Targeting HSPB1 Reduces Tumor Growth and Abrogates Treg-Mediated Tumor Immunosuppression

Colorectal cancer (CRC) shows limited responsiveness to immune-checkpoint blockade, highlighting the need for further investigation. The intratumoral Treg/CD8⁺ T-cell ratio serves as a predictive biomarker for therapeutic efficacy. Here, we first demonstrate that targeting HSPB1 lowers this ratio and confers therapeutic benefit in CRC. Methods. Candidate genes were identified by integrative single-cell transcriptomics and spatial transcriptomics, followed by survival analyses of TCGA cohorts. Functional interrogation was performed using CRISPR-Cas9 engineered knockout cell lines. Subcutaneous tumor models were established, and the immune microenvironment was characterized by multiparametric flow cytometry. Mechanistic validation was achieved through bulk RNA-seq and complementary functional assays. Results. Single-cell profiling identified HSPB1 as a determinant of the intratumoral Treg/CD8⁺ T-cell ratio, and TCGA analysis showed its prognostic relevance in CRC. Spatial transcriptomics revealed colocalization of HSPB1-expressing tumor cells with Tregs. Subcutaneous tumor models demonstrated that CRISPR-mediated HSPB1 deletion or pharmacologic inhibition markedly suppressed tumor growth and reprogrammed the Treg-dominated microenvironment. In vitro polarization assays confirmed that targeting HSPB1 selectively restrains Treg differentiation without affecting Th17. Integrated transcriptomic and functional studies further elucidated that HSPB1 orchestrates CCL20–CCR6 mediated Treg recruitment, thereby shaping the immunosuppressive milieu within colorectal tumors.Conclusions. Targeting HSPB1 exerts dual anti-tumor effects by directly suppressing neoplastic proliferation and concurrently alleviating Treg-mediated immunosuppression within the tumor microenvironment.