Cancer cell mechanics and cell microenvironment: An optical tweezers study

Since cancer metastasis is a complex process, lot of research has been carried out to identify different hallmarks for its diagnosis and cure. Mechanical alterations in cancer cells during cell spreading to adjacent tissues and other organs of the body emerged as a prominent hallmark in the last decade. In this thesis we employed a mechanistic approach and used stiffness (elasticity) as a marker to study cell’s mechanical response in varying microenvironmental conditions. Cell– microenvironment mechanical interaction is a blend of cell-matrix and cell-cell interactions. Therefore we adopted an approach to study cells in the presence of their neighbouring cells as well as on compliant substrates at small forces (<10pN) using optical tweezers. The elastic modulus was calculated using the Hertz-model. We considered three breast cell lines as model, showing three phases of cancer progression: MDA-MB-231, a highly aggressive cell line belonging to the Basal cell-like phenotype; MCF-7, a less aggressive cancer cell line, belonging to the Luminal A cell-like phenotype; and HBL-100, a non-neoplastic cell line, derived from the milk of a Caucasian woman, normal control for breast basal-myoepithelial cells. Cell elasticity can be locally measured by pulling membrane tethers, stretching or indenting the cell using optical tweezers. We introduce a simple approach to perform cell indentation by axially moving the cell against a trapped microbead. Our scheme is similar to the AFM vertical cell indentation approach and can help to compare the quantitative results and thus complement AFM in a low force regime and loading rates. The elasticity trend of the three cell lines in isolated conditions showed that the aggressive MDA-MB-231 cells are significantly softer as compared to HBL-100 and MCF-7 cells. We demonstrate that stiffness measurements are sensitive to the cellular sub-regions as well as the interacting microenvironment. We probed the cells at three cellular sub regions: central (above nucleus), intermediate (cytoplasm) and near the leading edge. In isolated condition, all cells showed a significant regional variation in stiffness: higher at the center and fading toward the leading edge. However, the regional variation become statistical insignificant when the cells were in contact with other neighboring cells. We found that neighboring cells significantly alter cell stiffness: MDA-MB-231 becomes stiffer when in contact, while HBL-100 and MCF-7 exhibit softer character. Furthermore, we have studied the influence of substrate stiffness on cell elasticity by seeding the cells on Collagen and Polydimethylsiloxane (PDMS) coated substrates with varying stiffnesses to mimic extracellular (ECM) rigidities in vitro. PDMS polymer to crosslinker ratio was adjusted to 15:1, 35:1 and 50:1 corresponds to 173kPa, 88kPa and 17kPa respectively. These results show that cells adapt their stiffness to that of the substrate. Flexible substrates leads to reduced cell spreading morphological changes. Cells on complaint substrates are softer as compared to stiffness substrates. Our results demonstrates that the substrate stiffness influence not only cell spreading and motility, but also cell elasticity. Finally, from the 3D tracking of the bead probe we analyzed the lateral forces arising during the vertical indentation of the cell membrane during cell-bead interaction. We calculated and compared the elastic moduli resulting from the total and vertical forces for two breast cancer cell lines: MDA-MB-231 and HBL-100, showing that the differences are important and the total force should be considered.