Matriptase expression promotes metastasis-associated behaviors: adhesion and clearance of mesothelial monolayer and invasion through mesothelial monolayer into submesothelial matrix. (A) Spheroid adhesion to mesothelial monolayer. ES-2 and NCI/ADR-Res Vec or Mat spheroids formed on agarose hydrogel and labeled with calcein-AM were seeded onto LP9 monolayers (formed on thin collagen-coated 24-well plates). Spheroids were allowed to adhere for 1 h and washed three times with PBS; adherent spheroids were visualized and counted before and after wash by EVOS (representative whole-well scans at 4× magnification of ES-2 experiment are shown in left panels; scale bars represent 1.4 mm). Right panel: Data represents the average fold change of the percentage of adherent spheroids relative to Vec from three independent experiments performed in triplicate (error bars are ± SEM, *P < 0.05, **P < 0.01). (B) Mesothelial clearance. ES-2 and NCI/ADR-Res Vec or Mat spheroids formed on agarose hydrogel (shown in “Spheroid [Brightfield]” panels) were seeded onto LP9 mesothelial monolayers labeled with calcein-AM (shown in “LP9 [Green]” panels). “Overlay” panels are Brightfield and Green channel views superimposed. Mesothelial clearance was visualized by EVOS, representative images of ES-2 experiment are shown at 4× magnification, scale bars represent 650 μm; top panel. The area of clearance was quantified by ImageJ and calculated as a percentage of the total spheroid area. Data in the bar graph (bottom panel) represent the average fold change of the percentage of clearance area relative to Vec from three independent experiments performed with at least three replicates each (error bars are ± SEM; *P < 0.05, **P < 0.01). (C) Migration and invasion. ES-2 Vec and Mat cells were seeded onto uncoated or Matrigel-coated Transwells, allowed to migrate or invade for 16–24 h, stained by Kwik-Diff, and quantified by ImageJ; data represents average migration/invasion relative to Vec from three independent experiments performed in triplicate (error bars are ± SEM, *P < 0.05, **P < 0.01). (D) Invasion of mesothelial monolayers. ES-2 and NCI/ADR-Res Vec or Mat cells labeled with calcein-AM were seeded onto Transwell filters coated with thin collagen and an LP9 mesothelial monolayer; after 24 h, invaded cells were fixed with PFA, visualized by EVOS, and quantified by ImageJ. A representative image of one field of invaded cells is shown (4× magnification; scale bars represent 650 μm). Data represents the average ratio of invaded cells relative to Vec from three independent experiments performed in triplicate (error bars are ± SEM, **P < 0.01, ***P < 0.005). (E) 3D collagen invasion. ES-2 and NCI/ADR-Res Vec or Mat spheroids formed on agarose hydrogel were embedded into a 3D type I collagen matrix in chamber slides and allowed to invade for 48 or 72 h respectively; spheroids were visualized by EVOS (representative images shown at 4× magnification; scale bars represent 650 μm). The average fold change of the percentage increase in spheroid size relative to Vec was quantified by ImageJ from three experiments performed with six to eight replicates (error bars are ± SEM, *P < 0.05 ***P < 0.005). All P values were calculated according to two-tailed unpaired Student’s t tests.