Matriptase activation of a PAR-2/MMP9 signaling axis cleaves E-cadherin to mediate loose spheroid formation. (A and B) ES-2 and NCI/ADR-Res Vec and Mat hanging-drop spheroids were formed in serum-free media for 4 d in the presence of vehicle control (DMSO), GB-83 (5 μM), and GM6001 (50 μM) or DMSO and JNJ 0966 (10 μM; C and D). Conditioned media was concentrated ∼10×, analyzed by SDS-PAGE, and immunoblotted for mouse-anti human E-cadherin recognizing the ectodomain region (HECD1-Invitrogen) or total protein Ponceau stain. Densitometric analysis was conducted using ImageJ and represents average soluble E-cadherin (sEcad) levels normalized to total protein (Ponceau staining) and relative to Vec DMSO (error bars are ± SEM of three independent experiments; *P < 0.05, ***P < 0.005, ns = not significant, two-tailed unpaired Student’s t tests). (E) Left panel: Conditioned media from IOSE397 and IOSE7576 cells, ES-2-Mat and ES-2-Vec spheroids, and clarified ascites fluid from mice bearing ES-2 Mat tumors were assessed for sEcad levels by quantitative ELISA (0.5–0.15 ng/ml). Right panel: Clarified ascites fluid from nine OvCa patients were assessed for sEcad levels by quantitative ELISA (10–125 ng/ml). Patient information is in the Materials and methods. (F) Matriptase (left panel) and HAI-1 (right panel) mRNA levels were determined by qPCR analysis of IOSE397 and IOSE7576 cells, ES-2 Vec and Mat spheroids, and tumor cells isolated from ascites of ES-2-Mat tumor-bearing mice. qPCR analysis was conducted in triplicate and mRNA expression was normalized to GAPDH. (G) Matriptase (left panel) and HAI-1 (right panel) mRNA levels were determined by qPCR analysis of tumor cells recovered from ascites fluid from nine independent patients. qPCR analysis was conducted in triplicate and mRNA expression was normalized to GAPDH. (H) Linear regression analysis of sEcad levels versus matriptase:HAI-1 ratios revealed a significant positive correlation (R2 = 0.6241, P <0.0001). Source data are available for this figure: SourceData F7.