March 1, 2012 (Vol. 32, No. 5)
Janet Anderl R&D Research Scientist for cell-based assay development EMD Millipore
Luke Armstrong, Ph.D. Senior R&D Manager for cell based assay development EMD Millipore
Jun Ma Product Manager EMD Millipore
Kit Provides Standardized Process for Visualizing Matrix Degradation
Fluorescently labeled gelatin has been an informative tool in the study of cell invasion and matrix degradation. The method involves plating cells on a culture surface coated with a thin layer of a fluorescently labeled matrix and then visualizing regions where the cell has degraded the matrix to cause a loss of fluorescence signal. Such assays have pinpointed cellular regions that initiate invasion and revealed that invasive cells extend small protrusions of localized protease activity, termed podosomes in nonmalignant cells and invadopodia in cancerous cells.
This invasion of cancerous cells through extracellular matrix layers is a key step in tumor metastasis, inflammation, and development. The process involves several stages, including adhesion to the matrix, degradation of proximal matrix molecules, extension and traction of the cell on the newly revealed matrix, and movement of the cell body through the resulting gap in the matrix. Each of these invasion stages is executed by a suite of proteins, including proteases, integrins, GTPases, kinases, and cytoskeleton-interacting proteins.
The classical method for analyzing this process involves the application of cells to one side of a layer of gelled matrix molecule and quantifying the relative number of cells that traverse across the layer. Though such methods are useful for analyzing invasion at the cell population-level, fluorescently labeled gelatin has allowed for more detailed analysis of subcellular events.
However, conjugating fluorescent molecules to gelatin is laborious and dependent on user technique to create a homogenously labeled matrix. Inconsistent application on glass substrates is also possible due to non-standardized protocols.
QCM™ Gelatin Invadopodia Assay kits from EMD Millipore address these issues, providing a simplified and standardized method for producing homo-genously fluorescent matrices. The kits provide the reagents necessary for affixing thin, consistent coatings of pre-labeled fluorescent gelatin (fluorescein- or Cy3-conjugated) on glass substrates. They also include fluorescently labeled phalloidin (TRITC- or FITC-conjugated) and DAPI for visualizing cytoskeletal F-actin and nuclei, respectively, to allow for co-localization of matrix degradation with cellular features.
To demonstrate the utility of these kits in the visualization and quantification of gelatin degradation, multiple cell types were tested at multiple time points and following treatment with modulators of invadopodia formation.
Preparation and Cell Seeding
The QCM Gelatin Invadopodia Assay kits can accommodate 8-well glass chamber slides, glass multiwell plates, and glass coverslips. In this study, 8-well glass chamber slides were used, coated with dilute poly-L-lysine in deionized water and rinsed with Dulbecco’s PBS (DPBS). Next, dilute glutaraldehyde in DPBS was added to each well to “activate” the poly-L-lysine surface for further protein attachment. Each well was again rinsed with DPBS. Finally, 200 µL of dilute gelatin in DPBS, mixed at a 1:5 ratio of fluorescently labeled:unlabeled gelatin, was coated onto each well, followed by additional DPBS rinses.
To prepare for cell plating, the gelatin substrates were disinfected with 70% ethanol. After ethanol removal and rinsing in DPBS, free aldehydes were quenched by the addition of amino-acid-containing growth media. Cell types of interest were detached using 0.25% trypsin-EDTA, pelleted, then resuspended in growth medium to a concentration of 28,000 cells/mL (20,000 cells/cm2). Cells were seeded in a volume of 500 µL/well and cultured for the desired duration of degradation, generally between 8–48 hours.
Fixation, Staining, and Imaging
At the desired time-point, growth media was removed from the chamber slides, and samples were fixed with 3.7% formaldehyde in DPBS. Samples were then rinsed with fluorescent staining buffer.
For immuno-co-localization studies, primary antibody in fluorescent staining buffer was added to each well for incubation. Samples were then rinsed with fluorescent staining buffer before proceeding on to incubation with fluorescent secondary antibody, fluorescently conjugated phalloidin, and DAPI in staining buffer. Primary and secondary antibodies were omitted for stains incorporating phalloidin and DAPI only. Samples were then rinsed with fluorescent staining buffer and DPBS.
Mounted cover glasses were allowed to hard-set before fluorescent imaging with illumination and filters appropriate for fluorescein/FITC, Cy3/TRITC, and DAPI excitation and emission wavelengths. Samples were imaged on an inverted wide-field fluorescent microscope at 20X objective magnification for quantification studies or at 63X objective magnification for co-localization experiments. Image analysis was performed utilizing free, downloadable ImageJ software distributed by the NIH.
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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.
Janet Anderl is an R&D research scientist for cell-based assay development, Luke Armstrong, Ph.D., is senior R&D manager for cell based assay development, and Jun Ma ([email protected]) is product manager at EMD Millipore. Web: www.millipore.com.