Abstract 22: Engineering KRAS-mutant-organoids from pancreatic ductal adenocarcinoma for advanced drug screening applications <i>in-vitro</i> and <i>in-vivo</i>

Abstract Background: Organoids are powerful tumor models for cancer research surpassing 2D-cultures in maintaining tumor heterogeneity and genetic fidelity. However, working with organoids encounters challenges in imaging, drug-screening, and in-vivo efficacy studies, due to their structural complexity. Additionally, the absence of other relevant cell types including cancer-associated-fibroblasts (CAFs) and immune cells, limits their ability to fully recapitulate the tumor-microenvironment (TME). To overcome these limitations, we developed GFP- or RFP-expressing KRAS-mutant-PDAC-organoids for efficient in-vitro and in-vivo drug-screening in mono- and co-culture systems. Methods: We developed KRAS-mutant-PDAC-organoids that overexpress GFP or RFP using lentiviral-transduction. The growth and gene expression profile of these engineered organoid lines were compared to the parental line using Incucyte, DRUG-seq, and immunoblotting. These modifications enabled in-vitro mechanistic studies and drug sensitivity assays via confocal-microscopy and cell viability assays, as well as in-vivo quantification of tumor growth, drug-response, and gene expression. Results: Antibiotic-sensitivity assays showed that 10 μM of antibiotic induces complete cell death within 5 days. Based on this, we stably overexpressed GFP or RFP in organoid lines, with strong GFP and RFP signals confirmed through microscopy. Optimizing culture conditions revealed that the addition of growth-factors (EGF and FGF) is crucial in initial phases of growth, though they can be removed later for drug-screening without impacting the organoids growth curve. This is important for accurate drug-screening, particularly for growth-factor pathway inhibitors. Further, we compared the growth rates and protein expression of the engineered organoids to their parental lines and observed no substantial differences. In high-throughput drug screening, we tested the effects of chemotherapy drugs (Gemcitabine and SN38) on organoids at varying seeding densities. While lentiviral transduction did not significantly affect the IC50 values, increasing the number of cells significantly raised the IC50 values (e.g., IC50 for Gemcitabine increased from 6-70 nM with 1k cells to 32-517 nM with 5k cells). Conclusion: We successfully engineered KRAS-mutant-PDAC-organoids, which will serve as invaluable tools for in-vitro and in vivo efficacy experiments. Engineered organoids will simplify detection in co-culture systems and eliminate the need for additional staining. Future studies will focus on evaluating the growth and gene expression of engineered lines in-vivo compared to their parental lines. Additionally, these novel co-culture systems are being used to investigate the effects of novel KRAS inhibitors on organoid growth in-vitro and in-vivo. Citation Format: Alireza Savadipour, Krishna Tummala, Eunsil Park, Vania Vidimar, Mike Veling, Mark Buchanan, Eric Muise, Gustav Hebert, Brian Long, Nicolas Solban. Engineering KRAS-mutant-organoids from pancreatic ductal adenocarcinoma for advanced drug screening applications in-vitro and in-vivo [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 22.