口腔癌酸化的臨床相關性及其對淋巴轉移的影響

Abstract

口腔癌在本國男性為第四常見之癌症，若轉移到淋巴，其五年存活率極低。根據實驗室先前研究成果得知，口腔癌微環境酸化與轉移密切相關。腫瘤微環境會透過不同因子影響腫瘤轉移，而胞外體作為生物體內訊息傳遞的工具，參與腫瘤和微環境之間的交互作用。然而，在口腔癌中，微環境對其胞外體分泌的影響尚未充分被了解。 本研究主軸是酸化微環境對口腔癌細胞的影響，除了實際搜集臨床口腔癌檢體以建立其即時測量酸鹼值方法外，我們也發現長期酸化之口腔癌細胞株具有大量釋放胞外體的特性，並進一步使用蛋白質體學和轉錄體學分析來研究其內容物。另一方面，我們將口腔癌胞外體添加至淋巴內皮細胞的培養基中進行刺激，使用跨膜電阻儀和免疫螢光細胞染色觀察內皮細胞之間的連結是否受到影響，並使用帶有螢光標記之葡聚醣測試內皮屏障之滲透度。根據實驗結果發現，酸化會造成口腔癌細胞釋放出大於正常酸鹼值的囊泡，且帶有不同蛋白質和 miRNA 內容物，這樣的細胞外囊泡具有破壞淋巴內皮細胞屏障的特性。結合臨床檢體的實際觀察和細胞實驗的證據，有助於更進一步了解酸化微環境所釋放的胞外體對口腔癌細胞轉移的潛力。

Oral cancer ranks as the fourth most common cancer among men in Taiwan. Once it metastasizes to lymph nodes, the five-year survival rate becomes even lower than the tumor in situ. Based on previous research in our laboratory, we found that the acidotic microenvironment correlates to cancer metastasis by establishing the acidotic animal and cell line model. Tumor metastasis is a complex process influenced by various factors, and one of these factors is the in vivo message transmission via extracellular vesicles, which participates in the reciprocal interaction between tumors and their metastatic site. However, the impact of acidosis on oral cancer metastasis remains largely unknown. This study aims to evaluate the effect of oral cancer acidosis and its relationship with extracellular vesicle secretion and metastasis. We collected clinical oral cancer specimens and developed a protocol to obtain reliable pH measurements at the operation site. We isolated extracellular vesicles secreted by our long-term acidosis oral cell line model and found increased secretion and differential population as compared to its neutral pH counterpart. Subsequent proteomic and transcriptomic analyses were employed to analyze the contents of these extracellular vesicles. Also, we stimulated lymphatic endothelial cells with extracellular vesicles, then the integrity of the endothelial cell barrier was tested by transmembrane resistance using trans-epithelial electrical resistance (TEER) and immunofluorescence staining (IF). Furthermore, we used fluorescent-labeled dextran to interrogate the change in endothelial layer permeability after extracellular vesicle treatment. In summary, our results show that acidosis oral cancer cells release more extracellular vesicles, which contain distinct protein and miRNA contents. Moreover, these extracellular vesicles exhibit the capability to disrupt the lymphatic endothelial cell barrier. The amalgamation of our clinical observations, omics analyses, and functional experiments contribute to a more comprehensive understanding on the role of oral cancer acidosis in lymph metastasis in the context of extracellular vesicles.