牙齦卟啉單胞菌的外膜囊泡透過牙齦蛋白酶加劇頭頸鱗狀細胞癌進展之研究

Abstract

頭頸癌是全球前十大常見癌症，並且有90%是位在口腔的鱗狀細胞癌（oral squamous cell carcinomas, OSCC），而轉移仍是導致患者死亡的重要原因。牙周病關鍵致病菌——牙齦卟啉單胞菌（Porphyromonas gingivalis, Pg）主要透過分泌奈米大小且富含磷脂質的外膜囊泡（outer membrane vesicles, OMVs），同時攜帶毒性因子牙齦蛋白酶（gingipains）來作為致病環境中，宿主與微生物之間跨界交流的關鍵參與者。因此目標在於探討富含gingipains的Pg OMVs是否為惡化頭頸癌轉移的因素。 使用即時定量聚合酶連鎖反應分析發現，Pg 16s-rRNA在口腔鱗狀細胞癌患者手術檢體的腫瘤部位略比腫瘤鄰近正常部位表現量高。在免疫組織化學染色中，中度惡性口腔鱗狀細胞癌患者組織切片比惡性患者gingipains表現強，且惡性患者淋巴轉移處有一定強度之gingipains表現。透過穿透式電子顯微鏡、奈米粒子追蹤儀分析，從口腔鱗狀細胞癌患者手術檢體中分離出約100 nm左右的奈米粒子後，利用蛋白水解酶活性試劑，發現晚期患者手術檢體分離之奈米粒子的精氨酸牙齦蛋白酶（arginine-gingipain）水解活性顯著較高（P < 0.05）。在細胞實驗，Pg OMVs顯著提升癌細胞遷移與侵襲的能力（P < 0.01），並使用抑制劑確認了遷移與侵襲的能力受gingipains調控（P < 0.01）。此外，動物實驗中，在給予含Pg OMVs的組別，發現較多的癌細胞發生頸部淋巴轉移。 在本研究中，能夠成功從頭頸癌患者手術檢體分離出含有Pg OMVs的奈米粒子，並且發現其中的蛋白水解酶活性與腫瘤的惡性程度有關。口腔微生物群外膜囊泡之於頭頸癌的關聯，在本研究掀起一頁序幕，而藉這冰山一角，或許在未來可以發展疾病預後預測的機制。而發現富含gingipains的Pg OMVs在頭頸癌轉移所扮演的角色，可以發展相關抑制藥物，作為治療後預防轉移的潛在目標。

Head and neck cancer ranks among the top ten most common cancers worldwide, with 90% being oral squamous cell carcinomas (OSCC). Metastasis remains a leading cause of death in these patients. The key periodontal pathogen, Porphyromonas gingivalis (Pg), primarily exerts its pathogenic effects through the secretion of phospholipid-rich outer membrane vesicles (OMVs) that carry the toxic factor gingipains. These OMVs act as crucial mediators of interkingdom communication between host and microorganisms in the pathogenic environment. This study aims to investigate whether gingipain-rich Pg OMVs contribute to the aggravation of head and neck cancer metastasis. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that Pg 16s-rRNA levels were slightly higher in tumor tissues than adjacent normal tissues in surgical specimens from OSCC patients. Immunohistochemical staining showed that gingipains expression was stronger in advanced OSCC tumors than mild-moderate tumors, with notable gingipains expression in the lymph nodes of patients with lymphatic metastasis. Meanwhile, we isolated approximately 100 nm nanoparticles from OSCC surgical specimens, which were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteolytic activity assays showed significantly higher arginine-gingipain activity in nanoparticles from advanced-stage patient specimens (P < 0.05). Pg OMVs significantly enhanced the migration and invasion abilities of cancer cells in vitro (P < 0.01). Moreover, these abilities were regulated by gingipains via using inhibitors (P < 0.01). Likewise, the group administered with Pg OMVs showed increased neck lymph node metastasis in vivo. In conclusion, this study successfully isolated Pg OMVs-containing nanoparticles from head and neck cancer surgical specimens and found that their proteolytic activity is associated with tumor malignancy. This finding opens a new chapter in understanding the relationship between oral microbiome OMVs and head and neck cancer. This initial insight might lead to future developments in disease prognosis prediction. On the other hand, identifying the role of gingipains-enriched Pg OMVs in head and neck cancer metastasis could help develop targeted inhibitors as potential therapeutic options to prevent poor prognosis post-treatment.