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Background Approximately 95% of patients with advanced colorectal cancer (CRC) harbor mismatch repair-proficient (MMRp) ‘cold’ tumors, unresponsive to PD-1 blockade monotherapy. In contrast, patients with MMR-deficient (MMRd) ‘hot’ tumors exhibit high microsatellite instability (MSI-H), elevated tumor mutational burden (TMB), increased neoantigen load, and cGAS-STING pathway activation, conferring high immunotherapy responsiveness. A critical unmet need is therapeutic conversion of MMRp to MMRd states to extend immunotherapy benefits to broader patient populations.Methods We designed a novel fluorescent reporter system to detect microsatellite frameshift mutations and integrated it into diverse tumor cell lines. This system enabled real-time monitoring of microsatellite instability (MSI) activity following treatment with bioactive compounds. After optimization, we selected the most sensitive cell line (QHT-CL) for high-throughput screening. Using QHT-CL, we screened chemotherapeutic libraries for compounds inducing MSI-H phenotypes.Results 1) Platform Development: A panel of tumor cell lines harboring an MSI reporter was established, with the QHT-CL line selected as the optimal platform. 2) Assay Validation: The QHT-CL system demonstrated exceptional sensitivity and robustness for evaluating drug efficacy in human tumor cells (6-days duration). 3) Compound Discovery: High-throughput screening identified QHT025—a novel, potent, selective small-molecule MMR modulator. QHT025 showed superior potency in driving MSI-H and converting MMRp→MMRd versus reference compounds (NP1867, TMZ).Conclusions This platform establishes an innovative cell-based assay for assessing MSI activity. Using this approach, we discovered QHT025: a small-molecule modulator that induces MSI-H and functional MMRp→MMRd conversion. Acting as an immune-priming agent, it remodels the TME to convert ‘cold’ tumors to ‘hot’ lesions. The platform and compound pioneer a novel therapeutic approach to sensitize MMRp tumors to immunotherapy.