Figure 1.
Increased matriptase:HAI-1 ratio correlates with poor patient survival and enhanced late stage peritoneal dissemination in an orthotopic xenograft model. (A) Left panel: Kaplan–Meier progression-free survival (PFS) analysis based on low or high HAI-1 expression in a cohort of OvCa patients stratified by matriptase mRNA expression above the median using publicly available data obtained from KMPlotter (N = 326; log rank P = 0.0024). Right panel: Kaplan–Meier PFS analysis based on low or high matriptase expression in a cohort of OvCa patients stratified by HAI-1 mRNA expression below the median (N = 326; log-rank P = 0.026). All patients also expressed CA125 mRNA below the lower quartile and biased arrays were excluded from analysis. (B) ES-2 Vec and Mat cells were analyzed for AEBSF-sensitive cell surface serine protease activity using the Boc-QAR-AMC fluorogenic substrate over 4 h; representative time course performed in triplicate is shown; error bars represent ±SEM of cell surface serine protease activity amongst triplicate wells (left panel). Right panel shows the average fold change of activity at the endpoint relative to Vec from three independent experiments (error bars represent ±SEM) performed in triplicate (*P < 0.05, two-tailed unpaired Student’s t test). (C) ES-2 Vec and Mat cells were injected i.p. into nude mice (n = 5 per group) and tumor burden was measured by IVIS over time. Mean photon intensity measured on the abdomen was quantified per mouse on the days indicated (error bars represent ± SEM; **P < 0.01, ***P < 0.005, two-tailed unpaired Student’s t test). (D) Volume of ascites fluid recovered from peritoneal cavity upon necropsy was measured per mouse (error bars represent ±SEM; *P < 0.05, two-tailed unpaired Student’s t test). Ascites fluid was undetectable in ES-2 Vec tumor-bearing mice. (E) Tumor samples recovered from metastatic sites in peritoneal cavities in mice upon necropsy were analyzed by qPCR for matriptase mRNA expression and normalized to luciferase mRNA expression to only account for tumor cells (error bars represent ±SEM; ****P < 0.001, two-tailed unpaired Student’s t test). (F) Photos of intraperitoneal tumor burden in mice bearing ES-2 Vec and Mat tumors on the diaphragms upon necropsy. Differences in tumor morphology are indicated by yellow arrows. (G) Tumorsphere formation. ES-2 and NCI/ADR-Res Vec or Mat cells were seeded at low density onto poly-HEMA coated 24-well plates in Mammocult media and allowed to form tumorspheres for 10 d. Tumorspheres were visualized by EVOS FL Auto Cell Imaging (representative images are enlarged images of 10× magnification). The total tumorsphere number was counted and the percentage of tumorspheres <200 or >200 μm2 (measured by gridlines on EVOS, scale bars represent 200 μm) was calculated per well. Data represent the average of three independent experiments (error bars are ±SEM) performed with four to six replicates each (*P < 0.05, **P < 0.01, two-tailed unpaired Student’s t test). (H) ES-2, NCI/ADR-Res, and SKOV3 Vec or Mat cells were seeded onto 0.75% agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Spheroid morphology was visualized by EVOS FL Auto Cell Imaging and representative images are shown at 4× magnification (top images; scale bar = 500 μm for ES-2 and SKOV3, 650 μm for NCI/ADR-Res). Graphs below the images show 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 < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, two-tailed unpaired Student’s t test). (I) Non-malignant IOSE397 cells, OvCa cell lines CAOV3, OVCAR3, and COV362 were seeded on agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Representative images are shown at 4× magnification (top images). Graphs below the images 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 < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, two-tailed unpaired Student’s t test). (J) Patient ascites-derived tumor cells from two patients (Patient 36 and Patient 37; see Materials and methods) were seeded on agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Representative images are shown at 4× magnification. Scale bars for H–J represent 650 μm.

Increased matriptase:HAI-1 ratio correlates with poor patient survival and enhanced late stage peritoneal dissemination in an orthotopic xenograft model. (A) Left panel: Kaplan–Meier progression-free survival (PFS) analysis based on low or high HAI-1 expression in a cohort of OvCa patients stratified by matriptase mRNA expression above the median using publicly available data obtained from KMPlotter (N = 326; log rank P = 0.0024). Right panel: Kaplan–Meier PFS analysis based on low or high matriptase expression in a cohort of OvCa patients stratified by HAI-1 mRNA expression below the median (N = 326; log-rank P = 0.026). All patients also expressed CA125 mRNA below the lower quartile and biased arrays were excluded from analysis. (B) ES-2 Vec and Mat cells were analyzed for AEBSF-sensitive cell surface serine protease activity using the Boc-QAR-AMC fluorogenic substrate over 4 h; representative time course performed in triplicate is shown; error bars represent ±SEM of cell surface serine protease activity amongst triplicate wells (left panel). Right panel shows the average fold change of activity at the endpoint relative to Vec from three independent experiments (error bars represent ±SEM) performed in triplicate (*P < 0.05, two-tailed unpaired Student’s t test). (C) ES-2 Vec and Mat cells were injected i.p. into nude mice (n = 5 per group) and tumor burden was measured by IVIS over time. Mean photon intensity measured on the abdomen was quantified per mouse on the days indicated (error bars represent ± SEM; **P < 0.01, ***P < 0.005, two-tailed unpaired Student’s t test). (D) Volume of ascites fluid recovered from peritoneal cavity upon necropsy was measured per mouse (error bars represent ±SEM; *P < 0.05, two-tailed unpaired Student’s t test). Ascites fluid was undetectable in ES-2 Vec tumor-bearing mice. (E) Tumor samples recovered from metastatic sites in peritoneal cavities in mice upon necropsy were analyzed by qPCR for matriptase mRNA expression and normalized to luciferase mRNA expression to only account for tumor cells (error bars represent ±SEM; ****P < 0.001, two-tailed unpaired Student’s t test). (F) Photos of intraperitoneal tumor burden in mice bearing ES-2 Vec and Mat tumors on the diaphragms upon necropsy. Differences in tumor morphology are indicated by yellow arrows. (G) Tumorsphere formation. ES-2 and NCI/ADR-Res Vec or Mat cells were seeded at low density onto poly-HEMA coated 24-well plates in Mammocult media and allowed to form tumorspheres for 10 d. Tumorspheres were visualized by EVOS FL Auto Cell Imaging (representative images are enlarged images of 10× magnification). The total tumorsphere number was counted and the percentage of tumorspheres <200 or >200 μm2 (measured by gridlines on EVOS, scale bars represent 200 μm) was calculated per well. Data represent the average of three independent experiments (error bars are ±SEM) performed with four to six replicates each (*P < 0.05, **P < 0.01, two-tailed unpaired Student’s t test). (H) ES-2, NCI/ADR-Res, and SKOV3 Vec or Mat cells were seeded onto 0.75% agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Spheroid morphology was visualized by EVOS FL Auto Cell Imaging and representative images are shown at 4× magnification (top images; scale bar = 500 μm for ES-2 and SKOV3, 650 μm for NCI/ADR-Res). Graphs below the images show 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 < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, two-tailed unpaired Student’s t test). (I) Non-malignant IOSE397 cells, OvCa cell lines CAOV3, OVCAR3, and COV362 were seeded on agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Representative images are shown at 4× magnification (top images). Graphs below the images 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 < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, two-tailed unpaired Student’s t test). (J) Patient ascites-derived tumor cells from two patients (Patient 36 and Patient 37; see Materials and methods) were seeded on agarose hydrogel in 96-well plates and allowed to form spheroids overnight. Representative images are shown at 4× magnification. Scale bars for H–J represent 650 μm.

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