title: Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak-Force Modulation Atomic Force Microscopy.
abstract: We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degrees of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/noninvasive), and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Young's modulus values up to 1 order of magnitude larger than those in regions where these structures are not observed, but these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines produced indistinguishable results.
authors.affiliation: Instituto de Microelectrónica de Madrid (IMM, CSIC) Isaac Newton 8, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Calzado-Martín
authors.affiliation: Instituto de Microelectrónica de Madrid (IMM, CSIC) Isaac Newton 8, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Encinar
authors.affiliation: Instituto de Microelectrónica de Madrid (IMM, CSIC) Isaac Newton 8, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Tamayo
authors.affiliation: Instituto de Microelectrónica de Madrid (IMM, CSIC) Isaac Newton 8, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Calleja
authors.affiliation: Instituto de Microelectrónica de Madrid (IMM, CSIC) Isaac Newton 8, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: San Paulo
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D001696:Biomechanical Phenomena; D001940:Breast / Q000473:pathology*; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D055119:Elastic Modulus; D005260:Female; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods
keywords: atomic force microscopy; cancer mechanics; cell mechanics; live cell imaging; peak-force