Radiation Biology Research Center, Institute for Radiological Research, Chang Gung University, Taoyuan 333, Taiwan; Division of Pulmonary Oncology and Interventional Bronchoscopy, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan 333, Taiwan.
Division of Gastroenterology, Cheng Hsin General Hospital, Taipei 112, Taiwan.
Int Immunopharmacol. 2022 Nov;112:109110. doi: 10.1016/j.intimp.2022.109110. Epub 2022 Aug 27.
Radiotherapy (RT) is applied to eradicate tumors in the clinic. However, hepatocellular carcinoma (HCC) exhibits resistance against RT. It is demonstrated that RT directly inhibits tumor growth but which induces type I interferons (IFNs) expression to phosphorylate STATs and increase STATs-downstream PD-L1 levels in the survival tumor cells. Since sorafenib is capable of suppressing STATs, we, therefore, hypothesize that sorafenib suppresses IFNs-mediated radioresistance and PD-L1 in the residual tumor cells and may synergistically enhance RT-mediated reactivation of CD8 T immunological activity to eradicate HCC cells. We found that combined RT, sorafenib, and PBMCs significantly suppress the colony formation in the HCC cells, whereas CD8 T cells expressed high granzyme B (GZMB) and perforin (PRF1) in co-cultured with RT-treated HCC cells. We demonstrated RT significantly inhibited HCC cell viability but induced IFNα and IL-6 expression in the RT-treated HCC cells, resulting in immune checkpoint PD-L1 and anti-apoptosis MCL1 and BCL2 overexpression in the non-RT HCC cells. We found that sorafenib decreased RT-PLC5 medium (RT-PLC5-m)-mediated cell growth by suppressing IFNα- and IL-6-mediated STAT1 and STAT3 phosphorylation. Sorafenib also reduced IFNα-mediated PD-L1 levels in HCC cells. Meanwhile, RT-PLC5-m reactivated CD8 T cells and non-CD8 PBMCs, resulting in high IFNγ and IL-2 levels in CD8 T cells, and cytokines IFNα, IFNγ, IL-2, and IL-6 in non-CD8 PBMCs. Particularly, CD8 T cells expressed higher GZMB and PRF1 and non-CD8 PBMCs expressed higher IFNα, IFNγ, IL-2, IL-6, CXCL9, and CXCL10 in co-cultured with RT-treated HCC cells compared to parental cells. Although we demonstrated that sorafenib slightly inhibited RT-mediated GZMB and PRF1 expression in CD8 T cells, and cytokines levels in non-CD8 PBMCs. Based on sorafenib significantly suppressed IFNα- and IL-6-mediated radioresistance and PD-L1 expression, we demonstrated that sorafenib synergized RT and immune surveillance for suppressing PLC5 cell viability in vitro. In conclusion, this study revealed that RT induced IFNα and IL-6 expression to phosphorylate STAT1 and STAT3 by autocrine and paracrine effect, leading to radioresistance and PD-L1 overexpression in HCC cells. Sorafenib not only suppressed IFNα- and IL-6-mediated PLC5 cell growth but also inhibited IFNα-mediated PD-L1 expression, synergistically enhancing RT-mediated CD8 T cell reactivation against HCC cells.
放疗（RT）用于临床根除肿瘤。然而，肝癌（HCC）对 RT 具有抗性。已经证明 RT 直接抑制肿瘤生长，但诱导 I 型干扰素（IFNs）表达，在存活的肿瘤细胞中磷酸化 STATs 并增加 STATs 下游 PD-L1 水平。由于索拉非尼能够抑制 STATs，因此，我们假设索拉非尼抑制残留肿瘤细胞中 IFN 介导的放射抗性和 PD-L1，并可能协同增强 RT 介导的 CD8 T 免疫活性的再激活以根除 HCC 细胞。我们发现，联合 RT、索拉非尼和 PBMCs 可显著抑制 HCC 细胞的集落形成，而在与 RT 处理的 HCC 细胞共培养时，CD8 T 细胞表达高颗粒酶 B（GZMB）和穿孔素（PRF1）。我们证明 RT 显著抑制 HCC 细胞活力，但诱导 RT 处理的 HCC 细胞中 IFNα 和 IL-6 表达，导致非 RT HCC 细胞中免疫检查点 PD-L1 和抗凋亡 MCL1 和 BCL2 过表达。我们发现，索拉非尼通过抑制 IFNα 和 IL-6 介导的 STAT1 和 STAT3 磷酸化，降低了 RT-PLC5 培养基（RT-PLC5-m）介导的细胞生长。索拉非尼还降低了 HCC 细胞中 IFNα 介导的 PD-L1 水平。同时，RT-PLC5-m 重新激活 CD8 T 细胞和非 CD8 PBMCs，导致 CD8 T 细胞中 IFNγ 和 IL-2 水平升高，非 CD8 PBMCs 中 IFNα、IFNγ、IL-2 和 IL-6 水平升高。特别是，与亲本细胞相比，在与 RT 处理的 HCC 细胞共培养时，CD8 T 细胞表达更高水平的 GZMB 和 PRF1，而非 CD8 PBMCs 表达更高水平的 IFNα、IFNγ、IL-2、IL-6、CXCL9 和 CXCL10。尽管我们证明索拉非尼略微抑制了 RT 介导的 CD8 T 细胞中 GZMB 和 PRF1 表达以及非 CD8 PBMCs 中的细胞因子水平，但我们证明索拉非尼通过协同 RT 和免疫监视抑制 PLC5 细胞活力。总之，本研究表明，RT 通过自分泌和旁分泌作用诱导 IFNα 和 IL-6 表达，导致 HCC 细胞发生放射抗性和 PD-L1 过表达。索拉非尼不仅抑制 IFNα 和 IL-6 介导的 PLC5 细胞生长，还抑制 IFNα 介导的 PD-L1 表达，协同增强 RT 介导的 CD8 T 细胞对 HCC 细胞的再激活。
