Abstract 5193: Optimization of tumoroid size and payload delivery methods for the engineering of patient-derived tumoroid models

Abstract Tumoroids, also known as cancer organoids, are three-dimensional (3D) in vitro cell cultures established from primary tissue samples. These next-generation cancer cell models largely recapitulate the spatial organization, gene expression, and mutational profiles of patient tumors. It may be beneficial to engineer tumoroids to generate reporter lines, model specific mutations involved with tumor progression, or modulate gene expression to help understand the mechanism of action of therapeutics. Here, we explore methods for genetic engineering of tumoroids established and cultured in Gibco™ OncoPro™ Tumoroid Culture Medium to facilitate these studies. We first examined the impact of tumoroid size on transfection efficiency via electroporation with the Neon™ NxT Electroporation System. Colorectal tumoroids were transfected with enhanced green fluorescent protein (eGFP) immediately after dissociation or after 4 or 7 days in culture in OncoPro medium, at which point tumoroid size had reached approximately 400 µm in diameter. Nearly 80% of dissociated tumoroids were successfully transfected, while efficiency dropped to 35-40% when using intact 4- or 7-day old tumoroids. Therefore, dissociated tumoroids were used for further experiments. We then compared lipofection, electroporation, and lentivirus-based transduction in a difficult-to-transfect model, focusing on reporter line generation. While lipofection is easy-to-use and cost effective, delivery of enhanced green fluorescent protein (eGFP) by lipofection led to transfection efficiencies of only 15%. Electroporation of eGFP was more efficient, transfecting 30% of tumoroid cells for the tumoroid line used. Given the importance of preserving the cellular heterogeneity found in patient samples and recapitulated in tumoroid models, these lower delivery efficiencies indicate that lipofection- and electroporation-based methods are not ideal for the generation of stably engineered tumoroid models. In contrast, lentiviral transduction at high MOI was the most efficient method for eGFP overexpression, initially transducing 67% of cells and enabling stable pool reporter line generation with nearly identical mutational profile and gene expression compared to the parental population. In follow up experiments, transduction also enabled generation of eGFP-expressing breast and endometrial reporter tumoroid lines. Overall, our results indicate that electroporation may be more suited for transient transfection across a wide range of payloads, and siRNA, gRNA, and Cas9 protein were able to be delivered by electroporation in follow up experiments. Lentivirus-based transduction is more suitable for generation of stable tumoroid pools. The insights gained from this study can be applied to tumoroids derived from various tissues, potentially accelerating advancements in cancer research and personalized medicine. Citation Format: Isha Dey, Xiaoyu Jenny Yang, Jacob Delgadillo, Garrett Wong, Colin D. Paul, Chris Yankaskas, Brittany Balhouse, Shyanne Salen, Sylvia Beam, Amber Bullock, Anthony Chatman, Pradip Shahi Thakuri, Kanika Singh, Jason Sharp, Matt Dallas, David Kuninger. Optimization of tumoroid size and payload delivery methods for the engineering of 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 5193.