Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

<div><p>Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.</p> </div

The migratory phenotype of MDA-MB 231T is similar to that of M4 cells.

<p>(<b>A</b>) Phase contrast images of MDA-MB-231T cells moving into open space after 12 h under control (Black), 5 ng/mL EGF (red), and 1 µM LPA (blue) treatments. Bar = 100 µm. (<b>B</b>) Right: The effects of EGF and LPA treatment on average speed (top) and directionality, CV (bottom) determined over all times and space, were compiled from 5–6 independent experiments and reported as mean ± SD. Left: Aggregate directionality profiles for control, EGF and LPA conditions. Statistical significance: *p<0.05 (Tukey-Kramer test, n = 3). (<b>C</b>) Representative spatiotemporal heat plots show speed responses in control, EGF, and LPA treated cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058859#pone-0058859-g004" target="_blank">Fig. 4</a> for details.</p

Cell lines of the MCF10A series show distinct migration speed and directionality.

<p>(<b>A</b>) PIV analysis enables the mapping of velocity fields associated with the underlying epithelial sheet motions captured by phase time-lapse imaging (scale bar  = 100 µm). Spatial profiles of directionality and speed are depicted with white vectors and a heat map, respectively (right panel). (<b>B</b>) Left: Aggregate speed distributions, determined over all times and space, were compiled from 5–6 independent experiments for each cell line. Right: Quantification of the mean of the average speed (mean ±95% CI) for each cell line; M1–M2 (benign, black circles) and M3–M4 (tumorigenic, red triangles). (<b>C</b>) Left: Rose plots depicting aggregate directionality distributions were compiled over all times and space for each cell line (n = 5–6). Right: Variability of the direction of motion was quantified by the coefficient of variation (CV) and reported as mean ±95% CI. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 (Tukey-Kramer test, n = 5–6). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p

Cell lines of the MCF10A series show distinct migration properties.

<p>(<b>A</b>) Migration potential of M1–M2 (benign, black circles) and M3–M4 (tumorigenic, red triangles) cell lines after 4 h was assessed with the transwell assay using collagen IV coated membranes and no biased stimulation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058859#pone.0058859.s001" target="_blank">Fig. S1A</a>). (<b>B</b>) Phase images of the M1–M4 cell lines after 0 and 12 h of unconstrained migration. The dash vertical line indicates the initial location of the sheet edge. Scale bar  = 100 µm. (<b>C</b>) Quantification of the net displacement (during the 3–12 h time frame) is presented as in panel A. (<b>D</b>) M1–M4 cells were first pretreated with 25 µg/mL Mitomycin C for 20 min and then allowed to migrate into open space under conditions identical to panel B (black bars). The net displacement (mean ± SD) is shown compared to control (w/o drug) conditions (white bars), n = 2. For panels A and C results are presented as mean ±95% CI of 6–7 independent experiments. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001 (Tukey-Kramer test, n = 6–7). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p

Cell lines of the MCF10A series show distinct responses to EGF and LPA.

<p>(<b>A–D</b>) M1–M4 cells were stimulated with 5 ng/mL EGF (red) or 1 µM LPA (blue) and perturbations of average speed and of directionality (angle distributions and CV) compared to controls (black) were assessed (mean ± SD). Rose plots depict controls (unfilled, black bars) and 5 ng/mL EGF (filled, red bars) or 1 µM LPA (filled, blue bars). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 (Tukey-Kramer test, n = 3 for all conditions except M2 with EGF where n = 2). All comparisons were made with M1 cells unless indicated by pairing-brackets.</p