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

Abstract BackgroundColorectal cancer (CRC) exhibits limited responsiveness to immune-checkpoint blockade, necessitating further investigation. The intratumoral Treg/CD8⁺ T-cell ratio serves as a predictive biomarker for therapeutic efficacy. Here, we demonstrate that HSPB1 targeting reduces this ratio and confers therapeutic benefit in CRC.MethodsCandidate genes were identified by integrative single-cell transcriptomics, TCGA 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.ResultsSingle-cell profiling and TCGA WGCNA analyze identified HSPB1 as a putative determinant of the intratumoral Treg/CD8⁺ T-cell ratio, and survival 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.ConclusionsTargeting HSPB1 exerts dual anti-tumor effects: it directly suppresses neoplastic proliferation and simultaneously alleviates Treg-mediated immunosuppression within the tumor microenvironment.