Detecting resistant subpopulations by profiling drug response heterogeneity in patient-derived cancer organoids (PDCOs) following first-line treatment.

e15603 Background: Colorectal cancer (CRC) frequently progresses due to resistance to standard first-line chemotherapy, such as irinotecan. Patient-derived cancer organoids (PDCOs) enable ex vivo drug sensitivity testing, but clinical impact requires detecting resistance linked to treatment failure in vivo . Conventional whole-well viability assays may fail to identify resistant tumor subpopulations. We report findings from Metsystem’s feasibility study (H-24038460) evaluating PDCO establishment from metastatic CRC (mCRC) and their ex vivo response to SN-38 (irinotecan’s active metabolite) using a whole-well readout and our proprietary methods. Methods: Adults with biopsy-confirmed mCRC and resectable hepatic metastases were enrolled in the study following informed consent. All patients had progressed after first-line chemotherapy (no regimen-specific eligibility criteria). PDCOs were established and expanded before drug screening. Ex v ivo dose-response testing was performed using SN-38, at increasing concentrations up to 5 uM. Drug response was assessed using a conventional whole-well viability assay (PrestoBlue, 7-day endpoint) and our proprietary methods. Results: PDCOs were successfully established and expanded from 7/8 patients. Drug-screening was performed for all 7, with 4 of them having paired evaluations using both methodological approaches. All evaluated drug screens showed viability changes when exposed to SN-38 regardless of the methodology used. Despite documented clinical resistance to irinotecan in some of the analyzed patients, whole-well viability measurements suggested near-complete sensitivity at higher SN-38 concentrations. In contrast, our method identified viable, treatment-resistant subpopulations persisting after high-dose SN-38 exposure. These resistant subpopulations were detected in all four samples and could comprise less than 15%, rendering them undetectable by whole well viability-based assays. Conclusions: In this feasibility study, PDCO establishment and multi-readout SN-38 testing were feasible in a clinical workflow. The study also indicates that irinotecan resistance in mCRC may be driven by small tumor subpopulations that can be undetectable by conventional whole-well ex vivo screening. Therefore, novel drug screening assays that can detect drug-resistant PDCOs may help in treatment or re-treatment decisions. Such findings underscore the need for integrating functional testing ex vivo into precision oncology workflows to improve therapeutic selection.