Abstract 1231: Exploring drivers of variability in patient tumor responses to HIF-2α inhibitor using the Xsphera patient-derived organotypic tumor spheroid (PDOTS) platform

Abstract Background: The hypoxia-inducible factor 2 alpha (HIF-2α) complex is a critical driver of clear cell renal cell carcinoma (ccRCC), with FDA-approved inhibitor PT2977 (belzutifan) demonstrating promising clinical efficacy. Xsphera’s patient-derived organotypic tumor spheroid (PDOTS) platform offers an innovative ex vivo approach to recapitulate the tumor microenvironment (TME) and assess patient-specific responses to cancer therapeutics. This study highlights how the PDOTS platform can bridge the gap between preclinical and clinical data, providing insights into patient-specific tumor responses and mechanisms of resistance. Methods: Resected patient RCC tumors (n=7) were processed into PDOTS and cultured within a microfluidic device to preserve native TME. Baseline immune cell profiles were characterized using flow cytometry on dissociated single-cell sub-fractions. PDOTS from each patient tumor were treated with the HIF-2α inhibitors PT2977 (7.5-750 ng/mL) and PT2385 (50-5000 ng/mL), or vehicle control. Tumor responses were evaluated at day 3 post-treatment using Hoechst Propidium Iodide (HPI) automated image analysis. RNA was extracted post-treatment, and gene expression was profiled using the NanoString IO360 panel. Integrated analyses of immune cell composition, cytotoxicity, and gene expression were performed to explore potential correlations with response. Results: Cytotoxicity: PT2385 induced dose-dependent cytotoxicity, in 4 of 7 samples (57%), while PT2977 demonstrated activity in 2 of 7 (28%) patient tumors without a clear dose-response relationship. Immune Profiles: No significant associations were observed between baseline immune cell composition and tumor response. Gene Expression: Preliminary NanoString analysis revealed distinct gene expression changes in responsive versus non-responsive tumors. Responsive tumors exhibited downregulation of pro-tumorigenic pathways, including cell proliferation and G2M checkpoint-related genes, relative to controls. Interestingly, markers associated with myeloid cells, such as CD163 and LILRB4, were elevated in responsive tumors following treatment, but remained unchanged in non-responsive tumors. Conclusions: Xsphera’s PDOTS platform effectively mirrors the clinical response rates to HIF-2α inhibitors, offering actionable insights into tumor variability and resistance mechanisms. By combining cytotoxicity, immune profiling, and transcriptomic analyses, the PDOTS platform provides a robust predictive and exploratory tool for oncology drug development. These findings underscore the utility of this platform in tailoring therapeutic strategies for ccRCC and other cancer types. Citation Format: Alyssa Martin, Chunxiao Cui, Aaron Goldman, Michael A. Perricone. Exploring drivers of variability in patient tumor responses to HIF-2α inhibitor using the Xsphera patient-derived organotypic tumor spheroid (PDOTS) platform [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 1231.