cell types identified.