Abstract 2710: Establishment of a co-culture model to investigate the effects of chemotherapy in pancreatic periampullary intestinal adenocarcinoma

Abstract Introduction: Periampullary adenocarcinoma (PAC) refers to cancers originating from the head of the pancreas (60%), ampulla of Vater (20%), distal common bile duct (10%), and duodenum (10%). The pancreatic tumor microenvironment (TME) plays a critical role in driving progression and therapeutic resistance in pancreatic cancer, with cancer-associated fibroblasts (CAFs) emerging as key contributors. CAFs, a heterogeneous population within the TME, are central to stromal remodeling through interactions with tumor cells and immune components. Understanding CAF phenotypes is essential to unravel their role in PAC biology and to identify novel therapeutic targets. Methods: Patient-derived tumor organoids (PDTOs) and CAFs were isolated from primary PAC specimens. Tissue was minced, digested, and dissociated into single cells. Tumor cells were seeded into 3D domes within a basement membrane extract (BME) and cultured in supplemented medium. CAFs were seeded in 2D and allowed to attach. KRAS mutations were analyzed using Sanger sequencing, and CAFs subtypes were characterized through immunofluorescence staining for key markers. Co-cultures of organoids and CAFs were formed at a 1:4 ratio, stained with CellTracker Dyes and their growth was monitored over 2 weeks by microscopy. Flow cytometry was used to characterize T cell subpopulations from PBMCs. Advanced co-cultures were used to measure cytotoxicity and immune efficacy. Results: PDTOs and CAFs were successfully derived and cultured from PAC specimens, both confirmed as KRAS wild type. All 3 isolated CAF lines expressed fibroblast markers Smooth Muscle Actin alpha (α-SMA) and vimentin. CAFs showed heterogeneous intensity and expression of specific myofibroblastic (myCAFs) markers like Platelet Derived Growth Factor Receptor (PDGFR) and inflammatory CAFs (iCAFs) markers, like Fibroblast Activation Protein (FAP). Co-culture of PDGFR+/FAP- myCAFs with PDTOs at increasing concentrations of cisplatin showed a 4.0 and 6.8-fold increase in the IC50 of the co-culture compared to PDTOs and CAFs alone, respectively. Co-culturing a heterogenous myCAF/iCAF population with PDTOs and treatment with cisplatin, showed no induction of resistance (IC50 co-culture 6.6 μM) compared to organoids alone (IC50 9.5 μM), and CAFs alone (IC50 9.4 μM). Enrichment of T cells from PBMCs revealed 95% T cell viability, of which 26.9% were cytotoxic, 63.1% were helper T cells, 4.3% were double positive (CD4+CD8+) and 5.7% were double negative (CD4-CD8-). Conclusion: PDTOs and CAFs serve as a useful model for studying the cellular heterogeneity of PAC in vitro. CAF subtypes based on protein markers exhibit both tumor-promoting effects and increased cisplatin resistance. High viability and enrichment of T cells for co-culture models will provide valuable insights into PAC biology and support testing strategies targeting the TME. Citation Format: Panagiotis Sarametidis, Jojanneke Stoof, Laura Ivers, Jean Murphy, Niall Swan, Luca Benigno, Fiona Hand, Kevin Conlon, Paul Mullan, Maeve Lowery, Naomi Walsh. Establishment of a co-culture model to investigate the effects of chemotherapy in pancreatic periampullary intestinal adenocarcinoma [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 2710.