Establishing diffuse gastric cancer patient-derived organoids for drug response analysis

Introduction: Gastric cancer (GC) is a major health problem in Chile, with high incidence and mortality rates, particularly among younger populations. Diffuse GC is the most aggressive subtype, characterized by a poorly cohesive and signet-ring cell features, matching the genomically stable molecular subtype. These tumors are frequently linked to advanced nodal metastasis and peritoneal dissemination. Response to chemotherapy is generally limited, although a subset of GC patients can achieve significant clinical efficacy from first-line immunotherapy (PD-1/PDL-1 inhibitors plus chemotherapy). Patient-derived organoids (PDOs), three-dimensional cultured generated from stem cells, can recapitulate tumor heterogeneity and have emerged as a powerful preclinical model for studying tumor biology and predicting response to anticancer treatments. The aim of this study was to characterize GC PDOs and evaluate their responses to drug treatment. Material and Methods: Fresh tumor tissues and ascites samples obtained from GC patients were enzymatically dissociated using collagenase/dispase solution and cultured following a modified protocol previously described. PDOs and their corresponding original tissues were characterized using H&E staining, immunohistochemistry, immunofluorescence, and flow cytometry. GC PDOs were then subjected to chemotherapy dose-response assays using oxaliplatin, 5-fluorouracil and irinotecan, and additionally screened against 103 FDA-approved anticancer drugs. After 72 hours of treatment, cell viability was assessed using the CellTiter-Glo 3D assay. Results: Eight GC PDOs were successfully established from endoscopic biopsies or ascites fluid samples. The PDOs preserved the histopathological features and epithelial polarity of their tumors of origin and retained key mutations, including TP53 and CDH1 . The EPCAM-positive organoid population displayed membranous PD-L1 expression ranging from 0.28-75% by flow cytometry. Based on normalized AUC analysis, the PDOs showed heterogeneous responses to standard chemotherapies, and several specific drugs reduced the organoid viability. Conclusions: The GC-PDOs effectively recapitulated the histological and molecular characteristics of their epithelium tumor of origin and demonstrated differential sensitivity to chemotherapeutic and targeted agents. These findings support the use of GC PDOs as a valuable tool for investigating tumor biology and provide a promising platform for precision medicine approaches in the treatment of diffuse gastric cancer. Funding: ANID-FONDECYT 1221253, 1221345. ANID-FONDAP-CECAN 152220002, ANID-FONDAP-ACCDIS 15130011.