Four representative cell lines—MDA-MB-231 human breast adenocarcinoma, RPMI-7951 and SK-MEL-28 human skin melanoma, and IC-21 mouse peritoneal macrophages—were plated onto fluorescein (green)-conjugated gelatin substrates at 20,000 cells/cm2 for a culture duration of 24 hours (Figure 1). F-actin and nuclei were stained, respectively, with TRITC-phalloidin (bottom panel, red) and DAPI (bottom panel, blue).
MDA-MB-231, RPMI-7951, and IC-21 gelatin proteolysis demonstrate the range of degradation patterns that may be observed due to invadopodia or podosome formation, including “punctate”, “linear”, or “blotchy” areas devoid of fluorescein-gelatin fluorescence.
Often, not all cells in a population will exhibit proteolytic behavior, and cellular movement between sites of degradation may frequently be observed. SK-MEL-28 cells, a noninvasive melanoma type, do not display gelatin degradation as expected.
The kit also allows the user to co-localize sites of gelatin degradation with phalloidin (F-actin) puncta and cortactin foci. Cortactin protein is strongly associated with actin assembly, and co-localization of this molecule with areas of proteolysis is indicative of dynamically “active” invadopodia formation.
RPMI-7951 cells were seeded onto Cy3 (red)-gelatin matrices, and cells were incubated with a primary antibody against cortactin, followed by detection with a Cy5-conjugated secondary antibody.
Secondary antibody incubation was performed concurrently with FITC-phalloidin and DAPI staining. White arrows in the gelatin, phalloidin, cortactin, and overlay images demonstrate an example of co-localization between matrix degradation, F-actin puncta, and cortactin foci (Figure 2).
Matrix degradation time courses can also be studied. Over 100 cells per condition were analyzed to obtain the percent degradation area of total cell area over time. For MDA-MB-231 and IC-21 cells, degradation percentage increased over time, with the most significant augmentation in proteolysis occurring between eight and 24 hours. No degradation by noninvasive SK-MEL-28 cells was observed at any time point.
To study the modulation of matrix degradation, cells were seeded onto fluorescein-gelatin matrices and simultaneously treated with focal adhesion kinase (FAK) inhibitor II or a DMSO control (Figure 3).
FAK inhibition, which has previously been shown to enhance invadopodia formation in certain cell types, was indeed observed to increase MDA-MB-231 degradation over the course of 24-hour treatment. The noninvasive phenotype of SK-MEL-28 cells was not altered by addition of FAK inhibitor II, but surprisingly, IC-21 degradation was decreased.
Such opposite effects seen between the MDA-MB-231 and IC-21 cells emphasize variations in proteolytic behavior between cell types, and may be due to differences in degradation signaling pathways between cell types in general, or between cancerous and normal cell phenotypes.
Simple and consistent production of homogeneously fluorescent matrices is a critical step in cell invasion studies. The new QCM Gelatin Invadopodia Assay kits provide the reagents necessary for generating thin coatings of fluorescently labeled gelatin on glass substrates for microscopic investigation of invadopodia formation and matrix degradation.
These kits allow for the visualization of degradation produced by multiple cell types, quantification of degradation by image analysis, characterization of proteolytic time courses, and exploration of modulator effects on invadopodia formation. Such assays provide a convenient system for monitoring matrix degradation and investigating key components of the proteolytic process.