以力學觀點探討正常或癌化上皮細胞間葉化

吳宗憲

doi:10.6844/NCKU.2014.01529

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以力學觀點探討正常或癌化上皮細胞間葉化

The Normal or Cancerous Cell Mechanical Properties Alteration After Epithelial-Mesenchymal Transition

吳宗憲 , Ph.D　　Advisor：葉明龍　　

英文 DOI： 10.6844/NCKU.2014.01529

轉化生長因子；上皮細胞間葉化；細胞力學；腫瘤復發；肺癌轉移； TGF-beta 1 ； EMT ； Cell Mechanics ； Tumor recurrence ； Lung cancer metastasis

Abstract │ Reference (213) │ Altmetrics

Abstract 〈TOP〉

上皮細胞的間葉化 (epithelial-mesenchymal transition, EMT)涉及到幾個眾所皆知的生理以及病理現象，而且能使癌症細胞擁有遷移和侵入的特性。然而，迄今間葉化過程之細胞力學改變仍未得到一致的結論，除此之外，基材軟硬與型態如何影響間葉化後細胞的爬行速度也尚待闡明。近期研究已經開始採用細胞力學區辨間葉化過程以及診斷癌症惡性分期，但在腫瘤環境中間葉化出現在癌症細胞侵入或穿出血管的時間僅僅維持數秒鐘，所以至今仍缺乏利用細胞力學預測腫瘤惡化或轉移的研究。
	因此，本研究目的為二：首先，發展一套微板量測系統 (microplate measurement system, MMS)，此系統利用彈性探針的偏折量去計算細胞的勁度和貼附力，提供奈米牛頓等級的力學解析度去探討間葉化過程的力學變化，同時探討間葉化前後細胞在特殊二維表面形態上的爬行特性改變，可調控的基材特性包含1微米或5微米線寬、溝槽狀或圓錐狀、軟(1.96±0.48 MPa)或硬(3.70±0.74 MPa) 之聚二甲基矽氧烷基材。第二個研究目的是藉由分析腫瘤老鼠身上取出之癌細胞力學特性，預測腫瘤復發以及轉移可能性，可以進一步建立癌症生理以及細胞力學間的關聯性。 
實驗結果顯示在長時間貼附後，間葉化後細胞有勁度高貼附力強的細胞力學特性，是因為具有較粗的肌動蛋白以及豐富的貼附蛋白表現，因此微板量測系統(MMS)能區辨間葉化前後細胞力學的改變，包含：壓縮勁度(500.0和616.6 Pa)、拉伸勁度(469.3和 517.5 Pa)和黏附力(153.2和173.9 nN)，一旦利用肌動(cytochalasin D)或肌凝蛋白阻斷劑(ML-7)將肌動蛋白或肌凝蛋白的合成或活化抑制，則間葉化前後的細胞力學差異將會消失。另外，間葉化之後的細胞因為α-平滑肌動蛋白與貼附蛋白高度共位性表現，所以具有較快的爬行速度，然而在2MPa的基材上間葉化後細胞會因為1微米線寬的椎狀基材效應而減速達0.13微米/分(較溝槽狀慢36%)，但在5微米線寬溝槽基材的加速效應反而使得間葉化後細胞爬行速度快0.18微米/分(增加100%)。
使用微板量測系統以及免疫染色分析顯示在將復發腫瘤中的細胞有非均質性的組成，包含：軟的癌症細胞、硬的間葉化癌症細胞和內皮細胞。同樣的結果也出現在原子力學顯微鏡所作的勁度分布圖，圖中顯示將復發腫瘤具有較高且複雜的勁度分布；進一步利用流式細胞儀能確定腫瘤主要由癌症細胞所構成，體外實驗結果亦發現間葉化後癌症細胞有較高力學特性，進而觀測到將復發腫瘤內有豐富血管新生，因而產生較硬的基質以及豐富的胞泌素導致腫瘤惡化與嚴重侵襲特性產生；從腫瘤萃取出的細胞力學與腫瘤預後因子的多重相關性分析，也發現了老鼠體重成長減緩以及腫瘤重量增加和細胞力學增加以及後續腫瘤復發或轉移有相關性。
	細胞在間葉化後會具有較高的勁度以及貼附力，的確會加強細胞爬行與侵襲能力，因此本系統MMS適合用於癌症診斷、藥物開發和細胞與基材效應之研究，在臨床應用方面，也可以在腫瘤切除後合併使用MMS搭配診斷，進一步預測腫瘤之復發性與轉移性。

Parallel Abstract 〈TOP〉

The epithelial-mesenchymal transition (EMT) is known to involve several physiological and pathological phenomena and endows cells with invasive and migratory properties. However, to identify the alteration of mechanical property of cell undergoing EMT needs further elucidation. Moreover, the effects of substrate stiffness and topography on the migration of cells before or after transforming growth factor-β1 (TGF-β1)-induced EMT are unknown. The cell mechanical properties (CMs) can be used to identify not only cancer EMT, but also the cancer malignant transforming process. Previous in situ study revealed that EMT taken place and affected cancer cells in seconds during intravasation and extravasation. However, there is a scarcity of defined tumor CMs with which to predict which noninvasive tumors will progress to malignancy and which cancers will metastasize. 
	Therefore, there were two purposes in this study. Firstly, this study utilized a microplate measurement system (MMS) approach based on the deflection of a flexible micro-cantilever was built to measure cell stiffness (in Pa) and adhesion force (in nN) of a single cell during EMT with nN resolution. Herein, the migration alteration of EMT cell model were measured on the 2D patterns consisted of 1 μm or 5 μm line-widths and groove or cone patterns on either soft (1.96±0.48 MPa) or stiff (3.70±0.74 MPa) polydimethylsiloxane (PDMS) substrates. Secondly, in bridging across physical and physiological disciplines, this study reconciled single-cell measurements in whether recurrent or not tumors and reveal CMs can be used to predict cancer malignant transformation.
	The results demonstrated that after EMT, mesenchymal cells became stiffer due to thicker and more abundant F-actin and displayed stronger vinculin accumulation after long-term cell-substrate adhesion. The MMS could distinguish differences in compressive stiffness (500.0 and 616.6 Pa), tensile stiffness (469.3 and 517.5 Pa), and adhesion force (153.2 and 173.9 nN) between cells before and after EMT. However, without proper development of the F-actin or myosin II structure following cytochalasin D (cyto-D) or ML-7 inhibition, the mechanical differences were diminished.
	The increased expression of α-smooth muscle actin (α-SMA) with vinculin in focal adhesion (FA) sites led to an acceleration of EMT cell motility. On the 2 MPa substrate, the most influenced substrate was the 1 μm, cone-patterned substrate, where the EMT cells motility decelerated by 0.13 μm/min (36% slower than the cells on groove pattern). However, on the 5 μm, groove-patterned substrate, where the EMT cells demonstrated the most rapid motility relative to the control cells, with a increment of 0.18 μm/min (100%).
	Using MMS combined immunostaining, the discrete results demonstrated the intrinsic heterogeneous composition of Rec tumor: including soft lewis lung cancer cell (LLC), stiff EMT LLC and endothelial cells. Also, using atomic force microscopy (AFM) force mapping, the identified stiffer and more complex composition in Rec tumors were observed. By flowcytometry differentiation, the tumor-retrieval cells were mainly identified as LLC. In vitro experiment showed LLC acquired stiffer properties after EMT as well. Additionally, the rich angiogenesis in Rec tumors offer the stiffer stroma and cytokines for tumor malignant transformation and aggressive invasion ability. A comprehensive effort to correlate the CMs of tumor-retrieval cells to specific tumor-prognosis markers were conducted in this study. Trivially, the decreased body weight gain ratio (BWG) and increased tumor weight (TW) correlated with the stiffer and adhesive CMs of tumors as well as the recurrence and metastasis possibility. 
	After EMT, the cells with increased stiffness and cell-substrate adhesion force were benefited by faster migratiion and higher invasiveness. Thus, this technology has the potential to benefit researches on cancer diagnosis, drug development, and cell-substrate interactions. In terms of clinical application, the local tumor excision followed by MMS analysis offer the predictability of tumor recurrence and metastasis.

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