Elevated matriptase:HAI-1 ratios increase cell surface serine protease activity and promote spheroid phenotype in three OvCa models
| Cell line . | Relative Matriptase:HAI-1 protein ratioa . | Cell surface serine protease activity (fold change ± SEM)b . | Spheroid morphology . | |
|---|---|---|---|---|
| Spheroid phenotypec . | % empty space (rel. to Vec)d . | |||
| ES-2 Vec | 0.0e | 1.00 | Tight | 1.00 |
| ES-2 Mat | 23.0 | 3.26 (±0.22) | Loose | 4.85 |
| NCI/ADR-Res Vec | 0.0e | 1.00 | Tight | 1.00 |
| NCI/ADR-Res Mat | 19.6 | 4.02 (±0.84) | Loose | 2.24 |
| SKOV3 Vec | 0.0e | 1.00 | Tight | 1.00 |
| SKOV3 Mat | 5.4 | 2.78 (±0.002) | Loose | 2.56 |
| Cell line . | Relative Matriptase:HAI-1 protein ratioa . | Cell surface serine protease activity (fold change ± SEM)b . | Spheroid morphology . | |
|---|---|---|---|---|
| Spheroid phenotypec . | % empty space (rel. to Vec)d . | |||
| ES-2 Vec | 0.0e | 1.00 | Tight | 1.00 |
| ES-2 Mat | 23.0 | 3.26 (±0.22) | Loose | 4.85 |
| NCI/ADR-Res Vec | 0.0e | 1.00 | Tight | 1.00 |
| NCI/ADR-Res Mat | 19.6 | 4.02 (±0.84) | Loose | 2.24 |
| SKOV3 Vec | 0.0e | 1.00 | Tight | 1.00 |
| SKOV3 Mat | 5.4 | 2.78 (±0.002) | Loose | 2.56 |
Matriptase and HAI-1 protein expression were determined by immunoblot analysis; levels were quantitated by densitometric analysis, normalized to β-tubulin, and the matriptase:HAI-1 ratio was calculated (see Fig. S1 B).
AEBSF-sensitive cell surface serine protease activity was measured using Boc-QAR-AMC fluorogenic peptide. Fold change of endpoint fluorescent values (normalized to cell number) was calculated relative to each respective Vec control.
Spheroids were allowed to form on nonadhesive agarose hydrogels overnight, visualized using the EVOS microscope and qualitatively scored as tight or loose as defined in the Materials and methods.
As a quantitative measure of spheroid morphology, the percentage of empty space in EVOS images of spheroids was determined using ImageJ as described in the Materials and methods and expressed relative to each respective Vec control.
Matriptase protein was undetectable in Vec cells by immunoblotting, thus resulting in a zero value for the relative matriptase:HAI-1 ratio (see Fig. S1 D).