Kobayashi Miho, Fujiwara Kashio, Takahashi Kazuki, Yoshioka Yusuke, Ochiya Takahiro, Podyma-Inoue Katarzyna A, Watabe Tetsuro
Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.
Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan.
Inflamm Regen. 2022 Sep 4;42(1):38. doi: 10.1186/s41232-022-00225-7.
During metastasis, cancer cells undergo epithelial-mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β), which is abundant in the tumor microenvironment, and acquire invasive and metastatic potentials. Metastasis to distant organs requires intravascular invasion and extravasation of cancer cells, which is accompanied by the disruption of the adhesion between vascular endothelial cells. Cancer cell-derived extracellular vesicles (EVs) have been suggested to induce the destabilization of normal blood vessels at the metastatic sites. However, the roles of EVs secreted from cancer cells that have undergone EMT in the destabilization of blood vessels remain to be elucidated. In the present study, we characterized EVs secreted by oral cancer cells undergoing TGF-β-induced EMT and elucidated their effects on the characteristics of vascular endothelial cells.
Induction of EMT by TGF-β in human oral cancer cells was assessed using quantitative RT-PCR (qRT-PCR) and immunocytochemistry. Oral cancer cell-derived EVs were isolated from the conditioned media of oral cancer cells that were treated with or without TGF-β using ultracentrifugation, and characterized using nanoparticle tracking analysis and immunoblotting. The effects of EVs on human umbilical artery endothelial cells were examined by qRT-PCR, cellular staining, and permeability assay. The significant differences between means were determined using a t-test or one-way analysis of variance with Tukey's multiple comparisons test.
Oral cancer cells underwent EMT in response to TGF-β as revealed by changes in the expression of epithelial and mesenchymal cell markers at both the RNA and protein levels. Oral cancer cells treated with TGF-β showed increased EV production and altered EV composition when compared with untreated cells. The EVs that originated from cells that underwent EMT by TGF-β induced endothelial-mesenchymal transition, which was characterized by the decreased and increased expression of endothelial and mesenchymal cell markers, respectively. EVs derived from oral cancer cells also induced intercellular gap formation which led to the loss of endothelial cell barrier stability.
EVs released from oral cancer cells that underwent TGF-β-induced EMT target endothelial cells to induce vascular destabilization. Detailed characterization of oral cancer-derived EVs and factors responsible for EV-mediated vascular instability will lead to the development of agents targeting metastasis.
在转移过程中，癌细胞会响应肿瘤微环境中丰富的转化生长因子-β（TGF-β）而发生上皮-间质转化（EMT），并获得侵袭和转移潜能。癌细胞转移至远处器官需要其进行血管内侵袭和外渗，这伴随着血管内皮细胞间黏附的破坏。癌细胞衍生的细胞外囊泡（EVs）被认为可诱导转移部位正常血管的不稳定。然而，经历EMT的癌细胞分泌的EVs在血管不稳定中的作用仍有待阐明。在本研究中，我们对经TGF-β诱导发生EMT的口腔癌细胞分泌的EVs进行了表征，并阐明了它们对血管内皮细胞特性的影响。
使用定量逆转录聚合酶链反应（qRT-PCR）和免疫细胞化学评估TGF-β在人口腔癌细胞中诱导EMT的情况。使用超速离心法从经或未经TGF-β处理的口腔癌细胞的条件培养基中分离口腔癌细胞衍生的EVs，并使用纳米颗粒跟踪分析和免疫印迹进行表征。通过qRT-PCR、细胞染色和通透性测定来检测EVs对人脐动脉内皮细胞的影响。使用t检验或带有Tukey多重比较检验的单因素方差分析来确定均值之间的显著差异。
如RNA和蛋白质水平上皮和间质细胞标志物表达的变化所示，口腔癌细胞响应TGF-β发生了EMT。与未处理的细胞相比，用TGF-β处理的口腔癌细胞显示出EV产量增加且EV组成改变。源自经TGF-β诱导发生EMT的细胞的EVs诱导了内皮-间质转化，其特征分别为内皮细胞标志物表达减少和间质细胞标志物表达增加。源自口腔癌细胞的EVs还诱导了细胞间间隙形成，导致内皮细胞屏障稳定性丧失。
经TGF-β诱导发生EMT的口腔癌细胞释放的EVs靶向内皮细胞以诱导血管不稳定。对口腔癌衍生的EVs以及负责EV介导的血管不稳定的因素进行详细表征将有助于开发针对转移的药物。
