| Summary: | 博士 === 國立交通大學 === 材料科學與工程學系奈米科技碩博士班 === 105 === 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.
|
|---|
