Establishment and characterisation of PDTOs and CAFs for the development of a co-culture system in pancreatic periampullary adenocarcinoma

Periampullary adenocarcinoma (PAC) constitutes a diverse group of malignancies originating from the pancreatic head (60%), the ampulla of Vater (20%), the distal common bile duct (10%), and the duodenum (10%). The pancreatic tumour microenvironment (TME) is a major driver of tumour progression and therapeutic resistance, with cancer-associated fibroblasts (CAFs) emerging as central contributors. Representing a heterogeneous population within the TME, CAFs actively mediate stromal remodelling through complex interactions with tumour cells as well as with immune and stromal components. Detailed characterisation of CAF phenotypes is essential to define their impact to PAC pathobiology and to facilitate the design of novel therapeutic strategies against this highly aggressive malignancy.Patient-derived tumour organoids (PDTOs) and CAFs were isolated from freshly obtained primary PAC specimens. CAFs subtypes were characterised by immunofluorescence staining for key CAF proteins and transcriptomic sequencing. PDTOs were characterised by genomic sequencing. Drug screening of PDTOs with talazoparib and olaparib was conducted to examine the correlation between an HRD mutation and sensitivity to PARP inhibitors.4 PDTOs and 4 CAFs were successfully derived and cultured from PAC specimens. CAF cell lines showed positive expression of fibroblast markers alpha-SMA and vimentin, and showed heterogeneous intensity and expression of CAF markers such as PDGFR and FAP. They were categorised into antigen-presenting (apCAFs), inflammatory (iCAFs) and myofibroblastic (myCAFs) subgroups based on the RNA expression of key genes. MyCAFs were grouped based on the expression levels of CTGF, COL1A1, POSTN and ACTA2 genes. CD74 and HLA genes were chosen for the characterisation of apCAFs, while iCAFs were characterised based on the expression of IL-1/6/11, FAP and CXCL1/2 genes. Mutational profiling of the PDTOs identified a missense ATM variant c.5558A>T (p.Asp1853Val) in an intestinal-type adenocarcinoma, which was sensitive to olaparib (1.66 µM) and talazoparib (0.45 µM).PDTOs and CAFs serve as a useful model for studying the cellular architecture and heterogeneity of PAC in vitro. Building on this, defined CAF heterogeneity will be assessed for its association with immunosuppressive versus immunostimulatory profiles, and T cells, along with immunotherapeutic agents, will be incorporated into the co-cultures. These models will provide valuable insights into PAC tumour-stroma-immune biology and form the basis for advanced co-cultures to test strategies targeting the TME.