High-throughput phenotypic profiling of patient-derived colon cancer organoids to reveal a chemoresistant, AKT-driven tumor subpopulation and treatment strategy.

Abstract

e15073 Background: Colorectal cancer (CRC) remains a significant clinical challenge due to its intrinsic heterogeneity, the paucity of personalized treatment options, and the commonality of chemoresistance. Methods: To better understand drug response dynamics at the single-cell level, we have developed a high-throughput, image-based phenotypic profiling pipeline using CRC patient-derived organoid (PDO) monolayer cultures. Diverse CRC PDOs representing multiple Consensus Molecular Subtypes were treated with a combination of 5-Fluorouracil and Oxaliplatin (FOX), imaged utilizing a biomarker panel identified using transcriptomic data, and analyzed using our newly developed phenotypic profiling pipeline. To further elucidate individual tumor response in vivo, we have also developed protocols to implant our PDOs into mice to create Patient Derived Xenograft (PDX) models. Results: Using our new analysis pipeline, we have identified a subpopulation of CRC cells that, after acute exposure to FOX treatment, exhibit elevated AKT signaling and increased expression of cancer stem cell markers. We observe this subpopulation across multiple PDOs, including across distinct CRC molecular subtypes, suggesting that chemotherapy itself may contribute to the enrichment of drug-resistant phenotypes driven by PI3K/AKT survival signaling. Targeting this survival phenotype, we also demonstrate that transient pre-treatment with the clinically utilized PI3K/mTOR dual inhibitor Dactolisib effectively sensitizes CRC PDOs to FOX, synergizing with this standard-of-care chemotherapy regimen and reducing the fraction of chemoresistant cells. Synergy score profiling revealed that this combination was broadly effective across diverse PDOs, with the strongest response noted in the organoid exhibiting the highest baseline AKT signaling. Preliminary data in our PDX models suggest this combination is synergistic in vivo as well. Conclusions: Our findings highlight the power of personalized organoid-based phenotypic profiling for dissecting molecular mechanisms of therapeutic resistance and support the rationale for transient PI3K/mTOR inhibition as a strategy to improve CRC treatments and outcomes.