Abstract
<title>Abstract</title> <p>Background Radiotherapy is an important treatment modality for colorectal cancer (CRC), but intrinsic and acquired radioresistance remain major barriers to durable tumor control. Increasing evidence suggests that therapy resistance is shaped by heterogeneous malignant epithelial states and their interactions with the tumor microenvironment. However, the epithelial cell states associated with radiotherapy non-response in CRC and their therapeutic vulnerabilities remain poorly defined. Objectives This study aimed to identify malignant epithelial populations associated with radiotherapy resistance in CRC, define their biological characteristics, and determine whether these resistant states can be therapeutically targeted. Methods Patient-derived CRC single-cell RNA sequencing data were integrated and analyzed to characterize cellular composition, malignant epithelial subclusters, copy-number variation, pathway activity, differentiation trajectory, and cell–cell communication. TROP2/TACSTD2-associated epithelial states were further evaluated using survival analysis, enrichment analysis, pseudotime reconstruction, and ligand–receptor inference. Functional validation was performed in CRC cell models using TROP2 knockdown, TROP2 overexpression, clonogenic survival assays, immunofluorescence, western blotting, cell-cycle analysis, apoptosis assays, drug–radiation synergy analysis, and xenograft models treated with irradiation and sacituzumab govitecan (Trodelvy). Results Single-cell analysis identified a distinct TROP2⁺ malignant epithelial population enriched in radiotherapy non-responsive tumors and associated with poor prognosis. These cells occupied a terminally remodeled, stress-adapted epithelial state characterized by enhanced translation, oxidative metabolism, keratinocyte-like remodeling, and fibroblast-associated paracrine signaling. Functionally, TROP2 expression correlated with intrinsic radioresistance. TACSTD2 depletion sensitized CRC cells to irradiation, whereas TROP2 overexpression promoted radioresistance. Irradiation further induced TROP2 expression, creating a therapeutic vulnerability to Trodelvy. Combined irradiation and Trodelvy enhanced apoptosis, altered cell-cycle distribution, and suppressed tumor growth in xenograft models, particularly in radioresistant CRC. Conclusion TROP2 defines a stress-adapted malignant epithelial state that contributes to CRC radiotherapy resistance while providing an actionable therapeutic vulnerability. These findings support combined radiotherapy and TROP2-targeted antibody–drug conjugate treatment as a potential strategy to overcome radioresistance in CRC.</p>