Targeting HSPB1 inhibits tumor growth and abrogates Treg-mediated tumor immunosuppression.

[BACKGROUND] Colorectal cancer exhibits limited responsiveness to immune-checkpoint blockade, primarily because an elevated intratumoral Treg/CD8 T-cell ratio extinguishes antitumor immunity. The molecular determinants that lock this immunosuppressive balance are unknown.

[METHODS] We integrated single-cell RNA-seq, TCGA WGCNA, and spatial transcriptomics to nominate candidate regulators of the Treg/CD8 ratio. CRISPR-Cas9 was used to generate HSPB1-knockout MC38 and SW480 cell lines. Subcutaneous models were employed to assess tumor growth. Immune profiling was performed by multiparametric flow cytometry; mechanistic dissection combined bulk RNA-seq, Transwell assays, western blot and in vitro T-cell polarization systems.

[RESULTS] Single-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 HSPB1 genetic deletion or pharmacological inhibition markedly suppressed tumor growth and abolish the Treg-dominated microenvironment. In vitro polarization assays confirmed that targeting HSPB1 selectively restrains Treg differentiation without affecting Th17. Integrated transcriptomic and functional assays further elucidated that HSPB1 orchestrates Treg recruitment via the CCL20-CCR6 axis, thereby shaping the immunosuppressive milieu within colorectal tumors.

[CONCLUSIONS] Targeting HSPB1 exerts dual anti-tumor effects: it directly suppresses neoplastic proliferation and simultaneously alleviates Treg-mediated immunosuppression within the tumor microenvironment.