| 摘要: | 肺癌是目前全球包含台灣在內的癌症致死率排名第一位的癌症。由於肺癌本身致死率高，加上肺癌病患因生活品質下降，而使病患生命週期益加縮短。在近代醫學研究中，慢性發炎狀態易造成器官組織硬化，進而增加罹患惡性腫瘤的危險性，如肝癌、子宮頸癌、鼻咽癌等。肺癌的演化也被認為和肺部纖維化息息相關。已知肺部纖維化導因於呼吸系統長期處於發炎所導致，如間質性肺炎或是慢性肺氣腫。臨床的觀察結果顯示，肺纖維化的病人肺癌發生率較高，且癌症預後亦較差,所以肺纖維化是肺腺癌不良預後因素之一。就細胞層面剖析，纖維化的肺實質會營造出一個較硬的細胞基質(extracellular matrix)環境，而這個較硬的環境會改變細胞的”生物物理效應”，是否因此而使細胞癌化，或是改變癌細胞的特性，包括生長速率、轉移能力、抑或是改變抗藥性，目前相關研究很少，分子機轉仍未知。在肺腺癌研究中，表皮生長因子受器(EGFR)及肝細胞生長因子受體(c-Met) 兩受體的活性與訊息傳導，與癌細胞生長及轉移能力有關，而組合蛋白(integrin) 則是細胞和基質之間的橋樑分子，被視為是細胞上接受基質訊息的受體，因此研究基質硬度(matrix rigidity)與肺癌的生物物理效應與EGFR、c-MET及integrin之間的關係於肺癌領域至為重要。
根據研究，生理性肺實質硬度為0.15-0.2 kPa，而纖維化的肺實質硬度約為7-25 kPa；然而，實驗室中的癌症研究 (in vitro cancer research)常將癌細胞培養於一般塑膠製或玻璃製的細胞培養皿(plastic or glass culture flask)，用以模擬及研究癌細胞的特性及分子機轉。塑膠製的細胞培養皿硬度約為106 kPa，已是生理性肺實質硬度的5 x 106倍；而玻璃製的細胞培養皿硬度更高達8 x 107 kPa，是生理性肺實質硬度的4 x 107倍。在如此超生理(Supra-physiological)的環境中實難解答纖維化的肺實質與肺癌細胞之間的生物物理效應。且近來研究顯示，不同癌細胞對基質硬度的敏感度不同，不同基質硬度亦會影響癌細胞的生長能力，但機轉尚不明瞭。
本研究目的在於探討纖維化的肺實質硬度與肺癌細胞生長、轉移及抗藥性的影響，從生物物理效應影響分子機轉的角度檢視肺癌細胞與基質間相互依存之關係。本研究將以不同基質硬度的細胞培養皿 (0.2, 2, 25 kPa) 培養肺腺癌細胞(A549)，研究肺實質硬度在生理性(0.2 kPa)、肺纖維化初期(2 kPa) 及嚴重肺纖維化 (25 kPa)時基質硬度對肺腺癌細胞分裂生長及轉移能力對影響，進一步以敏感度之差別找出關鍵受體(mechanoreceptor)及物理訊息傳導(mechanotransduction)，並探討基質硬度對抗藥性的影響。此外，在臨床肺腺癌組織中驗證肺纖維化對肺腺癌影響的活體機轉。
本研究發現基質硬度增加會經由增加β1組合蛋白(β1 integrin)及激活局部粘著斑激酶(Focal Adhesion Kinase, FAK)而將基質硬度變化的物理訊息傳入肺腺癌細胞(A549)內，因而促進A549細胞的增殖和增加表皮生長因子受體（EGFR），肝細胞生長因子受體（c-Met），和轉移因子(Snail)的表現量。 A549細胞生長在比生理性肺實質硬的基質上，會改變細胞的型態，朝向上皮間質轉化(epithelial-mesenchymal transition, EMT)，而誘發上皮間質轉化與肝細胞生長因子受體訊息之活化有關。此外，纖維化的肺實質硬度使A549細胞對EGFR抑製劑（Erlotinib）和c-Met抑製劑(PHA-66575)更具耐藥性;然而，只有高濃度的PHA-665752可逆轉基質硬度導致的形態上的多形性。另外，從病患身上取得的肺腺癌肺組織研究結果顯示，臨床以肺部電腦斷層判定肺纖維化的方式為時已晚，在人類肺癌組織中，間質中第1型膠原蛋白(type I collagen)的表現量，相較於α-平滑肌抗體(SMA)的表達，與局部肺組織纖維化的程度更加一致，且早於肺部電腦斷層中肺纖維化的變化。在較硬化基質之肺癌組織中，表現肝細胞生長因子受體和Snail的癌細胞，群聚在第1型膠原蛋白過量表現的肺實質內，而表現表皮生長因子受體的肺癌細胞與Snail或第1型膠原蛋白之組織表現較無群聚的現象。
類似的研究尚未在文獻中被探討。本研究成果將可應用在臨床醫師面對肺癌病患合併肺實質纖維化時，以生物物理效應的角度，提供一種新的治療策略，以達到改善病患預後的目標。
Lung cancer is the leading cause of cancer mortality in the world, including Taiwan. In addition to disease-induced mortality, lung cancer leads to a low life quality shorten the patient life cycle. In modern medical research, chronic inflammation in an organ or tissue results in tissue fibrosis, and which indicates increase in the cancer risk, such as liver tumor, cervical cancer, and nasopharyngeal cancer. Lung cancer evolution is considered to pulmonary fibrosis related. It is well known that long-term inflammation of respiratory system, such as interstitial pneumonia or chronic emphysema, contributes the pulmonary fibrosis.
The clinical observation shows that a patient with pulmonary fibrosis gets a high incidence of lung cancer and a poor prognosis. Pulmonary fibrosis serves as a poor prognostic factor for pulmonary adenocarcinoma. In a cell level, fibrotic lung parenchyma creates a rigid extracellular matrix, and such a hard environment may guide a "biophysical reaction" in a cell. The relationship between matrix rigidity and cancer formation as well as change from cancer behavior, including proliferation, metastasis, and drug resistance, is not clear so far. In lung cancer studies, epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor(c-Met) governed signaling transduction involve in cancer cell growth and cancer metastasis. The integrin, the bridge protein between cells and matrix molecules, is the mechanoreceptor receiving signals from the matrix. Therefore, study of matrix rigidity (stiffness) and biophysical effects on EGFR, c-MET and integrin are important in lung cancer research.
According to the previous studies, the physiological stiffness of lung parenchyma is 0.15-0.2 kPa, and the stiffness of fibrotic lung parenchyma fibrosis is about 7-25 kPa; however, the cancer research in laboratory(in vitro cancer research)usually cultured cancer cells in the plastic or glass culture flask/dishes, to mimic the characteristics of the cancer cells and to investigate their molecular mechanism. The stiffness of plastic cell culture dish is around 106 kPa, which is 5 x 106 folds harder than that of physiological lung parenchyma; and the stiffness of glass dish is 8 x 107 kPa, equal to 4 x 107 folds stiffness of physiology lung. In such a super-physiological environment, it is difficult to answer the real biophysical response between fibrotic lung parenchyma and lung cancer cells. Recent studies show that cancer cells present a variant sensitivities to matrix stiffness depends on the cancer cell type, and change of matrix stiffness affects a non-linear growth of cancer cells. The underlying mechanism is unknown.
The purpose of this study is to explore the growth ability, metastasis and drug resistance of lung cancer cells during lung fibrosis. The biophysical reaction, i.e., the molecular mechanism triggered by matrix rigidity, in lung cancer cells is investigated. We culture the lung adenocarcinoma cancer cells (A549 cells) on the dish with different matrix rigidity (0.2, 2, 25 kPa). 0.2 kPa serves as the normal matrix rigidity of lung parenchyma, 2 kPa mimics mild pulmonary fibrosis and 25 kPa is severe pulmonary fibrosis. We ask whether the matrix rigidity affects the ability of tumor growth, metastasis and drug resistance, and to identify the mechanoreceptor and relevant mechanotransduction. In addition, lung tissue samples are studied to confirm the in vivo effect of lung fibrosis on adenocarcinoma.
In this study, we discover that increased matrix rigidity triggers the expression of β1 integrin and activates focal adhesion kinase in A549 cells, which is the mechanoreceptor sensing the change of matrix rigidity and delivering the signals into the cells. It results in promotion of cell growth and upregulation of the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and Snail expressions in A549 cells. The morphology of A549 cells altered toward to epithelial-mesenchymal transition (EMT) change when grow on the matrix with fibrotic rigidity, and the EMT change is related to c-Met upregulation. A549 cells become more resistant to the EGFR inhibitor (Erlotinib) and c-Met inhibitor (PHA-665752) when increase in surrounded matrix rigidity; however, a high concentration of PHA-665752 reversed the rigidity-induced morphological pleomorphism. Clinically, lung CT scan is used for diagnosis of lung fibrosis. However, the sensitivity of fibrotic change in image study is far behind the sensitivity to detect the local fibrosis in tissue section. In human lung tissue, expression of type I collagen is more consistent with clinical fibrosis than the expression of alpha-smooth muscle antibody is. c-Met- and Snail-expressing tumor cells, rather than EGFR-expressing cells, are localized with lung parenchyma rich in type I collagen.
As we know, there is no study has been explored in this topic. Based on the biophysical viewpoints, the results of research will be potentially applied in the treatment of lung cancer patients with fibrotic pulmonary parenchyma. We provide the clinicians a new therapeutic strategy and policy to reach the goal of prolong lung cancer patient survival. |
