Abstract 177: Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system

Abstract

Abstract Tumoroid technology enables culture of patient tissue-derived cancer cells in 3D, with retention of key characteristics from the original patient tumor. However, current tumoroid culture relies on labor-intensive media formulations and culture workflows. To address these issues, we have developed the serum- and conditioned medium-free Gibco™ OncoPro™ Tumoroid Culture Medium to enable derivation and expansion of patient-derived lines from multiple cancer indications. The system has been optimized to retain key patient genotypic and phenotypic characteristics during in vitro culture in a Wnt agonist-free system that can be easily adopted and transferred between labs. To test the applicability of our system with previously established tumoroid/cancer organoid models, we procured publicly available cancer models from the National Cancer Institute Patient-Derived Models Repository (NCI PDMR). Multiple colorectal, lung, pancreas, and head and neck tumoroid models were tested, including tumoroids originally derived in conditioned medium containing Wnt-3A, R-spondin 3, and Noggin. Cells were assessed for survival, growth, and fidelity to starting material in both OncoPro Tumoroid Culture Medium and in PDMR-recommended homebrew media. Growth and morphology of tumoroid cells was monitored and was comparable between culture conditions. To test for genetic stability of cultures over time, the mutational status of organoids expanded in each condition was characterized using targeted NGS. Both SNV allelic frequency and ploidy values were conserved from initial starting material following expansion to cryopreservation-competent banks (>10e6 cells). Gene expression levels across over >20,000 human RefSeq genes were compared between culture methods and showed high (>0.9) correlation, and Wnt-related signaling pathways were not differentially regulated between media types. In select cases, tumoroid cells expanded in each condition were also analyzed by flow cytometry using optimized antibody panels and protocols. Total cell viability and expression of EpCAM, CD45, CD31, smooth muscle actin, vimentin, CDX2, CEACAM, and cytokeratin 7 was nearly identical across culture conditions. Taken together, our approach represents a simple and effective method to expand and maintain patient-derived human cancer organoids in vitro, with no clear adverse effects from moving to a Wnt-free system. Citation Format: Colin Paul, Brittany Balhouse, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 177.