Manipulation Of Cell Morphologies Through Mechanical Straining Or Micropatterns And Their Effects On Tumor Cell Behaviors

Malignant tumors are one of the biggest risks and threats for human health.Therefore,the development of an effective treatments for tumors is the main development direction in current medical technology.In modern medicine,the development of new treatments was always based on the clear understanding of tumor’s pathological mechanism.In recent years,numerous evidences in clinical research revealed that the occurrence and development of tumors was not only closely related to the patient’s genetic factors,but also strongly correlated to the long-term environmental factors.Therefore,clarifying the relationship between environmental factors and tumor pathological mechanisms is a key scientific issue in current cancer research.Tumor is not simple aggregates of malignant cells,but a complex solid tissue containing immune cells,cytokines,blood vessels,extracellular matrix(ECM)and other structures.The complex structures in these solid tissues were regarded as the tumor microenvironment.With the vigorous development of biomaterials,more and more researches have discovered the critical role of mechanical stimulation from the tumor microenvironment in development of tumors.In addition,previous data has shown that mechanical stimulation from biophysical environment has the ability to regulate cell morphogenesis and affect normal cells’ functions in the human.However,the relationship between cell morphological cues and tumor cell function remains unclear.Therefore,clarifying the mechanism by which cell morphological factors regulate tumor cell function is an urgent issue need to be solved in current research in biophysical factors and tumor cell behavior.Therefore,we simultaneously designed and constructed cyclic straining cell culture devices and micro-patterned materials to provide the cyclic straining and spatial restrictions on cancer cells from the tumor microenvironment and investigate the influence of mechanical stimulations on tumor cell behavior through manipulation of cell behaviors.The main research work of this dissertation is as follows:(1)Firstly,an in vitro cell culture device was designed and prepared by using PTFE and PDMS in this research to provide cyclic straining for tumor cells.In the results,cyclic straining was able to promote proliferation of melanoma cells through accelerate the elongation,spreading and vinculin assembly of melanoma cells.In addition,the regulatory morphogenesis of melanoma cells was also able to active mechanotransduction associated transpiration factor and further affected on the expression of tumor and mechanotransduction-related genes.Additionally,the morphology of melanoma cell morphology changing and response to cyclic straining were disappeared after treatment of cytoskeleton and mechanotransduction-related inhibitors.These results demonstrated the critical role of cellular morphogenesis and mechanotransduction in promotion of melanoma cells through cyclic straining.The results in this part provided theoretical basis in the strong correlation between the primary lesions’ location of acral melanoma and the mechanical stimulated area.(2)A reference-free traction force microscope(TFM)was prepared through photolithography to detect tumor cells’ contractility.Effects of different adhesion proteins on the contractility of osteosarcoma cells by regulating cell morphology was explored through chemical modification of the surface of reference-free TFM.The results showed that osteosarcoma cells had different adhesion behaviors on different protein coated substrates.Compared to poly-L-lysine and collagen type I coated surfaces,lower cell contractility was observed in osteosarcoma cells cultured on oxygen plasma treated or and fibronectin coated PDMS surfaces.In addition,osteosarcoma cells on poly-L-lysine and collagen type I coated surfaces showed more elongated cell morphology and assembled larger focal adhesions.These more elongated cells and larger focal adhesions lead to higher cell contractility and further activated mechanotransduction associated transcription factors.These results suggested that the components of substrates were able to regulate cell contractility and activate the mechanotransduction pathways through manipulation of tumor cell morphology.(3)The significantly difference in cell morphology and growth state between melanoma cells and osteosarcoma cells were observed during daily in vitro 2D experiments.Therefore,mechanism of cellular morphology in regulation of growth status of different tumor cells was explored by precisely control of tumor cell colony’s morphology by using micropatterned surfaces.In the results,melanoma cells were distributed randomly on the cell culture plate with homogeneous cell density.On the contrast,osteosarcoma cells always showed obvious aggregation in areas containing topologically defects.By using micropatterned materials,the spatial restrictions were applied to control morphology of tumor cell colonies and the generation of topological defects were artificially regulated.In the results,spatial heterogeneity was only observed within micropatterned osteosarcoma colonies.The phenomenon observed within micropatterned osteosarcoma cells was similar with the self-organization process in stem cells.Additionally,some cell aligned regions were observed near the edge region of micropatterned osteosarcoma colonies.Osteosarcoma cells were always moved from these aligned regions to the central region and finally formed central cell aggregates.Furthermore,the size of these aligned regions within the micropatterned structure has a clear positive correlation with the formation rate of these central aggregates.These results revealed the critical role of spatial restrictions and morphology of tumor cell colonies in regulation of selforganization and generation of intra-tumor heterogeneity in some specific tumors.Results in this part provided some supplementary information in understanding of the mechanism in spatial restriction and cell morphology regulated tumor development and generation of intra-tumor heterogeneity in vitro.(4)The influence of cell morphology factors on the cellular uptake of nanoparticles was also explored in this research.In particular,micropatterned pdms stencils containing holes with different sizes were prepared and applied to control the size of melanoma cell colonies.In the results,benefited with higher contacting possibility between micropatterned colonies and nanoparticles,more nanoparticles were uptake by the colonies with larger size.In addition,the cells located in the peripheral region of the micropatterned colonies were more likely to endocytosis the nanoparticles.This result was coursed by the concentrated cortical actin in the melanoma cell with circular morphology at peripheral region of micropatterned colonies.These cortical actin proteins are critical for endocytosis process of nanoparticles in tumor cells.These results revealed the important role of morphological cues in regulation of the ability of tumor cells endocytosis and provide new ideas for the development of tumor treatment strategies.(5)A novel approach for construction of 3D micropatterned tumor cell colonies was developed through photolithography.In ordered to make sure the tumor cells were only aggregated within micropatterned structures,the surface of 3D micropatterned templates were modified by PLL-g-PEG or PNIPAM.In the results,melanoma cells were only aggregated inside the micropatterned structure,and their three-dimensional structure was precisely controlled by the micropatterned template with a good cell viability.These results indicated a successful establishment in construction of an in vitro 3D tumor model with a controllable structure.The prepared 3D tumor colonies will provide a novel in vitro model in research of drug screening and exploration of tumorigenesis mechanism.In summary,effects of substrate components,cyclic straining and morphological cues on the cellular contractility,proliferation,self-organization and cellular uptake through preparation and application of reference-free TFM,cyclic straining cell culture device and micropatterned materials.Finally,a novel technique for construction of 3D micropatterned tumor colonies was also developed.The results of this study revealed the critical role of cell morphological cues in regulation of tumor cell behaviors.They will provide some supplementary information in understanding of the role of biophysical factors from tumor microenvironment in regulation of tumor pathological processes.