Promoting anti-tumor immune response in osteosarcoma by BETi through restoring Th17/Treg balance

The immune landscape of osteosarcoma (OS) remains incompletely characterized. We examined the Th17/Treg balance using public single-cell RNA-sequencing datasets, multiplex immunohistochemistry, peripheral-blood flow cytometry, an exploratory xenogeneic mouse model, and mechanistic assays. Human datasets and specimens were consistent with a Treg-enriched, lower Th17/Treg immune state, with the most pronounced imbalance in the peripheral blood of patients with metastatic disease. In the mouse model, BET inhibitor (BETi) treatment produced parameter-specific attenuation of osteolytic changes and shifts in Th17 and Treg frequencies. BRD4 chromatin-occupancy, expression, and migration assays identified the CXCL12/CXCR4 pathway as a candidate mechanism. B7-H3 was elevated in metastatic OS in the analyzed cohort, but none of the evaluated checkpoint-associated genes was significantly associated with overall survival. Under the tested schedules, full-dose BETi produced the greatest overall improvement in observed survival time and tumor-burden measures; the reduced-dose BETi and anti-B7-H3 combination remained active but did not outperform BETi monotherapy. These findings support that BRD4 can serve as a potential therapeutic target for OS to reverse its immunosuppressive microenvironment, and further in-depth research is warranted.