# br Cell shape morphology analysis br

Cell shape-morphology analysis

Cell aspect ratio is computed from maximal intensity projections of bright-field image stacks. From the image projection, we calculate the aspect ratio as the distance between the two points of the cell outline with the largest separation (major axis length), divided by the largest cell dimension found anywhere perpendicular to the long axis (minor axis length).

Internalization of latex beads

For increasing the steric effects imposed by the matrix, MDA-MB 231 165800-03-3 are loaded with 5-mm-diameter nondegradable carboxylated polystyrene beads that are larger than the average pore size of 3.8 mm for a 1.2 mg/mL collagen gel (3,12). The network pore size was evaluated using the covering radius transformation of confocal image stacks of the collagen gel as described in (16,18). The pore size distribution p(r) follows a Ray-leigh distribution (16), indicating that 74% of the network pores of a collagen gel are smaller than the bead size of 5 mm and thus cause steric hindrance.

2 105 beads (Thermo Fisher, Waltham, MA) are sonicated, added to MDA-MB 231 cells grown in a 35-mm cell culture dish, and incubated overnight (19–21). Cells are rinsed twice to remove unbound beads, har-vested with 0.5% trypsin/EDTA, mixed with unpolymerized collagen, and cultured for another 12 h before force measurements or for 24 h sin-gle-cell invasion experiments. For cell force and invasion assay analysis, we selected only cells carrying exactly one bead.

Statistical analysis

Unless otherwise noted, all experimental results are taken from at least three independent experiments. Differences between measurements are considered statistically significant at p % 0.05 using Student’s two-tailed t-test assuming unequal variances, including outliers.
Cell Forces under Steric Hindrance

RESULTS

Response of cell invasiveness to altered steric hindrance

We hypothesize that cells with higher cell stiffness experi-ence more steric hindrance when migrating through confining matrix pores. To explore how internal mechani-cal properties of cells influence cell invasiveness, we modulate the stiffness of MDA-MB 231 breast cancer cells with two different approaches. The first approach relies on overexpression of the nuclear protein lamin A using lenti-viral transduction, which leads to an increase in overall cell stiffness by 47% (8,12,15,16). In a second approach, we in-crease the apparent cell rigidity by binding or internalizing nondegradable 5-mm polystyrene beads. Polystyrene beads have been previously used to study remodeling processes of the cytoskeleton by tracking their spontaneous motion (19–21). MDA-MB 231 breast cancer cells readily inter-nalize these beads. After 30 min of bead incubation, 70% of beads are internalized by the cells (21). To in-crease the fraction of internalized beads further, we incu-bate the cells with beads overnight. Fluorescent imaging of cells expressing tandem tomato-farnesyl for labeling the cell membrane confirms that the beads are internalized (Fig. S10).

To quantify cell invasiveness, we track the movements of individual cells embedded in 1.2 mg/mL collagen gels over 24 h (Fig. 1 A). The resulting trajectories are classified into motile and nonmotile cells based on whether a cell moves away from its original position by more than 20 mm within the 24-h observation period. We find that 80% of MDA-con-trol cells are motile. The motile fraction decreases to 72% for MDA-lamA cells and 74% for MDA-beads cells (Fig. 1 B).

The migration speed of the motile cell fraction is decreased by 22% in MDA-lamA cells and by 8% in MDA-beads cells (Fig. 1 C). MDA-lamA cells compen-sate by an increased directional persistence, in contrast to the cells with internalized beads. Consequently, the mean invasion distance within 1 h of MDA-lamA and MDA-beads cells is decreased by 12 and 18%, respec-tively, compared to control cells. We conclude that increasing the steric hindrance has a surprisingly small ef-fect on cell invasion, suggesting that cells are able to partially compensate.

Adaptation of contractile forces to altered steric hindrance

We next test whether cells can actively counteract steric ef-fects by generating higher contractile forces. Cells of the three groups studied (MDA-control, MDA-lamA, and MDA-beads) are mixed with 1.2 mg/mL unpolymerized collagen. After initiating collagen polymerization, cells

Co´ndor et al.

FIGURE 1 Cell invasion. (A) 50 randomly selected trajectories over a time course of 4 h of con-trol cells (left), lam-A-overexpressing cells (mid-dle), and cells with 5-mm polystyrene beads (right). (B) Motile fraction of cells. The number of analyzed cells is noted in white. (C) Cell speed, measured between subsequent images (Dt ¼ 5 min). (D) Directional persistence, measured on a timescale of 1 h. (E) Mean Euclidean invasion distance over the course of 1 h. *p % 0.05, **p % 0.01, ***p % 0.001, c-square test of independence of variables in a contingency table for motile frac-tion and Student’s t-test assuming unequal variances including the outliers for all other variables; n.s., not significant (p > 0.05). To see this figure in color, go online.