P08.11 Integrative molecular and spatial analysis of endothelial cell heterogeneity in clear cell renal cell carcinoma

Background Accounting for 80% of all cases, the clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma. ccRCC exhibits resistance to common chemotherapies and a highly variable response to treatment with angiogenesis inhibitors, immune checkpoint blockade or a combination thereof. This, and its high tendency to metastasize necessitate advancements in therapy options. As a highly vascularized tumor type, ccRCC has been shown to exhibit different inter- and intratumoral vascularization patterns, high- and low-branching, which have been linked to therapy response and prognosis. 1–4 As endothelial cells are characterized by a vast phenotypic and functional heterogeneity, we aim to elucidate the molecular heterogeneity of endothelial cells in ccRCC and their potential role in primary or acquired resistance by identifying a possible molecular correlate to the tumors vascular architecture.Materials and Methods Matched and unmatched tumor and non-neoplastic tissue were collected from patients who underwent partial or total nephrectomies between 2022 and 2024 in the Department of Urology at the University Hospital Bonn and gave informed consent for biobanking. A protocol for the enrichment of isolated endothelial cells was established for single nuclei RNA sequencing as well as cultivation of the patient-derived endothelial cells. snRNA-sequencing was performed to identify a molecular correlate to the histology while tube-formation experiments were performed to characterize patient-derived endothelial cells‘ behavior in vitro. Xenium spatial transcriptomics experiments were performed to explore the previous findings in a spatial context.Results Six distinct endothelial cell clusters as well as two pericyte clusters were identified, including an endothelial cell cluster highly enriched in non-neoplastic tissue. Two of the identified clusters showed enrichment in samples with predominantly low-branching vascular architecture. Differences in tube-formation-behavior between endothelial cells isolated from the different patterns were observed. Using a custom-panel built from DEG identified in the snRNA-seq dataset, vessels corresponding to the disctint EC clusters were identified.Conclusions The results of this work indicate a link between molecular endothelial cell heterogeneity and vascular patterns in ccRCC and suggest a functional heterogeneity linked to the vascular architecture.References Cai Q, et al. Ontological analyses reveal clinically-significant clear cell renal cell carcinoma subtypes with convergent evolutionary trajectories into an aggressive type. EBioMedicine. 2020 Jan 1;51.Sabo E, et al. Microscopic analysis and significance of vascular architectural complexity in renal cell carcinoma. Clinical Cancer Research. 2001 Mar 1;7(3):533–7.Canete-Portillo S, et al. Vascular architectural patterns in clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. Virchows Archiv. 2021 Dec;479(6):1187–96.Toma MI, et al. Low-branching vessel architecture shapes immune cell niches and predicts immune responses in renal cancer. medRxiv. 2025 Jun 6:2025-06.