Single-cell transcriptomics reveals distinct molecular alterations of human renal cell carcinoma-derived endothelial cells that are stable in ex vivo primary culture [scRNA-seq]

Even though tumor endothelial cells are a key component of the tumor microenvironment and represent the main interface between tumor cells and the host's circulation and immune system, their phenotypes and functional properties are relatively poorly understood. To address this deficit, we purified tumor and matched normal endothelial cells from highly vascularized kidney cancer specimens and performed single cell RNA-seq to characterize endothelial cell gene expression patterns and heterogeneity. We found that tumor endothelial cells from multiple donors shared a common phenotype with increased expression of pathways related to extracellular matrix regulation, cell-cell communication and insulin growth factor signaling. We also found that while normal endothelial cells from kidney and liver were divergent, most tumor endothelial cells from kidney and liver cancer shared common gene expression signatures suggesting a congruence of tumor endothelial cell phenotypes. Using RNA-seq, we determined that many of the differentially regulated genes between tumor and normal endothelial cells were maintained in primary culture, which in turn permitted study of their functional phenotypes in vitro. Cultured tumor endothelial cells were resistant to apoptosis after trophic factor removal and displayed increased adhesiveness for CD+ CD25+ T regulatory cells. Our studies delineate the unique functional and immunophenotypic properties of kidney cancer tumor endothelial cells, which will serve as the basis to develop new therapies. Overall design: We performed scRNA-seq on TECs and NECs isolated by flow cytometry from 4 different donor nephrectomies. The RCC tumor type was confirmed as clear cell renal cell carcinoma (ccRCC) for all 4 patients on the final pathology report and confirmed by SA. The 4 donors were batched into two separate scRNA-seq libraries. In order to determine the purity of the isolated cell populations, primary TECs/NECs, we spiked in HUVECs and an aliquot of the entire tumor/normal adjacent tissue (NAT) single cell suspension