上皮–間質轉化 (epithelial–mesenchymal transition, EMT) 是一個上皮細胞獲取間質特性的細胞層次生理機轉，常發生於胚胎形成、器官發展、腫瘤侵犯及轉移等過程。歷經完全EMT而獲有間質特徵的癌細胞咸被認為較具幹細胞特性及抗藥性。此外，包括美國頭頸癌基因組圖譜 (TCGA-HNSC) 在內的多類癌症組織中，均可檢測到一種位於腫瘤組織侵襲前緣的獨特病理特徵，稱為partial EMT (p-EMT)。與進行單獨侵犯的EMT 癌細胞相比，集體移動的p-EMT 細胞尚需來自腫瘤微環境中其它成員的協助，如移動時軌道的清除、細胞外基質 (ECM) 重塑、逃避免疫系統攻擊等複雜且動態性之交互作用，方能於腫瘤基質 (stroma) 中做定向集體的移動，完成類似血管淋巴侵犯 (angiolymphatic invasion) 或神經周圍侵犯 (perineural invasion) 等不良預後病理現象。
為了深入探討口腔癌細胞進行侵襲時與微環境中各成員間的分子互動機轉，本論文針對具有 EMT (OC3) 及 p-EMT (TW2.6) 特性的口腔癌細胞株之十一例異種移植組織 (cell line derived xenograft, CDX) 進行轉錄體定序分析，清楚剖繪每一例 CDX 其源自口腔癌細胞及老鼠基質成分之基因表現情形，最後以免疫組織化學染色 (immunohistochemistry) 驗證生物資訊分析結果。我們發現與EMT 組別相比，p-EMT 腫瘤在小鼠體內生長得較快、腫瘤組織中偵測得到較豐富的宿主血管密度、巨噬細胞多聚集於腫瘤邊緣、以及測得較多的透明質酸沈積 (hyaluronan deposition)。此外，發炎性癌症相關成纖維細胞 (iCAF) 的特徵基因多富集於p-EMT 的異種組織基質中; 肌性癌症相關成纖維細胞 (myCAF) 的特徵基因則富集於EMT的組別裏。重要的是，異種組織基質基因的表現多能於人類口腔癌組織單細胞轉錄體數據庫 (scRNA-seq, GSE103322) 中得到驗證，包括肌成纖維細胞 (myofibroblast) 的IL6、ACTA2、MYL9、TAGLN; 肌性癌症相關成纖維細胞的TGFBI、TGFB2、FN1、THY1、COL12A1、FAP、HAS1、HAS2; 發炎性癌症相關成纖維細胞的FAP、CXCL12、GSN; 內皮細胞的 PECAM1、TGFBR2、HYAL1、HYAL2。
為了進一步探討EMT 與 p-EMT CDX 組織中之基質基因表現是否具臨床相關性，我們以一組參數完整的40例台灣口腔癌組織微陣列數據庫，針對20個基質基因表現與病人的總生存率進行Cox 比例風險模型評估。結果顯示其中6個基因在單變量分析中具統計顯著意義，包括TGFBI (HR 11, 95% CI 2.4–50, p=0.0021)、TGFB2、COL12A1、IL6、HAS2 和 HYAL1 (HR 0.15, 95% CI 0.042–0.55, p = 0.0039)。進一步多變量分析則闡明TGFBI及HYAL1的表現於該40例嚼食檳榔相關的台灣口腔癌組織中，分別為獨立的不良和具保護性生物標記。我們以同樣的方式針對美國TCGA 33種癌症組織，評估其基質基因表現對病人總生存率之Cox比例風險模型結果，發現TGFBI對於包括頭頸癌在內的七種癌症都是一個較差的指標 (BLCA, invasive BRCA, CESC, GBM, HNSC, KIRC, UVM)，而HYAL1則是其中四型癌症的良好指標 (KIRC, KIRP, Pheochromocytoma, UVM)。因此我們的結果強烈支持不同癌症類型中應存在共同基質效應的理論。綜合以上所述，本論文除揭示數個台灣口腔癌組織的新靶點，也提供了可行的臨床應用: 例如由術後檢體或復發活檢體 (biopsy) 組織中，進行TGFBI單一基因之免疫組織化學染色，依其結果提供兼顧存活風險及避免過度治療之臨床決策。

Epithelial-mesenchymal transition (EMT) is a physiological process that occurs in embryogenesis, organ development, tumor invasion and metastasis. Tumor cells undergoing sequential EMT processes are thought to associate with cell stemness and drug resistance. In addition, a distinct clinicopathological feature, designated as partial EMT (p-EMT), is prevalently detected in clinical tissues of various cancer types including the head and cancer of The Cancer Genome Atlas (TCGA-HNSC). In contrast to a fully mesenchymal cell that invades alone, p-EMT cells migrate collectively and directionally in the tumor stroma, notably the angiolymphatic and perineural invasions. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion, suggesting that cross talks between tumor cells and tumor microenvironment components are intricate and dynamic.
To gain further insights of the oral cancer ecosystem, transcriptomic analyses of xenograft tissues derived from oral cancer cell lines with EMT (OC3) and p-EMT (TW2.6) properties were used to delineate genes and pathways of human cancer cells and mouse stroma origins, followed by immunohistochemistry assays for validation. Compared to the EMT counterparts, p-EMT tumors grew faster, conferred higher microvascular density, cohabitated with more tumor-excluded macrophages, and displayed stronger hyaluronan deposition. Signature genes characteristic of inflammatory (iCAF) and myofibroblastic (myCAF) cancer-associated fibroblasts were enriched in the p-EMT and EMT xenograft stroma, respectively. Of importance, the expression of stromal genes identified by the aforementioned methodologies can be recapitulated in the scRNA-seq dataset of oral cancer (GSE103322), including myofibroblast (IL6, ACTA2, MYL9, TAGLN), myCAF (TGFBI, TGFB2, FN1, THY1, COL12A1, FAP, HAS1, HAS2), iCAF (FAP, CXCL12, GSN), and endothelial cell (PECAM1, TGFBR2, HYAL1, HYAL2).
To further corroborate the stromal genes with clinical relevance, a univariate Cox proportional hazards model was performed to compute the hazard ratios (HRs) of overall survival associated with each stroma-originating gene in the Taiwanese oral cancer cohort, NCKU-OrCA-40TN. Accordingly, 6 out of 20 selected genes revealed statistical significance, including TGFBI (HR 11, 95% CI 2.4–50, p=0.0021), TGFB2, COL12A1, IL6, HAS2, and HYAL1 (HR 0.15, 95% CI 0.042–0.55, p=0.0039). Multivariable Cox analysis further identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for the 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types (BLCA, invasive BRCA, CESC, GBM, HNSC, KIRC, UVM); HYAL1 was a good indicator for four tumor cohorts (KIRC, KIRP, Pheochromocytoma, UVM), suggesting common stromal effects existing in different cancer types. Thus, our results not only disclose novel targets for oral cancer control, but also provide feasible applications, e.g., a single immunohistochemical assay of TGFBI from treatment naive or recurred tumor biopsies, to assist clinical decision-making.

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