BioProject

Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of renal cancer, accounting for over 75% of cases. The asymptomatic nature of the disease contributes to late-stage diagnoses and poor survival. Highly vascularized and immune infiltrated microenvironment are prominent features of ccRCC, yet the interplay between vasculature and immune cells, disease progression and response to therapy remains poorly understood. Using droplet-based single-cell RNA sequencing we profiled 50,236 transcriptomes from paired tumor and healthy adjacent kidney tissues. Our analysis revealed significant heterogeneity and inter-patient variability of the tumor microenvironment. Notably, we discovered a previously uncharacterized vasculature subpopulation associated with epithelial-mesenchymal transition. The cell-cell communication analysis revealed multiple modes of immunosuppressive interactions within the tumor microenvironment, including clinically relevant interactions between tumor vasculature and stromal cells with immune cells. The upregulation of the genes involved in these interactions was associated with worse survival in the TCGA KIRC cohort. Our findings demonstrate the role of tumor vasculature and stromal cell populations in shaping the ccRCC microenvironment and uncover a subpopulation of cells within the tumor vasculature that is associated with an invasive phenotype. Overall design: Fresh ccRCC tumor (n=8) and healthy-adjacent (n=9) paired kidney tissues were obtained from the National Cancer Institute (Vilnius, Lithuania) with a bioethics committee approval No.2019/2˗1074˗586. No patient had received prior systemic therapy for their cancer. Samples were collected during an open or laparoscopic, partial or radical nephrectomy surgery, placed on ice and rapidly (<1 hour) transferred to the laboratory for dissociation. Briefly, patient derived tumor tissues were dissociated using Tumor Dissociation Kit (Miltenyi Biotec, cat.no.130-095-929) in an automated instrument gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) as per manufacturer’s instructions. Healthy-adjacent tissues were dissociated using Tissue Dissociation Kit I (Miltenyi Biotec, cat.no. 130-110-201). After dissociation, red blood cells were removed from the samples using RBC lysis reagent (Miltenyi Biotec, cat.no.130-094-183). After RBC lysis, cells were washed 3 times in ice-cold 1X DPBS (Gibco, cat.no. 14080-048) at 500g for 5 min. Cell viability and count was assessed using Trypan Blue dye (Gibco, cat.no. 15250061) on a hemocytometer. No further enrichment or selection of cells was performed. Cell suspension was immediately loaded onto inDrops platform for cell barcoding experiment.