Matriptase activity promotes the formation of smaller loose, disaggregated multicellular spheroids via disrupted homotypic cell–cell interactions, enhanced spheroid disaggregation, and increased tumorsphere formation. (A) Hanging-drop spheroids. ES-2 and NCI/ADR-Res Vec or Mat cells were seeded in 10 μl droplets (2 × 10⁴ cells/drop), inverted on the lid of a 10-cm dish, and allowed to form hanging drop spheroids for 4 d. Some were formed in the presence of matriptase-blocking antibody A11 (100 nM; see Fig. S3 C for titration of A11). Hanging drop spheroids were visualized by EVOS (representative images shown at 4× magnification; scale bars represent 650 μm; left images) and spheroid morphology was determined under each condition. Graphs show the quantitation of the spheroid morphology (% loose spheroids and % empty space) as described in the Materials and methods. Data represent the average of three independent experiments (error bars are ± SEM; **P < 0.01, ***P < 0.005, ****P < 0.001, ns = not significant). (B) Cell–cell adhesion. ES-2 and NCI/ADR-Res Vec or Mat cells were seeded onto a black-walled 96-well plate and allowed to form a monolayer for 48 h. Cells were labeled with CellTrackerOrange as per the manufacturer’s instructions and seeded on top of corresponding homotypic monolayers and allowed to adhere for 2 h, treated with either vehicle (PBS) or with 100 nM A11. Fluorescent cells were visualized by EVOS and fluorescence intensity was measured by FlexStation3 plate reader; cells were washed twice with PBS and remaining fluorescence of adherent cells was measured and visualized. Representative images of adherent cells (red) on homotypic monolayers (grayscale) are shown at 4× magnification; scale bars represent 650 μm. Data represents average percentage of remaining fluorescence from three independent experiments performed with at least three replicates each (error bars are ± SEM; *P < 0.05, ***P < 0.005, ns = not significant). (C) Spheroid disaggregation. LP9 mesothelial cells were seeded onto collagen-coated 96-well plates and allowed to form a confluent monolayer for 48 h. ES-2 and NCI/ADR-Res Vec or Mat spheroids were formed on agarose hydrogel overnight and labeled with calcein-AM. Labeled spheroids were seeded onto formed LP9 monolayers in the presence of vehicle control (DMSO) or 100 nM A11 and visualized for 72 h by EVOS; representative images are shown at 4× magnification, scale bars represent 650 μm. The percentage increase in spheroid area from starting point (0 h) was quantified by ImageJ; data represents average of three independent experiments performed with six replicates each (error bars are ± SEM; *P < 0.05, **P < 0.01, ***P < 0.005, ns = not significant). All P values were calculated according to two-tailed unpaired Student’s t tests.