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Investigation of Morphology and Mechanical Properties of NSCLC Cells Based on Nano-indentation

Author: LiYa
Tutor: DiWenJie
School: Harbin Institute of Technology
Course: Mechanical Design and Theory
Keywords: Nano-indentation NSCLC mechanical properties evaluation method mathematical model cytoskeleton
CLC: R73-3
Type: PhD thesis
Year: 2013
Downloads: 4
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Abstract

Cancer has become an important public health issue in humanity with its highincidence and fatality rate. Cancer could undergo a hidden unlimited proliferation,invasion and metastasis, therefore accurate early diagnosis of cancer is critical forcarrying out treatment in a timely manner. Histopathology, a definitive diagnosismethod of cancer, needs preparation of tissue slices. Not only these producing processare complicated and high costing which make cells loss physiological structure, andmany other factors will affect the correct judgment of the slice, such as the degree oftumor cell differentiation, cell heterogeneity, non-specific staining etc.. These methodsare restricted to the qualitative and semi-quantitative stage. Effective early diagnosis ofcancer has become a problem demanding prompt solution. The current pursuit of goal isquantitative diagnosis and prognosis of cancer, especially.Atomic force microscope (AFM) can detect the ultrastructural surface morphologyof living cells with nanometer level resolution in the liquid phase, it also can obtain themechanical characteristics of cell through appling a weak force on cell, and thus toobtain the cell internal structural changes. AFM has evaluated viscoelasticity of manydifferent kinds of cells including fish corneal cells, tumor cells, stem cells etc. whoseelastic modulus ranged from100Pa to100kPa. Such large difference is not onlyattributed to the difference between the cellular sub-structures, but also can be affectedby the impact of the detection conditions, methods and models, etc.. Based on the needof accurate analysis of non-small cell lung cancer (NSCLC) cell mechanical properties,this article aimed at the establishment of accurate and viable cancer cell mechanicalproperties evaluation method from surface imaging parameters setting, testingconditions, model selection perspective. Analysis of the relationship among themechanical properties, internal cytoskeleton structure and cellular metastatic potentialof NSCLC cells were performed. This is the first comparative analysis of themechanical properties and cytoskeletal fibers of NSCLC cells with different malignancydegree. This research mainly includes the following aspects:A novel method was proposed to optimize the quality of AFM imaging of livingcells and this layed a foundation for the detection of the mechanical properties of livingcells.A quadratic regression orthogonal design was made to simulate a mathematicalmodel for cell surface scanning. The clarity of cell image surface was defined as thedependent variable, while the AFM scanning setpoint, scanning rate and proportionalgain were defined as the independent variable in this mathematical model. Theoretically,the optimal cell image can be obtained at the scan setpoint of1.31V, the integral gain of1.93, and the proportional gain of3.96. The topography images of NSCLC cells with different malignancy degree were taken by this optimal scanning conditions. Thesurface structure of the cell can be observed clearly, such as cell pseudopod, protrusion,secretory granules, cytoskeletal fiber, cellular connection, nucleolus etc.. Greatdifference exists among the skeleton fibers of NSCLC cells with different degree ofmalignancy.Impact of a variety of detection factors on NSCLC cell elastic modulus (E) wereanalyzed in AFM experiments. Thus a new evaluation method was established for theaccurate identification of mechanical properties of lung cancer cells. Spherical tip isessential to avoid elasticity overestimation which might be caused by pyramidal tip.Mechanical detection at cell center and edge can be accurately evaluated by thesemi-infinite and finite thickness Hertz model, separately. Experiment environmentshould be similar to the physiological environment of37°C. No significant influenceof cell morphology has on cell elasticity. Loading rate should be lower than1Hz toavoid the cellular viscous effect. This evaluation method provided technical support forthe accurate detection of cancerous cell viscoelasticity.A new method was proposed for the identification of lung cancer cells withdifferent malignancy degree by external mechanical characteristics. A new means wasprovided for identification of cancerous cell. Three NSCLC cell lines were investigatedin this study. The elastic modulus relationship of the three NSCLC cells yielded Elowmalignant> Emedium malignant> Ehighly malignant. The viscous characteristics of NSCLC cellswith different malignant degree were established based on the AFM stress relaxationtesting. Compared with the low metastatic A549cell, high-metastatic NCI-H1299cellhad a26%lower relaxation modulus ER,26%lower Kelvin spring constant K1,23%lower apparent viscosity coefficient μ. These data proved that compliance andinvasiveness of lung cancer cell was positive correlation. The reduced viscoelasticitymakes cell softer and more conducive to the release of metastatic cancer cells from theprimary tumor, through the capillary endothelial gap endosmosis and extravasation andmetastasis to regional lymph nodes or distant organs.Elasticity and viscosity characteristics of lung cancer cells were investigated bynanoindentation testing system. The Oliver-Pharr method was used to calculate theelastic modulus of cells and yielded ENCI-H1299<ENCI-H520. Creep phenomenon of cancercells at constant force was observed. Nonlinear fitting between the creep data and Voigtmodel, Maxwell model and Kelvin model was carried out. Kelvin model, which hadperfect fitting with creep data, was used to calculate the creep parameters of both celllines. It was proved that the creep parameters E1, E2, η1and η2of the high-metastaticNCI-H1299cells were significantly lower than the counterparts of NCI-H520cells (P<0.05). The cellular cytoskeleton images were transformed into binary images and fractal box counting was used to calculate the fractal dimension of the binary images. Itwas proved that the fractal dimension can evaluate the complexity of the cytoskeletontexture and can be used as an indicator of quantitative description of the cytoskeletonimage. Thus an ancillary method was presented to identify cancer cells by fractaldimension counting of cytoskeleton. Eventually a quantitative relationship wasestablished, which focused on the cytoskeleton structure, cell mechanical properties andcarcinogenesis.

Investigation of Morphology and Mechanical Properties of NSCLC Cells Based on Nano-indentation

Abstract

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