Abstract 2573: Single-cell multi-omics analysis identifies a novel GPRC5B-marked endothelial cell subset and its mechanistic role in tumor stemness in colorectal cancer

Abstract Introduction: Tumor endothelial cells (ECs) support the tumor microenvironment and regulate the status of tumor cells. GPRC5B, enriched in ECs, is linked to poor prognosis in colorectal cancer (CRC). Yet, the influence of GPRC5B on ECs and its regulatory mechanism on CRC stemness are still elusive. Therefore, this study aims to clarify the impact of GPRC5B on ECs states, the specific role of GPRC5B-marked ECs (GPRC5B+ ECs) in tumor/stromal interactions and the mechanisms regulating CRC stemness. Methods: We analyzed scRNA-seq data from 18 CRC samples to decode the endothelial heterogeneity. We used an original SVM-based algorithm to find EC subsets linked to CRC stemness, ultimately identifying the GPRC5B+ ECs. By deconvoluted bulk RNA-seq data, we evaluated stemness level in GPRC5B+ EC-enriched samples. Cellular interactions and spatial omics analyses were employed to examine the signaling axis between GPRC5B+ECs and CRC stem cells. ChIP-seq and hdWGCNA algorithm pinpointed upstream regulators in the axis. We also developed a GPRC5B-overexpressing EC line and co-cultured it with CRC cells, whose stemness was then assessed by spheroid formation assays and western blot. Inhibitors targeting this signaling axis were administered, and changes in stemness phenotype and key signaling molecules were measured in co-cultured CRC cells. Finally, we constructed a CT26 subcutaneous tumor mouse model and treated the mice with siRNA targeting GPRC5B (siGPRC5B) to monitor tumor growth. Tumor tissues were collected to detect the expression of stemness markers using IHC and western blot. Results: GPRC5B+ ECs exhibited high angiogenic potential, with significant ERK pathway activation. ScRNA and bulk analyses showed GPRC5B+ ECs enhance CRC stemness, which was further confirmed by the increased spheroid formation ability and enhanced expression of KLF4 and SOX2 in CRC cells after co-culturing with GPRC5B-high ECs. Mouse models showed that high GPRC5B expression in ECs fosters tumor stemness and progression. Mechanistically, multi-omics and in vitro analyses revealed that GPRC5B in ECs activated the ERK-SP1 axis to promote the expression of ADAM17, which cleaved and activated JAG1, ultimately enhancing stemness of CRC cells via JAG1/NOTCH1 interaction. Application of ERK pathway or ADAM17 inhibitors to ECs decreased tumor stemness, respectively. Besides, application of siGPRC5B effectively inhibited tumor growth in vivo. Conclusion: Through single-cell multi-omics analyses, we identified a novel GPRC5B-marked EC subset, promoting angiogenesis and CRC progression, and revealed that GPRC5B+ ECs enhance tumor stemness via the ERK/SP1/ADAM17/JAG1-NOTCH1 axis. The study offers insights into CRC endothelial heterogeneity and tumor/stromal interactions, and future targeted therapy will be conducted to elucidate its clinical application potential. Citation Format: Mengying Suo, Hongyu Huang, Tianyou Li, Ziqi Meng, Xueqian Zhang, Shanshan Jiang, Na Li. Single-cell multi-omics analysis identifies a novel GPRC5B-marked endothelial cell subset and its mechanistic role in tumor stemness in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2573.