Abstract 6490: Patient-derived organotypic tumor spheroids (PDOTS) optimize donor selection in allogenic cell therapy development
Authors: Chunxiao Cui, Raluca Dumitru, Danielle Hagee, Anthony Attardo, Kirti Khardenavis, Wassim Basheer, Sabrina D. Munnik, David C. Lopez, Gemma Moiset, Ketki Bhise, Sinal Patel, Alyssa Martin, Michael Perricone, Aaron Goldman
Journal: Cancer Research (2025)

Abstract Introduction: Donor-to-donor variability significantly impacts the process development of cell therapies for cancer. Characterizing the donor-specific functional variances enables donor selection for successful cell therapy development. Ex vivo human models provide unique opportunities to evaluate the functionality of cell therapies in the patient’s native tumor microenvironment (TME). Here, using the Xsphera human tumor system, we assessed the specificity, migration, anti-tumor efficacy, and immune modulation of CAR-T cells from different donors to identify the optimal donor characteristics. Methods: HER2-targeting CAR-T cells were generated from the peripheral blood mononuclear cells (PBMCs) of two healthy donors (CAR-D1 and CAR-D2). HER2+ (ZR-75-30) and HER2- (MDA-MB-468) cell lines were used to generate tumor spheroids. Fresh tumor samples collected from patients with ovarian cancer were partially dissociated into spheroids (PDOTS) ranging from 40-100 µm. Tumor spheroids and PDOTS were loaded into microfluidic devices containing extracellular matrix (ECM) and exposed to CAR-D1 and/or CAR-D2. CAR-T cell migration, proliferation and anti-cancer efficacy were measured and quantified using image-based analysis. RNA transcription was analyzed via NanoString to study tumor response and immune modulation. Results: Both CAR-D1 and CAR-D2 showed time-dependent tropism through the ECM towards HER2+ tumor spheroids, but not HER2- tumor spheroids. Increased cytotoxicity was observed in HER2+ tumor spheroids with both CAR-T cells, with CAR-D2 showing greater cytotoxicity. CAR-D1 also induced off-target tumor killing in HER2- spheroids, which was not observed in CAR-D2. Nanostring analysis revealed higher cytotoxic signals in CAR-D1, whereas CAR-D2 showed higher antitumor cytokine signaling in HER2+ tumor spheroids and lower anti-tumor activity in HER2- tumor spheroids. In PDOTS derived from ovarian patient tumors (N=3), CAR-D2’s cytotoxic effect appeared to be correlated with tumor cells’ HER2 expression levels. In high HER2 PDOTS, CAR-D2 led to downregulation of tumor-related genes (ERBB2/HIF1A/EPCAM/EGFR), upregulation of CAR-T activation associated genes (IFNg/GZMB/PRF1/CXCL9/CXCL10/IL1A), and increased CD8 T cell abundances (CD8A/CD8B). In summary, CAR-D2 showed higher target specificity, slower ECM migration, faster spheroids uptake, greater anti-tumor efficacy, and lower off-target cytotoxicity than CAR-D1. Its efficacy in PDOTS appeared to be HER2 expression dependent. Conclusion: The use of Xsphera’s platform to study cell therapies provides a valuable tool for uncovering donor-to-donor variability by differentiating the variability in performance of CAR-T cells derived from different donors. This unique approach will support optimal donor selection and accelerate cell therapy development. Citation Format: Chunxiao Cui, Raluca Dumitru, Danielle Hagee, Anthony Attardo, Kirti Khardenavis, Wassim Basheer, Sabrina D. Munnik, David C. Lopez, Gemma Moiset, Ketki Bhise, Sinal Patel, Alyssa Martin, Michael Perricone, Aaron Goldman. Patient-derived organotypic tumor spheroids (PDOTS) optimize donor selection in allogenic cell therapy development [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 6490.