Patient- derived gallbladder cancer organoids mirror tumor genetics and resistance phenotypes, enabling ex vivo testing of adjuvant chemotherapy response

Abstract Background Advanced gallbladder cancer (GBC) is an aggressive malignancy with poor prognosis, high recurrence rates, and limited treatment options. Patient-derived organoids (PDOs) have emerged as promising preclinical models that faithfully mimic the epithelial architecture of tumors, offering valuable insights into cancer biology, drug resistance, and potential therapeutic strategies. Despite advances in GBC research, well-characterized GBC PDOs are lacking, and their potential to reflect patient-specific adjuvant chemotherapy outcomes and identify resistance-associated pathways remains underexplored. Methods In this study, we established and characterized eight PDOs from a cohort of Chilean patients with resectable advanced (stage IIIA and IIIB) and metastatic (stage IV) GBC. Histological analysis, whole-exome sequencing, and confocal microscopy were used to compare PDOs with their parental primary tumors. Ex vivo drug response assays with 5-FU, cisplatin, and gemcitabine were performed and compared with clinical response from patients. Gemcitabine-resistant PDOs were characterized by Tandem Mass Tag (TMT)-based quantitative proteomic, coupled with screening of 103 FDA-approved compounds to identify alternative therapeutic vulnerabilities. Results We successfully generated GBC-PDOs from 66.7% (8/12) of patient samples, and these organoids closely recapitulated the histopathological and genetic profiles of the parent tumors. In a preliminary proof -of-concept analysis (n=4), in vitro sensitivity to 5-FU showed a promising biological trend that aligned with patient disease-free survival and tracked with overall survival following adjuvant capecitabine treatment. Gemcitabine-resistant PDOs harbored concurrent APC , TP53 , and JAK2 mutations. Proteomics analysis of these chemoresistant PDOs identified enriched pathways related to epithelial-mesenchymal transition (EMT), KRAS signaling, apical junction, IL2-STAT5 signaling and hypoxia. Six overexpressed proteins were related to the acquisition of malignancy: Vimentin (VIM), Fibronectin 1 (FN1), Clusterin (CLU), Caveolin 1 (CAV1), Nestin (NES), and TGF-β. Furthermore, gemcitabine-resistant organoids displayed sensitivity to Ixazomib citrate, Panobinostat, Lapatinib and Bosutinib, suggesting potential therapeutical alternatives. Conclusions This study identifies resistance pathways and key proteins associated with gemcitabine resistance in GBC. These findings provide potential therapeutic targets that warrant investigation to validate their role in guiding treatment strategies for gallbladder cancer.