探討微環境酸化對腫瘤進程之影響

Abstract

腫瘤微環境酸化是瓦式效應所帶來的影響，原因是糖解作用提高，乳酸和氫離子產生，腫瘤活化乳酸質子雙向轉運蛋白的活性，維持腫瘤細胞內pH值來適應酸性環境。此外，酸化導致細胞外基質重塑，刺激血管或是淋巴管生成，有利於腫瘤擴散，這些都是轉移的先決條件。轉移是腫瘤從原位腫瘤脫離並進入血管或淋巴管，循環腫瘤細胞在循環系統存活，最後在新環境殖民生長。目前常見轉移動物模組，有自發性轉移模型、實驗性轉移模型、基因工程小鼠轉移模型三種。但腫瘤如何殖民，一直沒有異種移植可以了解，腫瘤殖民模型是需要被建立。另外需要提及的是，酸化造成的影響是特定癌症才有?還是廣泛對各種癌症有影響?因此，我們建立多種長期酸化細胞株，以及可觀察腫瘤殖民的模型，了解其中影響，有機會透過治療阻止腫瘤轉移。在本研究中，我們利用長期肺癌酸化細胞株，成功建立腫瘤殖民模型。轉錄組定序揭露出酸化主要影響細胞外基質路徑。在臨床組織切片中，ITGA4和HAS3在有轉移潛力的原位癌高度表現以及影響肺癌進程。此外，體內以及體外實驗中，酸化增加擬態血管數目，讓腫瘤有效率在新環境中殖民。這些結果指出我們能觀察腫瘤殖民過程，酸化誘導腫瘤重組細胞外基質，增加轉移潛力、提升發生率，並穩定擬態血管能力以及有害代謝物的清除，在新環境中殖民生長。另外，我們也建立長期酸化口腔鱗狀細胞癌來驗證酸化並不是腫瘤特異性。同時，轉錄組定序揭露出酸化在口腔鱗狀細胞癌主要影響細胞和細胞之間訊息路徑。我們也找出SAS和CLS1細胞共同酸化基因 FGF5，在異種移植實驗中，酸化造成FGF5高表現量。酸化誘導 FGF5 在細胞的表達和重新分佈。在臨床組織切片中，FGF5在有轉移潛力的原位癌高度表現並且有差存活率。最後，FGF5表現可能與吸菸危險因子有相關。綜合以上的結果，我們建立的轉移殖民模型，結合酸化研究，能了解腫瘤殖民過程，提供新的腫瘤治療觀點，阻止腫瘤轉移殖民。

The acidosis of the tumor microenvironment is caused by the Warburg effect. The reason is that glycolysis increases, resulting in the production of lactic acid and hydrogen ions. Tumors activate the activity of the lactate proton bidirectional transporter to maintain the pH value in tumor cells and adapt to the acidic environment. In addition, acidosis leads to the remodeling of the extracellular matrix, stimulates the formation of blood vessels or lymphatic vessels, and facilitates the spread of tumors, all of which are necessary for metastasis. Metastasis occurs when a tumor detaches from the primary tumor and invades either blood vessels or lymphatic vessels. Circulating tumor cells survive in the circulatory system and eventually colonize and grow in a new environment. Currently, there are three common animal models for studying metastasis: the spontaneous metastasis model, the experimental metastasis model, and the genetically engineered mouse metastasis model. However, there is a lack of a suitable model to understand how tumors colonize. A novel animal metastatic model should be established to assess the colonization of circulating tumor cells after extravasation. Another thing that needs to be mentioned is, does the impact of acidification only affect specific cancers? Or does it affect a wide range of cancers? Therefore, we have established a variety of long-term acidified cell lines and models that can observe tumor colonization. This allows us to understand the effects and provides an opportunity to prevent tumor metastasis through treatment. In this study, we successfully established a tumor colonization model using long-term acidosis lung cancer cell lines. Long-term acidosis increased the incidence of tumors. RNA-Seq revealed that acidosis induced extracellular matrix (ECM) reorganization. Identify the acidosis-responsive genes ITGA4 and HAS3, which exhibited high expression at both the RNA and protein levels. In clinical tissue sections, ITGA4 and HAS3 were upregulation expression in primary tumors with metastatic potential. In addition, long-term acidosis increases vasculogenic mimicry, which provides nutrition and enables tumors to efficiently colonize new environments. These results indicated that Long-term acidosis reprograms the transcriptome of lung cancer cells, characterized by extensive changes in extracellular matrix (ECM) composition. The upregulation of ITGA4 and HAS3 is associated with the stability of the simulated vascular structure and the elimination of toxic metabolites, thereby facilitating the formation of new metastatic colonies following CTC extravasation. In addition, we also established long-term acidosis oral squamous cell carcinoma (OSCC) cell lines to verify that acidosis is not specific to cancer. Acidosis mainly affects the cell-cell signaling pathway in OSCC, as revealed by RNA-Seq, which was found the common acidosis-responsive gene FGF5 in OSCC and CLS1 cells. In vivo, acidosis increased the high expression of FGF5 in tumors, and it also induced the expression and redistribution of FGF5 in cells. In clinical tissue sections, the presence of primary tumors with metastatic potential and high FGF5 expression was associated with poor survival. Finally, the expression of FGF5 was found to be related with cigarette consumption. Based on the above results, we believe that the establish of metastatic colonization model, combined with acidosis study, can help us to understand the mechanism of tumor colonization and provide new perspectives on tumor treatment.