Spatial and molecular profiling of tumor initiation in ccRCC

Clear Cell Renal Cell Carcinoma (ccRCC) is the most common and aggressive type of kidney cancer. ccRCC originates from proximal tubule (PT) epithelial cells in the nephron. Its initiation is characterised by a linear evolution from the loss of one copy of chromosome 3p to the inactivation of the second VHL allele on the remaining copy of 3p. Computational studies established that 3p loss occurs several decades before diagnosis. This offers an unprecedented window of opportunity for early detection, cancer prevention and for broader pan-cancer learning. However, the biological mechanisms driving the pre-cancerous expansion of PT cells harboring these events remain elusive. One of the major unmet needs in ccRCC initiation is the identification of molecular biomarkers for the initially quiescent tumor-initiating cell. Previous studies established that the putative ccRCC cell of origin (COO) is a subtype of PT cells characterized by VCAM1 expression, a marker of tubular injury in human kidneys. Therefore, we hypothesized that VCAM1 can be used as a marker to enrich for cells that have lost a copy of chromosome 3p. Preliminary single-cell whole genome sequencing (WGS) of VCAM1+ PT cells revealed a high incidence of aneuploidies, including chromosome 3-related aneuploidies, indicating that these cells represent a chromosomally unstable epithelial subpopulation within morphologically normal kidney tissue. Therefore, this data supports VCAM1 as a candidate marker for the study of ccRCC initiation in human kidneys.Despite its quasi-ubiquitous role in ccRCC initiation, several studies show that VHL inactivation is insufficient for tumorigenesis in mammalian kidneys. To study this, we used histological analysis of VHL patient-derived normal kidney tissues, where 3p loss occurs on the background of a germline VHL mutation. We demonstrated that VHL inactivation (marked by CAIX expression) occurs in all major cortical epithelial cell types. Surprisingly, only the proportion of CAIX+ distal tubule (DT) cells showed a significant correlation with the age of the patient at the time of tissue collection. In addition, there is a significantly higher proportion of multicellular DT CAIX+ foci compared to CAIX+ PT foci, suggesting clonal expansion after VHL inactivation is favored in DT cells. Only a minority of CAIX+ PT foci were multicellular, indicating that unknown cell-intrinsic or extrinsic factors are necessary for clonal expansion. Results from our cohort show a positive association between the density of VCAM1+ PT cells and CAIX+ PT in VHL patient-derived normal kidney tissues, indicating that tissue stress levels may potentiate the selection and expansion of VHL inactivation in the human kidney. Our data offers novel insight in the putative COO of ccRCC and the mechanisms driving the earliest stages of ccRCC. Moving forward, we plan to expand our cohort and molecularly profile VCAM1+ and CAIX+ cells using multi-omic approaches.