Abstract 1227: Cancer cell-specific killing by reduced dose of chemotherapeutic agents in combination with immune cell therapy may be optimized in 3D BioSPHEER™ cancer models

Abstract 3D cancer models consisting of cancer and fibroblast cell lines (BioSPHEERs™) exhibit complex morphology depending on the cell lines used. We recently reported that such 3D cancer models can be used for drug testing by measuring cell killing and cell survival [Nielsen et al 2024, doi: 10.3389/fonc.2024.1386097]. Chemotherapeutic agents, like 5-FU, cisplatin and cyclophosphamide, at a certain dose, kill the majority of cancer cells, but also normal cells in the body, thus causing unappreciated discomfort and side effects. In contrast, therapeutic immune cells, like TILs and CAR-T cells perform cancer cell specific killing. Importantly, our 3D cancer models also enable studies on T cell infiltration and T cell directed cell killing. PBMC-derived T cells added to heterospheroids infiltrated the structures without excessive killing, unless the T cells had been activated by CD3/CD28 beads, or primed with the respective cancer cell line ahead of mixing. In this study we first combined low (sub-lethal) dose of the chemotherapeutic agent 5-FU with subsequent administration of PBMCs. 5-FU was added to spheroids, consisting of mCherry-engineered HT29 cells and 1BR3G fibroblast, at day 7 after seeding and after 24 hours the 5-FU was removed and PBMCs were added. 5-FU added at 500 µM (high dose) caused dramatic killing measured by a luciferase assay, whereas the drug at 100 µM (low dose) appeared quite harmless to the cells in the spheroids. We next treated spheroids with the low dose of 5-FU and after 24 hours added T cells. After additional 72 hours, histological examination showed that the T cells themselves partly infiltrated the vehicle-treated spheroids, but caused little killing. However, the same T cells added to the 5-FU-treated spheroids caused excess killing, suggesting a synergistic effect of the two agents. We are currently testing more PBMC donors in combination with other therapeutic agents, including antibody drug conjugates (ADC). Our observations suggest that well-known chemotherapeutic agents applied at sub-lethal dose may enhance the effect of immune cell therapies. The combination treatment may retain or improve drug efficacy and at the same time reduce severe side effects caused by the chemotherapeutic agent. We propose that the use of 3D BioSPHEER™ cancer models to study combination treatment, will allow better optimization of dosage and timing of combined chemo-immuno-therapy. Citation Format: Boye Schnack Nielsen, Natasha H. Madsen, Isabella Skandorff, Jesper Larsen, Kim Holmstrøm, Monika Gad. Cancer cell-specific killing by reduced dose of chemotherapeutic agents in combination with immune cell therapy may be optimized in 3D BioSPHEER™ cancer models [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 1227.