Abstract A013: CRISPR-engineered human organoid-based pancreatic cancer model recapitulates early tumor features through TP53 inactivation

Background: Pancreatic adenocarcinoma is one of the deadliest solid tumors, with a two-year survival rate below 5%. Progress in developing effective treatments has been limited, largely due to the absence of physiologically relevant disease models that accurately recapitulate the tumor microenvironment. Such models are critical for understanding disease mechanisms and evaluating therapeutic strategies. Organoids have recently emerged as promising platforms for modeling various diseases, including pancreatic cancer, as they retain the structural and functional features of their tissue of origin. Moreover, CRISPR-Cas9 gene editing allows precise introduction of cancer-associated mutations, providing a powerful approach for generating genetically defined models. Methods: We established normal pancreatic organoids from human pancreatic tissue samples and validated their identity through qPCR and histological analyses, confirming their resemblance to native pancreatic epithelium. These organoids demonstrated stable growth over several passages and retained viability after cryopreservation and thawing. To model pancreatic cancer, we used CRISPR-Cas9 technology to introduce TP53 mutations, a key tumor suppressor gene altered in over 70% of pancreatic ductal adenocarcinoma (PDAC) cases. Following Nutlin-3a selection, successful TP53 knockout was confirmed by Sanger sequencing. The engineered TP53-knockout (KO) organoids were expanded and characterized to assess cancer-like phenotypes. Results: Human-derived pancreatic organoids retained their structural and functional identity across passages and after thawing. Following TP53 gene editing, the TP53 KO organoids exhibited increased proliferation and morphological changes consistent with early tumorigenesis. Importantly, qPCR analysis revealed significant upregulation of cancer-associated markers, including MUC5AC, KRT19, and CEACAM5, in TP53-mutated organoids compared to controls, further supporting their malignant transformation. These findings represent a critical step toward building a genetically defined pancreatic cancer model that closely reflects human disease. Conclusion: We successfully generated a pancreatic cancer organoid model by editing TP53 in normal human pancreatic organoids. The TP53 KO organoids showed enhanced growth, morphological transformation, and elevated expression of key cancer markers, recapitulating early features of pancreatic cancer. This model mimics critical aspects of pancreatic cancer biology and provides a robust platform for studying tumor initiation, progression, and drug response. Unlike conventional animal models or stem-cell-derived systems, our approach offers a human-based, physiologically relevant, and reproducible system for pancreatic cancer research and therapeutic screening. Citation Format: Eden Demere Amare, Soraya SALAS-SILVA, Ji Hyun Shin, Dongho Choi. CRISPR-engineered human organoid-based pancreatic cancer model recapitulates early tumor features through TP53 inactivation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3): nr A013.