Figure 1

The ROCK inhibitor Y-27632 enhances 3D colon cancer cell invasiveness. (A) Left, representative micrographs of SW620 human colon cancer cells in control condition (vehicle 0.1% DMSO, CTL) or treated for 24 h, 48 h or 72 h in presence of 30 µM TTX or 10 µM Y-27632. Scale bar, 30 µm. Right, a cell circularity index was calculated from micrographs taken in absence or presence of Y-27632 at the three different times (n = 100 cells for each condition). This was performed using the Fiji software after having manually delineated the shape of cells. ***Statistical difference at P < 0.001 (Mann–Whitney rank sum test) versus CTL condition of corresponding time. NS stands for not statistically different. (B) Representative phase contrast micrographs (× 10 amplification objective) taken from SW620 colon cancer cells grown as spheroids in a 3-dimension matrix composed of Matrigel™. Pictures of spheroids were taken at different incubation times (0 h, 24 h, 48 h, 72 h, 96 h ) in control condition (CTL, vehicle) or treated with Y-27632 (10 µM), NaV channel inhibitor tetrodotoxin (TTX, 30 µM), or both (Y-27632 + TTX). Scale bar, 100 µm. (C) A spheroid circularity index was calculated over time from time-lapse micrographs in the four experimental conditions indicated above, CTL (vehicle, black line), Y-27632 (10 µM, red line), TTX (30 µM, green line) and the combination Y-27632 + TTX (30 µM, blue line) (n = 12, 8 and 5 spheroids per condition, respectively). Only the TTX group showed as significant reduction of circularity compared to the CTL condition, starting at time 36 h (**P < 0.01, Mann–Whitney rank sum test). There was no other statistical difference between groups. (D) Spherical volumes were calculated over time from time-lapse micrographs in the three experimental conditions indicated in B, CTL (vehicle), Y-27632 (10 µM), TTX (30 µM) and Y-27632 + TTX (30 µM) (n = 12, 8 and 5 spheroids per condition, respectively). The co-treatment with Y-27632 and TTX significantly reduced the volume of spheroids as compared to the treatment with Y-27632 alone, or with TTX alone, at times indicated on the figure (*P < 0.05, Mann–Whitney rank sum test). There was no other statistical difference. (E) The surface of extracellular matrix (ECM) invasion by SW620 cancer cells, at distance from spheroids, was calculated over time in the four experimental conditions indicated in C. The treatment with Y-27632 significantly increased the invasion area as compared to the CTL condition (^#P < 0.001, Mann–Whitney rank sum test). The co-treatment with Y-27632 and TTX significantly reduced the surface of invasion as compared with the treatment with Y-27632 alone (**P < 0.01; ***PP< 0.001, Mann–Whitney rank sum test), but did not differ from the CTL condition.