Quantitative biophysical metrics for rapid evaluation of ovarian cancer metastatic potential

Abstract

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous sub-mesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here, we fabricated extracellular-matrix mimicking suspended fiber networks: crosshatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying inter-fiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease, while force-disease biphasic relationship exhibited f-actin stress-fiber network dependence. However, unique to suspended fibers, coiling occurring at tips of protrusions and not the length or breadth of protrusions displayed strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on time scale of hours. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Coiling of cellular protrusions around extracellular fibers

“…Experiments studying the membrane dynamics at the leading-edge of cellular protrusions, have found indications for coiling (wrapping) dynamics around the extracellular fibers. In (26, 27), protrusions of metastatic cancer cells (breast and ovarian) were observed to coil and rotate around the fiber’s axis in a curvature-dependent manner, while in (28) similar coiling dynamics of leading-edge ‘fin’-like protrusions were observed for several cell types (fibroblasts, epithelial, endothelial). These protrusions are important during the cell’s adhesion and spreading, and play an important role in maintaining the cell polarity and migration.…”

Actin filaments couple the protrusive tips to the nucleus through the I-BAR domain protein IRSp53 for migration of elongated cells on 1D fibers

“…To quantitate protrusive activity, we used our approach of depositing large diameter (∼2 μm), suspended “base fibers” orthogonal to smaller diameter suspended “protrusive fibers” ( Fig. 2A(i, ii) ) [7,8,12] . As previously reported, this configuration allows bulk cell body migration to be constrained along with the base fiber, while individual protrusive events are elicited and studied along the protrusive fibers.…”

“…Previously, we have demonstrated that cells coil (wrap-around the fiber axis) at the protrusion tip ( Fig. 3A(i, ii) ) [8,12] . Based upon our findings of delayed protrusive activity in IRSp53 KO cells, we naturally inquired if these differences translated to differences in the coiling cycle occurring at the protrusion tip ( Fig.…”

“…Since delayed cell spreading is associated with a loss of force exertion [39] , we inquired if depletion of IRSp53 resulted in a loss of contractility. We used nanonet force microscopy (NFM) to quantify the forces exerted by single cells [11,12,40–42] , as they spread on two parallel fibers of the same diameters ( Movie M5 ). NFM estimates forces by establishing force vectors that originate at focal adhesion clusters (FAC) and are directed along the actin stress fibers ( Fig.…”

“…Cell attachment and migration on the suspended 1D fibers begins with characteristic protrusions that coil around the fiber axis [8,12] . In 2D and 3D, filopodial protrusions are mediated by the actin cytoskeleton and the inverse Bin/Amphiphysin/Rvs (I-BAR) domain-containing proteins [13–15] .…”