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|Title:||Local measurement of breast cancer cells mechanical properties||Other Titles:||LOCAL MEASUREMENT OF BREAST CANCER CELLS MECHANICAL PROPERTIES||Authors:||Coceano, Giovanna||Supervisore/Tutore:||Bonin, Serena
Cojoc, Danut Adrian
|Issue Date:||24-Mar-2015||Publisher:||Università degli studi di Trieste||Abstract:||
In the last decades cell mechanics has been increasingly associated to cell health and function. Elasticity is one of the most investigated mechanical properties of cells and is now considered as a potential label free marker of cancer progression.
In this Thesis I report on the characterization of cells based on their mechanical properties. Three different biophysical micromanipulation tools have been used: Optical Tweezers (OT), Atomic Force Microscopy (AFM) and Speckle Sensing Microscopy (SSM).
We chose three breast cell lines selected as a model to study cancer progression: MDA-MB-231, a highly aggressive cell line belonging to the Basal cell-like phenotype; MCF-7, a less aggressive tumour cell line, belonging to the Luminal A cell-like tumour subtype; and HBL-100, a non neoplastic cell line, derived from the milk of a Caucasian woman, normal control for breast basal-myoepithelial cells.
The viscoelastic properties of the three cell lines have been measured using complementary approaches, thus allowing a thorough characterization: OT membrane tether pulling, OT and AFM vertical cell indentation and speckle interferometry with SSM.
With AFM and OT techniques we performed local measurements on specific parts of the cell; while with SSM we considered the cell as a whole viscoelastic body and we analyzed groups of cells at the same time.
OT membrane tether pulling uses a microbead trapped by the laser beam to pull cellular membrane tethers; from the resultant Force-Elongation (FE) curve, some viscoelastic parameters of the cell itself have been extracted and compared. The experimental approach results to be inefficient and time consuming and it has been, therefore, substituted by OT vertical indentation.
The new approach uses the OT in a similar way of the AFM technique, i.e. indenting the cell with a micron sized bead trapped by the laser. The elastic modulus has been therefore measured by vertical cell indentation, employing AFM and OT as two complementary techniques: with AFM we applied nN forces at high loading rates, while with OT we operated at pN forces at low loading rates.
OT has been implemented in an inverted optical microscope and the elastic modulus of the three cell lines results to be: 23.4 (HBL-100), 31.2 (MCF-7) and 12.6 (MDA-MB-231) Pa.
AFM indentation approach has been performed using the Bioscope Catalyst in Peak Force Quantitative Nanomechanical Mapping (PF-QNM) mode. Bioscope is able of applying nN forces by means of a nano-sized tip attached at the end of a cantilever. This new AFM mode allows mapping different mechanical properties of the cell under scan. The elastic modulus of the three cell lines has been extracted, providing more information about the mechanical alterations undergoing tumorigenesis. The mean values measured near the cell nucleus were: 91.1 (HBL-100), 81.8 (MCF-7), 57.6 (MDA-MB-231) kPa.
These results show that there is an inverse correlation between cell stiffness and breast cancer cell aggressiveness, since MDA-MB-231, the most aggressive cell line, has an elastic modulus significantly lower than the other two cell lines, both with OT and AFM measurements.
The difference values obtained by AFM and OT are the result of the different regimes used by these techniques: AFM applies higher forces and higher loading rates in comparison to OT. Nevertheless, the trend of the values between the cell lines was the same, showing that the aggressive cells were much softer than the other two. The combination of the two techniques is proposed for a more complete characterization of the mechanical properties of cells in different mechanical conditions.
Moreover we show that the stiffness of the substrate influences the elasticity of the cells; OT vertical indentation has been applied to HBL-100 cells cultured on bare and collagen coated substrates and their elastic modulus was 26±9 for bare and 19±7 Pa for collagen. These results show that cells adapt their structures to that of the substrate and demonstrate the potential of this setup for low-force probing of cell mechanics.
SSM has been originally proposed by our group in an international collaboration for fast diagnosis of malaria making available the analysis of thousand of cells per minute. It is based on the analysis of the speckles formed by light scattered by the cells when illuminated by a tilted laser beam. Speckle dynamics reflects the thermal vibration of the cell, which is linked to its stiffness. In this work SSM has been applied to MCF 7 cell line for cell mechanics characterization.
The final goal of this PhD Thesis is the characterization of the mechanical properties of cancer cells, by means of an integrated method based on rigorous biophysical techniques to understand the disease progression and differentiation towards metastasis.
|Ciclo di dottorato:||XXVII Ciclo||Insegnamento:||SCUOLA DI DOTTORATO DI RICERCA IN NANOTECNOLOGIE||Description:||
|Keywords:||cell mechanics; breast cancer; optical tweezers; atomic force microscopy; speckle sensing microscopy||Type:||Doctoral Thesis||Language:||en||Settore scientifico-disciplinare:||FIS/03 FISICA DELLA MATERIA||NBN:||urn:nbn:it:units-13691|
|Appears in Collections:||Scienze fisiche|
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