Abstract 6504: Generation of stable reporter pools in established patient-derived tumoroid models

Abstract Tumoroids are patient-derived cancer cells grown as 3D, self-organized multicellular structures. Recently, tumoroids have emerged as powerful and reliable platforms for preclinical cancer drug discovery due to their ability to retain key characteristics from the original tumor. However, traditional tumoroid culture relies on complex media formulations and labor-intensive culture workflows. To tackle this issue, we have developed the serum-free, conditioned medium-free GibcoTM OncoProTM Tumoroid Culture Medium for derivation and expansion of tumoroids. Our previous data demonstrated that OncoPro medium enabled the transition of 18 out of 19 previously established tumoroid models of various cancer indications from the National Cancer Institute Patient-Derived Models Repository (NCI PDMR) and another commercial source and preserved their genetic and transcriptional characteristics. Here, we extend this work demonstrating the compatibility of OncoPro medium with many previously established tumoroid lines and present a stable reporter pool generation custom project for a leading pharmaceutical company using a client-provided colorectal tumoroid line. We first tested the ability for the tumoroids to adapt to OncoPro medium and a suspension culture protocol. Tumoroids displayed higher growth rate when cultured in OncoPro medium compared to client-provided media (CM) in both embedded and suspension culture. To enable scalability, suspension culture was selected for downstream reporter pool generation. Firefly luciferase (fLuc)- and green fluorescent protein (GFP)-expressing tumoroids were generated by lentivirus transduction followed by antibiotic selection in OncoPro medium and CM. 5 days after transduction, >90% of cells were GFP-positive, minimizing the selection bias from editing and preserving population heterogeneity. 6 weeks after selection began, GFP-expressing pools evaluated by flow cytometry showed >97% GFP positive cells, while fLuc-expressing pools showed a cell number-dependent increase of luciferase activity in both media systems. Notably, OncoPro medium displayed a considerable advantage for reporter pool expansion, saving >50% of media and hands-on time compared to CM. To test for genetic and transcriptional stability, stable pools were compared with parental cells grown in CM using targeted NGS and bulk RNA sequencing. High correlation coefficients (r ≥ 0.99 for targeted NGS and ≥ 0.90 for RNA sequencing) were observed in pre- and post-edited pools, suggesting minimal genetic and gene expression drift over the engineering process. Taken together, these findings represent the ability of OncoPro medium to support cell engineering workflows on established tumoroids while preserving characteristics of the parental tumoroids. These tumoroid reporter pools serve as effective and clinically relevant models for evaluating cancer therapies in downstream applications. Citation Format: Xiaoyu Yang, Jacob Delgadillo, Garrett Wong, Matthew Woods, Jakhan Nguyen, Colin Paul, Chris Yankaskas, Pradip Shahi Thakuri, Shyanne Salen, Sylvia Beam, Matt Dallas, David Kuninger, Jason Sharp. Generation of stable reporter pools in established patient-derived tumoroid 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 6504.