利用結構式照明奈米測繪術(SINAP)觀測癌細胞在不同硬度下絲狀偽足的差異

Abstract

本論文利用結構式照明奈米繪測術(SINAP)觀察肺癌細胞在不同基材硬度刺激下絲狀偽足的生長與動態變化。 細胞的移動能力在許多生理反應過程中都扮演著重要的角色，例如組織發育、傷口癒合、腫瘤血管新生、過敏反應以及癌症轉移。之前的研究主要是集中在環境中化學信號對於細胞移動能力的影響，但近年來，有越來越多的研究指出，細胞所生存的微環境會給予細胞許多物理性的刺激，如環境硬度被認為可以影響細胞的移動能力。 以往對於細胞移動能力的觀察，是利用長時間追蹤同一顆細胞，並計算出他在一定時間中可以移動的距離做為此細胞的移動能力，但這種記錄方式需要長時間的累積，在實際醫療的應用上並沒有辦法給予即時的資訊，並且在實體內的實行上也有一定的困難。因此我們希望可以利用細胞外觀的特徵來和細胞的移動能力做連結。在本篇論文中，我們將觀察細胞絲狀偽足的生長及動態和基材硬度的關係，有許多研究已經發現絲狀偽足的生長和細胞的移動能力有正相關，並且我們所使用的細胞株CL1-5相對於其同種類細胞株CL1-0有較高的轉移能力，之前的研究也指出，這種具有高轉移能力的CL1-5其絲狀偽足的數目要比其他低轉移能力的細胞株更多，因此我們相信絲狀偽足的生長及動態會受到基材硬度的影響。 實驗中主要是利用李超煌博士所研發的超解析顯微術-結構式照明奈米繪測術(SINAP)觀察絲狀偽足結構。絲狀偽足的直徑大約只有100-300奈米，超越一般光學顯微鏡的繞射限制。而經由SINAP顯微術所得到的影像，其橫向解析度可以達140奈米，而縱像解析度更可以到達6奈米，加上搭配廣視野顯微鏡，不需要利用掃描方式取得二維影像，大幅加快了取像速度，且不需要事先對樣品進行螢光染色，非常適合用來長時間觀察絲狀偽足的動態變化。 由於SINAP顯微術需搭配正立顯微鏡來進行樣品的成像，因此所觀察之樣品其底下基材必須具有高折射率的特性，然而先前對於基材硬度對細胞影響的實驗所用可調硬度之基材材料大多為水膠，而水膠的折射率和細胞生存環境相近，並沒有辦法在SINAP系統中成像，因此我們實驗的第一步必須要找出一種折射率高，且可以調整其硬度的基材以符合我們實驗的需求。在本實驗中我們所使用的PVC具有高折射率，並可藉由加入塑化劑DINCH來調整其硬度。實驗結果證實PVC可以成功的應用到SINAP系統中，而其硬度符合生理環境硬度之範圍，且其生物相容性和傳統常被使用的生物材料差異不大，因此做為本實驗所選用的基材。 觀察絲狀偽足在不同硬度刺激下生長的實驗結果發現，在軟基材上，細胞具有較多且較長的絲狀偽足；而在硬基材上抑制了肌球蛋白(myosin II)的活性同時抑制張力絲(stress fiber)收縮能力，亦可以發現絲狀偽足在數量及長度上都有明顯的增加，模擬了在軟基材上的效果。因此判斷基材硬度可能是藉由影響張力絲的形成，而張力絲和絲狀偽足的形成機制又互相抗拮，進而影響絲狀偽足的生長。另一方面，我們也觀察絲狀偽足在不同硬度下其伸縮速度。實驗結果發現在不同硬度下所記錄的伸縮速度並無不同，可能和取像時間太長有關，之後必須對此做改善。 藉由此實驗的結果，可以了解到基材硬度對於絲狀偽足數量和長度上的影響，進一步研究其調控機制將對於未來在癌症轉移的治療及診斷上能有所幫助。

In this thesis, we observed the filopodia formation and dynamics of lung cancer cells (CL1-5) on substrates with different stiffness by structured illumination nano-profilometry (SINAP). Cell migration plays a key role in various physiological and pathological processes, such as tissue development, wound healing, angiogenesis, inflammation, and cancer metastasis. Most of the previous studies focused on the effects of environmental chemical cues on the cell migration. Recently, some studies have reported that the cell migration could also respond to the environmental mechanical stimuli; for example, the substrate stiffness is one of the most important mechanical stimuli. The traditional method to measure the cell motility is to trace the moving distance of a cell for an accumulated time. This method however should take a long time thus hard to be exerted in vivo and in real-time. Therefore, a way to improve is to observe the cell morphology and relate it to the cell motility. Evidences have shown that the formation of filopodia is related to the cell migration. Furthermore, the CL1-5, which has been shown to be the high invasive lung cancer cell line, has more filopodia than CL1-0, which is low invasive lung cancer cell line. In this study, we made the correlation between the formation of filopodia and the substrate stiffness. We speculated that the substrate stiffness could modulate the filopodia formation and dynamics through the effects on cell adhesions. The structured illumination nano-profilometry (SINAP), which is developed by Dr. Lee’s group, has lateral resolution of 140 nm and depth resolution of 6 nm. This super-resolution microscopy is advanced in the observation of filopodia, whose diameters are only between 100 and 300 nm. Without fluorescence labeling and two-dimensional scanning, the imaging of SINAP is high speed and very suitable for live-cells observation. However, these techniques require culturing cells on materials of refractive index close to that of glass, while most studies regarding the effects of mechanical cues on cellular dynamics were conducted on hydrogel-based substrates. Here we report the development of culturing substrates of tunable rigidity and refractive index suitable for SINAP studies. Polyvinyl chloride (PVC)-based substrates were mixed with a softener called Di(isononyl) Cyclohexane-1,2-Dicarboxylate (DINCH). The volume ratios of PVC to DINCH were varied from 1:1 to 3:1. The Young’s moduli of the resulting substrates ranged from 20 kPa to 60 kPa. Human lung adenocarcinoma cells CL1-5 were cultured on the composite substrates and cell viability was examined using the MTT assay. The results showed that the PVCs were successfully applied to the SINAP system and had high biocompatibility. Thus in this thesis, the observation of filopodia formation and dynamics were conducted on the PVC substrates. The results of cells on different stiffness showed that the cells on soft substrates had more filopodial density and length than those on the stiff substrates. Inhibiting the contractility of the stress fibers with blebbistatin treatment increased the density and length of filopodia on stiff substrates, and mimicked the situation of the cells on the soft substrates. Therefore, a possible but indirect mechanism of the effects of the substrate stiffness on filopodia formation might be the formation of the stress fibers, which antagonize to the formation of the filopodia. On the other respects, we also measured the stretching rate of filopodia on different stiffness. The results showed no difference in the protrusion rate and retraction rate between the soft and stiff substrates. This might resulted from the interval times we took between images. Thus in the future, we should shorten the interval time to observe the filopodia with higher stretching speed. From this study, we can understand the effects of the substrate stiffness on the filopodial density and length. Further studies are needed to determine the underlying mechanism of these effects. With their medical importance, the results would shed new light on the therapy and diagnosis of the cancer disease.