Abstract A006: Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue

Abstract

Introduction. We aim to elucidate the role of lymphatic cells in the progression of clear-cell renal cell carcinoma (ccRCC). Objective. Inactivation of tumor suppressor gene von Hippel–Lindau (VHL) is the main cause of ccRCC, whose progression is extensively influenced by the tumor microenvironment (TME). It has been shown that tissue inflammation, with extensive infiltration of lymphocytes, is a feature of ccRCC tissue. Here, the role of the lymphatic cells in the inflammatory microenvironment was analyzed in the kidney with or without VHL inactivation. Methods. The conditional Vhlh knockout mouse (Hoxb7-Cre-GFP; Vhlh loxP/loxP) was established as a ccRCC model. Single-cell RNA sequencing (scRNA-seq) of podoplanin-positive (Pdpn+) cells revealed heterogeneity of lymphatic cells in mouse kidney. Immunostaining of 3-dimensional tissue was used to visualize lymphatic outgrowth from lymphatic vessels. Protein marker expression in mouse kidney and human ccRCC sections was detected by immunofluorescence. Relevant molecular mechanisms were quantified by qPCR, Western blot, flow cytometry, and ELISA. Results. The lymphatic vasculature is responsible for draining interstitial fluid, mediating lymphocyte recruitment, and participating in immune responses, and is a major route for metastasis. Immunofluorescence of the classical lymphatic markers such as Lyve1, Prox1, CD31, and VEGFR3 was observed in the lymphatic vessels including collecting lymphatics, lymphatic valves, and lymphatic capillaries. Moreover, immunostaining of 3-dimensional tissue showed sprouting Pdpn+ cells from lymphatic vessels, more numerous in Vhlh mutants. In Vhlh-deficient kidney tissue, the Pdpn+ cells form linearly arrayed clusters with no or small lumen and exhibit cuboidal cell shape instead of the flattened endothelial shape. Similar PDPN+ clusters of cells were also present in ccRCC tissues, compared with normal adjacent tissues. Interestingly, scRNA-seq identified a subpopulation of the progenitor marker CD133+ cells within the Pdpn+ population, providing evidence for the differentiation of novel lymphatic endothelial progenitor cells (LEPCs) in postnatal tissues in the kidney. Especially, there was a concomitant threefold increase in CD45+ lymphocyte populations in Vhlh conditional knockout mice compared to wild-type. Interestingly, the chemokine CXCl13 was highly expressed in LEPCs and the receptor CXCR5 was expressed in Vhlh-deficient tubule cells and in infiltrating immune cells. These results suggest a crosstalk between LEPCs and Vhlh-deficient cells and immune cells to modulate metastasis and immune responses. Double knockout (Hoxb7-Cre-GFP; VhlhloxP/loxP; Cxcl13-/-) will be designed for further experiments to investigate the role of CXCL13/CXCR5 axis in the progression of ccRCC. Conclusion. These investigations provide novel insights into CXCL13/CXCR5 axis that may orchestrate LEPCs and Vhlh-deficient tubule cells interactions that promote lymphocyte infiltration and potential metastasis within the tumor microenvironment.

Citation Format: Tuong-Vi Nguyen, Hieu-Huy Nguyen-Tran, Thi-Ngoc Nguyen, Sheng-Chieh Lin, Tien Hsu. Identification of a potential lymphatic progenitor cell population in Vhlh conditional knockout mouse and in ccRCC tissue [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 A006.