TBX3 shapes an immunosuppressive microenvironment and induces immunotherapy resistance

Background: Identifying biomarkers that predict immunotherapy efficacy and discovering new targets for combination therapies are critical elements for improving the prognosis of bladder cancer (BLCA) patients. Methods: Firstly, we explored the expression patterns of TBX3 in normal and pan-cancer tissues and the correlation between TBX3 and the immune microenvironment using data from multiple public databases. Then, we combined various techniques, including bulk RNA sequencing, single-cell RNA sequencing, high-throughput cytokine arrays, functional experiments, ProcartaPlex multiplex immunoassays and TissueFAXS panoramic tissue quantification assays, to demonstrate that TBX3 shapes an immunosuppressive tumor microenvironment (TME) in BLCA. Results: We identified TBX3 as a key factor associated with the immunosuppressive microenvironment in BLCA through a systematic multi-omics analysis. We found that TBX3 is primarily expressed in malignant cells, where TBX3^high tumor cells increase the secretion of TGFβ1, which promotes the infiltration of cancer-associated fibroblasts (CAFs), thereby forming an immunosuppressive microenvironment. We further demonstrated that TBX3 enhances TGFβ1 expression by binding to the TGFβ1 promoter, and blocking TGFβ1 counteracts the immunosuppressive effects of TBX3. Moreover, TBX3 reduced the cancer-killing efficiency of CD8⁺ T cells by decreasing the proportion of GZMB⁺ CD8⁺ T cells, and knocking down TBX3 combined with anti-PD-1 treatment increased CD8⁺ T cell infiltration and reduced CAFs in vivo. We also validated the inverse relationship between TBX3⁺ malignant cells and CD8⁺ T cells and the positive relationship with CAFs in tissue microarrays. Lastly, we found that TBX3 predicted immunotherapy efficacy in our real-world immunotherapy cohort and multiple public cohorts. Conclusion: In summary, TBX3 promotes BLCA progression and immunotherapy resistance by inducing an immunosuppressive microenvironment, and targeting TBX3 could enhance the efficacy of immunotherapy for BLCA.