The NANOFORCELLS team has studied the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degree of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/non-invasive) and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy was 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 one order of magnitude larger than in regions where these structures are not observed. The combination of AFM with immunofluorescence confocal microscopy determined 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.