Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
IUBMB Life. 2022 Sep;74(9):908-917. doi: 10.1002/iub.2655. Epub 2022 Jun 17.
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.
骨肉瘤（OS）是最常见的骨科肿瘤，尽管手术和化疗，但其转移率高，预后不良。免疫疗法为癌症患者带来了一线希望，但它们对 OS 的影响令人失望。本研究旨在评估 CTLA-4、PD-L1 和 TIM3 单、双和三联阻断对 OS 细胞活力、凋亡和迁移的影响。将 siRNA 特异性针对 CTLA-4、PD-L1 和 TIM3 的单、双和三联组合转染到 MG-63 和 U-2 OS 细胞系中。通过 qRT-PCR 评估转染效率后，分别应用 MTT assay 和流式细胞术评估 siRNA 转染细胞的细胞活力和凋亡率。最终，通过划痕愈合试验测量转染细胞的迁移。首先，qRT-PCR 分析显示，在 siRNA 转染的 OS 细胞中，CTLA-4、PD-L1 和 TIM3 下调。MTT assay 和流式细胞术结果证实，双或三组合中的这些免疫检查点的沉默，但不是单药阻断，显著降低细胞活力并分别增加凋亡。当进行三重沉默时，这些效果更为显著。最后，划痕愈合试验表明，免疫检查点的双和三重沉默显著抑制细胞迁移，三重沉默表现出更大的效果。我们的研究结果表明，CTLA-4、PD-L1 和 TIM3 的三重阻断是抑制 OS 中肿瘤细胞进展和迁移的有效策略，需要进行大规模的临床研究才能将其转化为 OS 患者广泛的治疗应用。
