Cancer combination therapies by silencing of CTLA‐4, PD‐L1, and TIM3 in osteosarcoma
Amin Daei Sorkhabi, Aila Sarkesh, Ali Fotouhi, Hossein Saeedi, Leili Aghebati‐Maleki
Abstract
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.