<scp><i>PI3</i></scp> expression predicts recurrence after chemotherapy with <scp>DNA</scp>‐damaging drugs in gastric cancer

Despite recent advances in gastric cancer therapy, chemotherapy resistance and lack of methods for selecting combination regimens remain major problems. Organoids, which provide a culture system that more closely resembles tumor cell organization than traditional cell lines, can be established from surgical specimens with a high success rate and are widely used for drug sensitivity assays. In this study, we aimed to identify a novel biomarker for predicting multidrug resistance using gastric cancer organoids (GCOs). We evaluated 5-fluorouracil or oxaliplatin-resistant GCOs to find novel biomarkers that reflect multidrug resistance in gastric cancer. To examine the resistance mechanisms, RNA-sequencing analysis and ex vivo drug sensitivity testing were performed. The association of biomarkers with patient prognosis and chemotherapy efficacy was evaluated using three original cohorts with a total of 230 cases. The results were also validated with two independent public cohorts and single-cell RNA sequence data. Increased expression of peptidase inhibitor 3 (PI3) was detected in all 5-fluorouracil or oxaliplatin-resistant GCOs. Our findings suggest a potential association of PI3 expression with ribosome biosynthesis and RNA metabolism under organoid conditions. We also found that PI3 overexpression promoted 5-fluorouracil/oxaliplatin/cisplatin resistance but not paclitaxel resistance. Immunohistochemical evaluation of PI3 expression revealed that the PI3-positive gastric cancer group had a poorer outcome, especially in terms of time to recurrence. PI3 positivity was also an independent predictor of relapse after chemotherapy with DNA-damaging agents. PI3 promotes DNA-damaging drug resistance through multiple downstream regulations related to RNA and ribosomal metabolism. PI3 may be useful as a biomarker for the therapeutic selection of non-DNA-damaging agents. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.