The relationship between metastatic potential and in vitro mechanical properties of osteosarcoma cells.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.

A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device.

Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cell-substrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both "soften" and "stiffen" the cell cytoskeleton with either 0.5 µg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5 ± 0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5 ± 0.5%, p < 0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nano-indentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.