Abstract 3985: Optimizing co-culture of tumoroid and immune cells for recapitulation of the tumor microenvironment

Abstract Immunotherapies for solid cancer treatment aim to potentiate immune cell recognition of cancer cells within the tumor microenvironment or recruit peripheral immune cells (native or engineered) to the tumor. Patient-derived tumoroids, or cancer organoids, are multicellular structures that closely mimic the architecture and genetic and transcriptomic landscape of tumors. When co-cultured with immune cells, these models enable a more comprehensive investigation of the tumor-immune microenvironment than traditional cancer cell lines. However, culture conditions to prolong survival of both epithelial cancer cells and immune cells remain poorly defined. Here, we explored methods to supplement Gibco™ OncoPro™ Tumoroid Culture Medium to promote immune cell survival for better recapitulation of the tumor microenvironment. In initial experiments, healthy donor PBMCs were thawed and cultured in suspension in OncoPro medium for 5 days. Total viable cells were counted at the end of the culture period and revealed loss of 80±2% of the cells initially present. Similarly, RNA sequencing comparing freshly dissociated colorectal and endometrial tumors with cells cultured for 7 days in OncoPro medium indicated downregulation of genes related to immune cell signaling pathways, and flow cytometry of tumoroid models showed loss of immune cell populations over time compared to the initial material. Therefore, designed experiments were used to screen and optimize cytokine cocktails that maximized PBMC survival while minimizing the change in the percentage of monocytes, B cells, NK cells, helper T cells, and cytotoxic T cells between the original (day 0) donor material and in vitro PBMC cultures. Cytokine addition led to an increase in PBMC cell number after 5 days in culture of 2.2±0.8x compared to culture in non-supplemented OncoPro medium across three independent donors, with <10% change in the relative proportion of cells in the immune compartments examined over the culture period. Follow up experiments are focused on determining conditions that support the survival of immune cells present in cryopreserved dissociated tumor cell (DTC) samples using cytokines identified during PBMC screening experiments. In preliminary work, the addition of cytokines to potentiate immune cell survival did not negatively affect the culture of epithelial tumoroid cells. Co-culture of immune cells with tumoroids will facilitate the examination of immune checkpoint inhibitors and other immunotherapy strategies in vitro to enable higher throughput and more controlled screening of immunotherapy candidates. We envision that this approach will provide critical insights into the mechanisms of immune evasion and resistance and potentially lead to more effective treatment options for cancer patients. Citation Format: Shyanne Salen, Sydney Hawkins, Sylvia Beam, Colin D. Paul, Chris Yankaskas, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Optimizing co-culture of tumoroid and immune cells for recapitulation of the tumor microenvironment [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 3985.