Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A011.