bioRxiv2026
BackgroundFluoropyrimidines, specifically 5-fluorouracil (5-FU), remain the cornerstone of colorectal cancer (CRC) therapy. However, intrinsic and acquired resistance, alongside dose-limiting systemic toxicities, often result in treatment failure and disease relapse. There is a pressing clinical need for next-generation fluoropyrimidines that can retain the antitumor activity in 5-FU-refractory CRC models while maintaining a favorable safety profile. MethodsWe evaluated the antitumor efficacy of CF10, a novel polymeric fluoropyrimidine designed for the sustained delivery of FdUMP, against equimolar 5-FU. We utilized a diverse panel of six patient-derived CRC organoid (PDO) models to assess 3D growth inhibition under both normoxic ([~]20% O2) and physioxic (5% O2) conditions. Mechanisms of action were investigated via {gamma}H2AX signaling (DNA damage), Annexin V/PI flow cytometry (death kinetics), and ALDEFLUOR assays (stem-like populations). Functional suppression of metastasis-associated phenotypes was evaluated using 3D Matrigel invasion assays. Finally, the therapeutic index and overall survival were validated in vivo using two independent patient-cell-derived xenograft (PCDX) models (TX-CC-199 and TX-CC-201). ResultsCF10 demonstrated significantly greater suppression of organoid growth compared to equimolar 5-FU across all patient-derived lines, regardless of morphological heterogeneity or oxygen tension. In 3D invasion assays, CF10 achieved superior anti-invasive activity even at a 10-fold lower molar dose than 5-FU. This functional advantage was mirrored by a marked depletion of the ALDH-high stem-like subpopulation, which was largely recalcitrant to 5-FU. Mechanistically, CF10 induced intensified replication stress, DNA damage and repair signaling ({gamma}H2AX, Top1cc/pRPA32, FANCD2), and pushed the CRC to irreversible/terminal, PI-positive death states. In vivo, CF10 treatment resulted in profound tumor growth inhibition and a robust survival advantage in two patient cell-derived xenograft (PCDX) models (Log-rank P<0.01) without inducing systemic weight loss or noticeable toxicity. ConclusionsBy integrating 3D patient-derived modeling with in vivo validation, we demonstrate that CF10 effectively overcomes the biological and pharmacological limitations of 5-FU. CF10 targets the aggressive, invasive, and stem-like subpopulations of CRC that drive clinical relapses. These findings provide a compelling translational rationale for the clinical development of CF10 as a superior alternative to standard fluoropyrimidines in both treatment-naive and refractory CRC. Significance StatementDespite the foundational role of 5-fluorouracil (5-FU) in colorectal cancer (CRC) therapy, resistance and systemic toxicity remain major barriers to curative outcomes. This study identifies CF10, a novel polymeric fluoropyrimidine, as a superior alternative that overcomes 5-FU resistance in biologically diverse patient-derived organoids and xenograft models. Crucially, CF10 demonstrates a unique capacity to suppress the invasive, aldehyde dehydrogenase (ALDH)-high stem-like subpopulations that likely survive standard chemotherapy (5-FU) by maintaining efficacy under physiological oxygen levels and providing a significant survival advantage in vivo with improved tolerability. CF10 represents a promising translational candidate for the treatment of both treatment-naive and refractory CRC.