[Ferroptosis in laryngeal squamous cell carcinoma and its regulation by M2 macrophage-derived exosomes].

Objective: To investigate ferroptosis in laryngeal squamous cell carcinoma (LSCC) and its regulation by M2 macrophage-derived exosomes. Methods: LSCC and adjacent noncancerous tissue samples were collected from 32 patients treated in the Department of Otorhinolaryngology, Head and Neck Surgery of the Second Affiliated Hospital of Harbin between September 2018 and April 2021, including 26 males and 6 females, aged 43-79 years. The expressions of ferroptosis marker glutathione peroxidase 4(GPX4) in LSCC and adjacent noncancerous tissues were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction(RT-PCR). The correlations between GPX4 expression and clinicopathological factors in LSCC were analyzed. Biological changes of TU212 cells after treated with ferroptosis-induced agent erastin were detected by transmission electron microscope, cell counting kit-8(CCK-8), clone test, reactive oxygen species(ROS), malondialdehyde(MDA), glutathione(GSH), JC-1, RT-PCR and western blot. Exosomes were isolated from the supernatant of M0/M2 macrophages (M0-exos/M2-exos) and co-incubated with erastin-treated TU212 cells to detect the change of ferroptosis in cells of each group. The data were analyzed by SPSS software of version19.0. Results: GPX4 expression in LSCC tissues was significantly higher than that in adjacent noncancerous tissues (2.04±0.65 vs. 0.99±0.09, F=30.36, P<0.001), and was closely related to T stage and clinical stage (Ⅰ-Ⅱvs.Ⅲ-Ⅳ: 1.75±0.39 vs. 2.18±0.71, F=2.25, P<0.05; T1-2 vs. T3-4: 1.71±0.42 vs. 2.20±0.69, F=2.06, P<0.05). In TU212 cells treated with erastin, mitochondrial crest became smaller, membrane density increased, proliferation rate decreased, intracellular ROS level increased, mitochondrial membrane potential depolarized, GSH content decreased, intracellular MDA level increased and expressions of GPX4 mRNA and protein decreased. Change of M0 into M2 macrophages was induced by IL-4 stimulation. When erastin-treated TU212 cells were incubated with M2-exos, cell proliferation was partially restored and GPX4 expression was enhanced, and also with the recoveries of levels of ROS, MDA and GSH (all P<0.05). Conclusions: Ferroptosis is one of the cell death ways of LSCC. M2-exos may inhibit ferroptosis of LSCC cells.目的： 验证喉鳞状细胞癌（简称鳞癌）铁死亡的变化以及探讨M2巨噬细胞来源的外泌体对喉癌铁死亡的调控作用。 方法： 收集哈尔滨医科大学附属第二医院耳鼻咽喉头颈外科2018年9月至2021年4月诊治的32例喉鳞癌和邻近非癌组织标本，其中男性26例，女性6例，年龄43~79岁。通过免疫组化和反转录聚合酶链反应（RT-PCR）检测组织铁死亡标志物谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）的表达并分析其与临床病理因素的关系。通过透射电镜、细胞增殖-毒性检测试剂盒（cell counting kit-8，CCK-8）、克隆试验、活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathione，GSH）、线粒体膜电位、RT-PCR和蛋白免疫印迹法（WB），检测铁死亡诱导剂erastin处理后喉鳞癌TU212细胞的变化。分离M0/M2巨噬细胞上清液中的外泌体（M0-exos/M2-exos），并与经过erastin处理的TU212细胞共同孵育，检测各组铁死亡变化情况。数据采用SPSS 19.0软件统计分析结果。 结果： 铁死亡标志物GPX4在喉鳞癌组织中的表达水平明显高于邻近非癌组织（2.04±0.65比0.99±0.09，F=30.36，P<0.001），且与临床分期及T分期密切相关（Ⅰ-Ⅱ期比Ⅲ-Ⅳ期：1.75±0.39比2.18±0.71，F=2.25；T1-2期比T3-4期：1.71±0.42比2.20±0.69，F=2.06，P值均<0.05）。TU212细胞经erastin处理后，出现铁死亡的改变，表现为细胞线粒体嵴变小、膜密度增大、增殖率降低、细胞内ROS和MDA水平升高、GSH含量下降、线粒体膜电位发生去极化，细胞内GPX4的mRNA和蛋白的表达降低。在白介素4（IL-4）诱导下，M0巨噬细胞极化为M2型，分离M2巨噬细胞上清液，获得外泌体（M2-exos），发现M2-exos能够诱导erastin处理的TU212细胞高表达GPX4和GSH，下调ROS和MDA的表达水平，逆转细胞的增殖能力（P值均<0.05）。 结论： 铁死亡是喉鳞癌细胞死亡的方式之一，M2巨噬细胞源性外泌体能够抑制喉鳞癌铁死亡的发生。.