利用氧化鉭人造微環境探討腫瘤微環境之組成及其調控

Abstract

腫瘤微環境(tumor microenvironment, TME)具有高度複雜性，其中由致瘤性、非致瘤性、異質性的細胞群體與構成細胞外基質的周圍蛋白所組成。腫瘤微環境與細胞之相互作用與控制癌症的生長、侵犯(invasion)、轉移(migration)、對化療有抗性等具有相關性。然而，鑑於腫瘤微環境的多樣性，其觸發癌症發病或調節癌細胞行為的確切作用仍是未知的。因此在這項研究中採用一種體外的方法來突顯奈米尺寸之腫瘤微環境與周圍細胞的生長、行為和功能。

首先，我們開發一種重新定位藥物之平台，用來闡述M2-巨噬細胞(macrophages)於各種非小細胞肺癌(Non-Small-Cell-Lung-Cancer, NSCLC)之作用。我們將單核球(monocytes)分化成巨噬細胞，進而研究非小細胞肺癌對M2-巨噬細胞的培養基之遷移與侵犯的能力。接著使用次世代定序(Next Generation Sequencing, NGS)來研究非小細胞肺癌響應於M2-巨噬細胞的培養基之特異基因的數量與程度，同時並行使用Connectivity map (C-map)篩選具有正調控或負調控基因之藥物。透過此研究，我們不僅證明非癌細胞可以調控癌細胞之行為，還能突顯不同藥物在限制轉移中的療效。

其二，我們設計具有奈米結構的人造微環境，其構成氧化鉭奈米點(Tantalum oxide nanodots)，奈米點的直徑範圍為10至200nm，此微環境可用來研究奈米形貌對細胞生理之調節。我們種植心肌細胞(H9c2 cells)於不同的奈米點微陣列，並研究奈米點的直徑與分泌一氧化氮(nitric oxide, NO)之調節作用。我們不僅發現奈米點可調節一氧化氮，還可負責控制一氧化氮分泌之遺傳途徑。此外，我們還發現調節一氧化氮分泌的是eNOS。因此透過此研究，我們可利用奈米形貌來控制細胞生理並可提出對於心臟植入物之理想設計。

其三，我們研究奈米點直徑、高度、點與點之間距等不同奈米點參數對細胞行為的影響。我們設計了兩個尺寸，其一為奈米點50nm直徑、20nm間距、40nm高度;其二為奈米點100nm直徑、70nm間距、100nm高度。我們種植成骨細胞(MG-63 cells)於奈米點上一段時間。我們發現具有不同直徑與間距的奈米點可調節細胞的型態、面積、存活率，此外還顯示奈米表面尺寸與細胞外基質蛋白之間有相互作用的可能性。我們觀察到由於更大的高度，蛋白質未能與100nm直徑、70nm間距的奈米點有所接觸。因此我們得到的結論為較大的奈米點高度由於無法建立具有奈米表面的局部粘附(focal adhesion)，導致細胞在100nm直徑、70nm間距的奈米點處發生細胞凋亡。另外我們還顯示奈米點50nm直徑，20nm間距可引起骨形成蛋白有較佳的釋放。因此透過此研究，我們了解不同奈米點尺寸對於細胞行為之影響，並可提出理想成骨之植入物設計。

其四，我們研究奈米形貌與體內參數(如剪切應力)之相互作用。我們在奈米點50nm 直徑與100nm 直徑上種植成骨細胞(MG-63 cells)，並將其施於0.5至2達因的剪切應力，該剪切應力值與細胞由於血液流動所致的力相當。首先我們證實奈米點可調節細胞型態，並研究奈米點直徑與固定剪切應力(2達因)在不同整合素表達之調節，這些整合素與細胞外基質之結合起關鍵作用。我們觀察到在奈米點100nm直徑上的整合素表達比在奈米點50nm直徑上大許多倍。進而我們將奈米點50和100nm直徑上的細胞進行各種剪切應力，並固定時間以研究細胞之密度。我們發現奈米點100nm直徑具有較佳的整合素與細胞密度之誘導。

其五，根據先前之研究結果，我們提出氧化鉭奈米點之微陣列平台設計，可用於開發一種無標誌物以達到監測卵巢癌之方法。可根據不同來源或階段的卵巢癌樣品並研究此微陣列對生存力、局部黏附、微絲束(microfilament bundle)與細胞面積之探討。我們的研究結果有兩個主要的發現，其一為奈米點用於調節細胞之特徵有明顯之趨勢，這意味可得知癌症的起源並可描述其階段；其二為此人造微環境能夠調節癌細胞之行為。

其六，我們顯示該微陣列平台可透過EMT過程觸發上皮細胞之轉移。我們在這些人造微環境中種植兩種不同來源的細胞，並研究了上皮(epithelial)與間質(mesenchymal)關鍵基因之調控。然而我們觀察到奈米點100nm和200nm直徑對間質基因具有正調控，其奈米點10nm和50nm直徑保留了上皮原本之性質。此外，我們也響應這些人造微環境對於癌細胞型態與特異基因之調節，且突顯腫瘤微環境給予良性癌細胞轉移性質之作用。因此我們開發一個可觸發癌細胞轉移之平台，並應用於藥物檢測與癌症研究之領域。

最後，其設計核心為觀察細胞型態之趨勢，因此我們顯示此奈米結構與奈米點如何結合並應用於心肌植入物或作為無標誌物之卵巢癌監測工具，甚至可探討癌症之轉移.

The tumor microenvironment (TME) displays a highly complex scenario, consisting of tumorigenic, non-tumorigenic components, heterogeneous population of cells, and the surrounding proteins making up the Extracellular matrix. The cellular interactions with other TME components controls the cancer growth, invasion, migration and resistance to Chemotherapy. However, given the diversity of TME, the precise role of its components in triggering the cancer onset or modulating the cancer cell behavior is unknown. In this study we have adopted an in-vitro approach to highlight the spatial and temporal control of cancer cell growth, behavior and function by not only their neighboring cells but also by the nano-sized components of TME.

First, we developed a drug repositioning platform to elucidate the role of M2-macroplages on a variety of Non-Small-Cell-Lung-Cancer (NSCLC) cells. We biochemically differentiated monocytes into macrophages and then studied the migratory and invasive abilities of NSCLC cells in response to M2-macrophage conditioned medium. We used Next Generation Sequencing (NGS), to study the number and extent of NSCLC cells genes in response to the M2-conditoned medium and then used The Connectivity map (C-map) to isolate the drugs which have had a history in up or downregulating the genes studied through NGS. Through this study, we not only proved that non-cancerous cells such as macrophages can modulate the cancer cell behavior but also highlighted the efficacy of different drugs in limiting the metastasis.

Second, we engineered nanostructured artificial microenvironments comprising of Tantalum oxide nanodots, ranging in size from 10 to 200nm in diameter to study the modulation of cellular physiology by the nanotopography. We seeded cardiomyocytes (H9c2 cells) on different nanodot arrays and studied the modulation of nitric oxide (NO) secretion as a function of nanodot diameter. We found not only that nanodots regulated the NO secretion profile but also the genetic pathway responsible for controlling the NO secretion. Besides, we also elucidated that the key gene regulating the No secretion was eNOS. Through this study we showed the control of cellular physiology by the nanotopography and also proposed an ideal design of cardiac implants.

Third, we studied the effect of different nanodot parameters such as nanodot diameter, height and inter-dot spacing on the cell behavior. We engineered nanodots of 50 and 100nm having a spacing of 20 and 70nm, respectively and seeded MG-63 cells (Osteoblasts) for a fixed period of time. We found that nanodots with a different diameter and inter-dot spacing modulated the cell morphology, area and viability. Besides, we also showed the possible nature of interactions between nanosurface dimensions and ECM proteins. We observed that proteins failed to come in contact with all dimensions of 100nm diameter nanodots due to a greater height. We concluded that cells suffered apoptosis on nanodots of 100 diameter and 70nm spacing due to failure to establish focal adhesions with the nanosurface owing to a greater nanodot height. We also showed that nanodots of 50nm diameter and 20nm height triggered a higher release of bone-forming proteins. Having understood the effect of different nanodot dimensions on cell behavior, we proposed the design of ideal orthopedic implants.

Fourth, we studied the role of nanotopography in conjugation with in-vivo parameter such as shear stress. We seeded MG-63 cells (osteoblasts) on 50, 100nm nanodots and subjected them to a shear stress from 0.5 to 2 dynes. This value of shear stress is comparable to the force that cells bear in-vivo due to the blood flow. First we confirmed that nanodots modulated the cellular morphology and then studied the the role of nanodot diameter and fixed shear stress (2 dynes) in modulating the expression of different integrins which play a vital role in making the cell-ECM junctions. We observed that the integrin expression on 100nm nanodots was many folds greater than on 50nm nanodots. In the next step, we subjected the cells on 50 and 100nm nanodots to a variety of shear stress values and studied the cell density after a fixed amount of time. We found that overall, 100nm was a preferable choice of nanodot diameter which induced a higher expression of Integrins and cell density.

Fifth, utilizing the results of all of our previous studies, we proposed the design of a platform, comprising of Nanochips of Tantalum oxide nanodots to develop a method for marker-less monitoring of ovarian cancer. We obtained ovarian cancer samples of different origin and in different stages and defined 4 parameters, namely, viability, focal adhesion number, microfilament bundle number and cell area and studied the modulation of these parameters by the Nanochips, comprising of nanodots from 10 to 200nm in diameter. Our results had two major findings, first there was a clear trend in the modulation of cell characteristics by the nanodots, meaning, having known the origin of cancer, the stage can be depicted, and second, these artificial microenvironments were capable of modulating the cancer cell behavior.

Sixth, in the final step, we showed that this platform is capable of triggering metastasis in Epithelial cells through the EMT process. We seeded cells lines of two different origins on these artificial microenvironments and studied the modulation of the key Epithelial and Mesenchymal genes. We observed that nanodots of 100 and 200nm diameter upregulated the mesenchymal genes while those of 10 and 50nm retained the Epithelial nature. In addition, for the first time, we also identified the transition in modulation of cancer cell morphology and genes signatures in response to these artificial microenvironments. This platform also highlights the possible role of TME components in the nano realm in imparting metastatic properties to benign cancer cells. Thus, for the first time, we engineered a platform capable of artificially triggering metastasis in cancer cells, thereby opening its applications in the fields of drug discovery, drug testing and cancer research.

In summary, I have shown how nanostructured tantalum oxide nanodots can have a variety of applications ranging from cardiac to bone implants. I have also shown how Nanochips of Tantalum oxide nanodots can be used as artificial microenvironments to monitor ovarian cancer’s progressiveness, marker-lessly. Finally, for the first time I have shown the importance of identifying the transition step in the modulation of cellular characteristics. In the end, I have achieved the proposed aim of my thesis: to elucidate the role of Nano sized components of TME in modulating the cancer metastasis.