Cancer combination therapies by silencing of CTLA-4, PD-L1, and TIM3 in osteosarcoma.

Osteosarcoma (OS) is the most common orthopedic neoplasm, with a high metastasis rate and a dismal prognosis despite surgery and chemotherapy. Immunotherapies have offered cancer patients a ray of optimism, but their impact on OS has been disappointing. The objective of this study is to assess the effect of mono, dual, and triple combinations of CTLA-4, PD-L1, and TIM3 blockade on OS cell viability, apoptosis, and migration. The MG-63 and U-2 OS cell lines were transfected with mono, dual, and triple combinations of siRNAs specific for CTLA-4, PD-L1, and TIM3. After evaluation for transfection efficacy by qRT-PCR, MTT assay and flow cytometry were applied to assess cell viability and apoptosis rate in siRNA-transfected cells, respectively. Ultimately, the migration of transfected cells was measured by wound-healing assay. First, the qRT-PCR analysis revealed that in siRNA-transfected OS cells, CTLA-4, PD-L1, and TIM3 were downregulated. The MTT assay and flow cytometry results confirmed that silencing of these immune checkpoints in dual or triple combinations, but not in the single-agent blockade, significantly decreases cell viability and increases apoptosis, respectively. These effects were more significant when triple silencing was performed. Finally, the wound-healing assay revealed that dual and triple silencing of immune checkpoints significantly inhibits cell migration, with triple silencing exhibiting a greater effect. Our findings suggest that triple blockade of CTLA-4, PD-L1, and TIM3 is an effective strategy for inhibiting tumor cell progression and migration in OS, which requires large-scale clinical investigations to be translated into broad therapeutic applicability for OS patients.