Atomic force spectroscopy in melanoma and keratinocytes cells.

• Ano de defesa: 2019
• Autor(a) principal: Reinoza, Nataly Zaribeth Herrera
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: AFM; elastic modulus; force volume; keratinocytes; melanoma; módulo de elasticidade; queratinócitos
• Link de acesso: http://www.teses.usp.br/teses/disponiveis/43/43134/tde-22042019-094701/

In this work, we used atomic force spectroscopy to obtain the elastic modulus of melanoma and keratinocytes fixed cells, with the purpose to determine the initial conditions for studies of confluente cultures of these cells in the future. The cell lines used were HaCaT cells and WM1366 melanoma cell, the last one is derived from a radial growth melanoma and both were analyzed, parental WM1366 cells (WM1366 shSCR cells) and galectin-3 silenced WM1366 cells (WM1366 shGal3). Cells were located and images of them were obtained by AFM contact mode under liquid conditions. Single force curves acquired in the central region of cells were used to determine the elastic modulus by the Hertzian contact model for the pyramidal tip, allowing to establish a comparison patter between cancer and normal cells. It was found that the melanoma cell (21.8 ± 0.5 kPa) exhibit smaller elastic modulus than keratinocytes cells (31.9 ± 0.4 kPa). For WM1366 shGal3 was found a elastic modulus of 16.1 ± 0.6 kPa, therefore, we found that for large indentation depth it is possible to distinguish between the same melanoma cell line, which represents general alterations in the organization of the cytoskeleton induced by the presence or absence of the galectin-3 protein. On the other hand, to detect local elastic modulus variations along the cell and to identify subcellular regions characterized by specific stiffness associated with local structures, we took elasticity maps in which a single force curve is acquired in each probe position. In order to interpret these maps, the cell was sliced into several different heights, curves of each height section were analyzed and represented in histograms, adjusted by the binomial distribution function. It was observed that the gradient of elastic modulus in cells from the nuclear region towards the cell periphery is more pronounced in cells devoid of galectin-3 than parental cells. The increased elastic modulus in the pericellular region of cells devoid of galectin-3 suggests that the organization of the extracellular matrix in these areas is different than those observed around HaCaT and shSCR WM1366 cells.