Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Medical Faculty Mannheim of Heidelberg University, D‑68167 Mannheim, Germany.
Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Ulm, D‑89075 Ulm, Germany.
Int J Oncol. 2023 Sep;63(3). doi: 10.3892/ijo.2023.5550. Epub 2023 Jul 28.
Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand‑1 (PD‑L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD‑L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD‑L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM‑SCC‑11B, UM‑SCC‑14C and UM‑SCC‑22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD‑L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un‑)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD‑L1 were detected using western blotting and semi‑quantitative levels were calculated. The functional impact of exosomes from the (un‑)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM‑SCC‑11B and UM‑SCC‑22B showed strong expression of pERK1/2 and PD‑L1, respectively. RT upregulated the PD‑L1 expression in UM‑SCC‑11B and UM‑SCC‑14C and in exosomes from all three cell lines. CT alone induced PD‑L1 expression in all cell lines. CRT induced the expression of PD‑L1 in all HNSCC cell lines and exosomes from UM‑SCC‑14C and UM‑SCC‑22B. The data indicated a potential co‑regulation of PD‑L1 and activated ERK1/2, most evident in UM‑SCC‑14C. Exosomes from irradiated UM‑SCC‑14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell‑mediated regulation of PD‑L1 upon platinum‑based CRT in HNSCC and in exosomes. A co‑regulation of PD‑L1 and MAPK signaling response was hypothesized.
尽管检查点抑制剂 (CPI) 最近扩展了晚期头颈部鳞状细胞癌 (HNSCC) 的治疗选择并提高了临床反应，但治疗效果仍难以预测。程序性死亡配体 1 (PD-L1) 是免疫治疗的关键因素。肿瘤细胞及其衍生的外泌体是 PD-L1 的载体，并能有效抑制免疫反应。本研究旨在分析既定治疗方法对头颈部鳞状细胞癌细胞系及其外泌体中 PD-L1 表达的影响。对头颈部鳞状细胞癌细胞系 UM-SCC-11B、UM-SCC-14C 和 UM-SCC-22C 进行了分次放疗 (RT; 5x2 Gy)、顺铂 (CT) 和西妥昔单抗 (Cetux) 单药治疗、联合治疗、放化疗 (RT 和 CT) 或放免治疗 (RT 和 Cetux)。使用蛋白质印迹法、免疫组织化学法和活组织培养模型评估 PD-L1 表达和作为放射抗性介质的磷酸化 (p)ERK1/2。通过尺寸排阻色谱法从未经处理和经处理的头颈部鳞状细胞癌细胞系的浓缩上清液中分离出外泌体。使用蛋白质印迹法检测外泌体中 PD-L1 的蛋白表达水平，并计算半定量水平。测量并比较未经处理和经处理的头颈部鳞状细胞癌细胞系的外泌体对未经处理的头颈部鳞状细胞癌细胞系增殖 (MTS 测定) 和凋亡 (Caspase 3/7 测定) 的功能影响。HNSCC 细胞系 UM-SCC-11B 和 UM-SCC-22B 分别表现出强烈的 pERK1/2 和 PD-L1 表达。RT 上调了 UM-SCC-11B 和 UM-SCC-14C 中的 PD-L1 表达，并上调了所有三种细胞系的外泌体中的 PD-L1 表达。CT 单独诱导所有细胞系中 PD-L1 的表达。CRT 诱导所有 HNSCC 细胞系和 UM-SCC-14C 和 UM-SCC-22B 外泌体中 PD-L1 的表达。数据表明，PD-L1 和激活的 ERK1/2 之间存在潜在的共同调节，在 UM-SCC-14C 中最为明显。来自辐照的 UM-SCC-14C 细胞的外泌体通过下调 Caspase 3/7 来保护未辐照的细胞免于凋亡。本研究表明，HNSCC 中铂类 CRT 后肿瘤细胞介导的 PD-L1 调节以及外泌体中的 PD-L1 调节。假设 PD-L1 和 MAPK 信号转导反应的共同调节。
