Resolving cell invasion

When it comes to investigating the sub-cellular mechanics of cell invasion – new techniques are needed. Here we learn of one such method   Invasion of cells through layers of extracellular matrix is a key step in tumour metastasis, inflammation, and development. The process of invasion 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 matrix1. Each of these stages of invasion is executed by a suite of proteins, including proteases, integrins, GTPases, kinases, and cytoskeleton-interacting proteins.

[caption id="attachment_25625" align="alignright" width="300" caption="Figure 1. Fluorescent gelatin degradation and phalloidin/DAPI staining of multiple cell types. Fluorescein-gelatin matrices (top row) were coated onto 8-well glass chamber slides as described. F-actin and nuclei were stained, respectively, with TRITC-phalloidin (bottom panel, red) and DAPI (bottom panel, blue)."][/caption]

Classical methods for analysing cellular invasion involve application of cells to one side of a layer of gelled matrix molecule and quantifying the relative number of cells that have traversed across the layer. Such methods are extremely useful for analysing invasion at the cell population-level, but analysis of the subcellular events mediating the stages of invasion requires techniques with higher resolution.

The method that has been most informative for pinpointing regions of the cell that initiate invasion involves plating cells on a culture surface coated with a thin layer of fluorescently-labelled matrix, and visualising regions where the cell has degraded the matrix to create an area devoid of fluorescence 2. Such assays have revealed that invasive cells extend small localised protrusions that preferentially degrade the surrounding matrix. These protrusions are termed invadopodia in cancerous cells, and podosomes in non-malignant cells such as macrophages3. Many molecules orchestrate the formation and function of invadopodia; a few of the key molecular events include Src phosphorylation of scaffolding protein Tks54, N-WASP activation and cortactin regulation of the Arp2/3 complex to induce actin polymerisation5, 6, generation of reactive oxygen species by NADPH oxidases7, and cortactin-mediated localisation of membrane-type and secreted MMPs to the invadopodia8.

The current drawback to using fluorescently-labelled gelatin to study cell invasion is that the process is highly laborious and dependent on user technique to create a homogeneously-labelled matrix. Furthermore, there exists the possibility of inconsistent and uneven application on glass substrates due to non-standardised protocols.

[caption id="attachment_25626" align="alignleft" width="300" caption="Figure 2. Co-localisation of degradation with invadopodia-related puncta. RPMI-7951 human skin melanoma cells were seeded onto the gelatin substrates for 24 hours. 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."][/caption]

QCM Gelatin Invadopodia Assay kits provide the reagents necessary for affixing thin, consistent coatings of pre-labelled fluorescent gelatin (fluorescein- or Cy3-conjugated) on glass substrates. These kits also include fluorescently-labelled phalloidin (TRITC- or FITC-conjugated) and DAPI, for visualising cytoskeletal F-actin and nuclei, respectively, to allow for co-localisation of matrix degradation with cellular features.

Results demonstrate the ability of the kits to allow visualisation of degradation produced by multiple cell types, quantification of degradation by image analysis, characterisation of proteolytic time courses, and exploration of modulator effects on invadopodia formation.

Simple, rapid, 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-labelled gelatin on glass substrates for microscopic investigation of invadopodia formation and matrix degradation.

Data presented here demonstrates the utility of these kits in the visualisation and quantification of gelatin degradation by a variety of cell types at multiple time points and following treatment with modulators of invadopodia formation. Such assays provide a convenient, flexible system for monitoring matrix degradation and investigating key components of the proteolytic process, both at the single cell and subcellular levels.

[caption id="attachment_25628" align="alignright" width="300" caption="Figure 3. Time-course of gelatin degradation. Multiple cell types (images are of MDA-MB-231) were plated onto Cy3-gelatin substrates (top image panel, red) and cultured for 8, 24, or 48 hours. Following staining with FITC-phalloidin (bottom image panel, green) and DAPI (bottom image panel, blue) cells were imaged at 20X objective magnification at 5 fields of view per well. Bar = 100 µm."][/caption]

Methodology

Substrate Preparation and Cell Seeding To facilitate attachment of fluorescent gelatin, 8-well glass chamber slides were chosen and first coated with 250µL/well of dilute poly-L-lysine in deionised water for 20 minutes at room temperature (the kits can also accommodate glass multi-well plates and glass coverslips, depending on user preference). The poly-L-lysine was then removed, and the slides rinsed three times with 500µL/well of Dulbecco’s PBS (DPBS). Next, 250µL of dilute glutaraldehyde in DPBS was added to each well for 15 minutes at room temperature to “activate” the poly-L-lysine surface for further protein attachment. Following removal of the glutaraldehyde, each well was again rinsed three times with 500µL of DPBS.  Finally, 200µL of dilute gelatin in DPBS, mixed at a 1:5 ratio of fluorescently-labelled:unlabelled gelatin, was coated onto each well for 10 minutes at room temperature, followed by three rinses in DPBS. All steps including, and subsequent to, fluorescent-gelatin coating were performed so as to protect the glass slides from photobleaching due to excessive exposure to light.

To prepare for cell plating, the gelatin substrates were disinfected with 500µL/well of 70% ethanol for 30 minutes at room temperature. After ethanol removal and rinsing in DPBS, free aldehydes were quenched by the addition of 500µL/well of amino-acid-containing growth media and incubated at room temperature for 30 minutes. Cell types of interest were detached using 0.25% trypsin-EDTA, pelleted, then re-suspended 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. For some experiments, a modulator of invadopodia formation, focal adhesion kinase inhibitor II (5µM final concentration, or 0.4% DMSO control), was added simultaneously with plating.

[caption id="attachment_25629" align="alignleft" width="300" caption="Figure 4. Modulation of gelatin degradation by a focal adhesion kinase inhibitor. Fluorescein-gelatin matrices (top image panel, green) were seeded with cells and simultaneously treated with 5 µM FAK inhibitor II or a 0.4% DMSO control. Following 24 hour treatment, cells were fixed and stained for F-actin and nuclei with TRITC-phalloidin (bottom image panel, red) and DAPI (bottom image panel, blue). Samples were imaged at 20X objective magnification at 5 fields of view per well. Bar = 100 µm."][/caption]

Sample Fixation and Staining At the desired time-point after plating, growth media was removed from the chamber slides and the samples were fixed for 30 minutes at room temperature with 250µL/well of 3.7% formaldehyde in DPBS. Samples were then rinsed twice with 500µL/well of fluorescent staining buffer (DPBS with 2% blocking serum and 0.25% Triton X-100 for cell permeabilisation). For immuno-co-localisation studies, 200µL of primary antibody in fluorescent staining buffer was added to each well for one hour incubation at room temperature. Samples were then rinsed three times with 500 µL/well of fluorescent staining buffer before proceeding on to one hour room temperature incubation with fluorescent secondary antibody, fluorescently-conjugated phalloidin (2µg/mL) and DAPI (1µg/mL) in staining buffer. Primary and secondary antibodies were omitted for stains incorporating phalloidin and DAPI only. Finally, samples were rinsed twice each with fluorescent staining buffer and DPBS before removal of culture chambers and cover-slipping. Slide mounting media contained anti-fade reagent and appropriate-thickness cover glasses were selected for imaging magnification of choice.

Imaging and Analysis 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 (five fields of view per well) or at 63x objective magnification (oil immersion) for co-localisation experiments.

Image analysis was performed utilising free, downloadable ImageJ software distributed by the NIH10. DAPI signal was thresholded for high intensities, then analysed as “particles” for determination of a nuclear (cell) count. Similarly, phalloidin signal was thresholded for high intensities to allow for measurement of total cell area per field of view. Conversely, fluorescent gelatin signal was thresholded for low intensities to enable quantification of total degradation area per field of view.

Figure 1 depicts example degradation observed from 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 plated onto fluorescein (green)-conjugated gelatin substrates at 20,000 cells/cm2 for a culture duration of 24 hours. F-actin and nuclei were stained, respectively, with TRITC-phalloidin (bottom panel, red) and DAPI (bottom panel, blue). Cells were imaged at 63X objective magnification (bar = 25µm).

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 behaviour, and cellular movement between sites of degradation may frequently be observed. SK-MEL-28 cells, a non-invasive melanoma type, do not display gelatin degradation, as expected.

As shown in Figure 2, RPMI-7951 cells seeded onto Cy3 (red)-gelatin matrices demonstrate the ability of the kit to co-localise sites of gelatin degradation with phalloidin (F-actin) puncta and cortactin foci.

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, as detailed. Cells were imaged at 63x objective magnification.

Cortactin protein is strongly associated with actin assembly, and co-localisation of this molecule with areas of proteolysis is indicative of dynamically “active” invadopodia formation. White arrows in the gelatin, phalloidin, cortactin, and overlay images demonstrate an example of co-localisation between matrix degradation, F-actin puncta, and cortactin foci.

Over 100 cells per condition were analysed to obtain the “percent degradation area of total cell area” data depicted in Figure 3. For MDA-MB-231 and IC-21 cells, degradation percentage increased over time, with the most significant augmentation in proteolysis occurring between the 8-hour and 24-hour time points. No degradation by non-invasive SK-MEL-28 cells was observed at any time point. Of note is that although the theoretical maximum of “percent degradation area of total cell area” is 100%, higher amounts of degradation were observed here, likely due to cellular movement during proteolysis. Such “historical” degradation is recorded using this assay, resulting in degradation areas larger than the area of a cell itself (particularly for longer time points or highly motile cell types).

Cells were seeded onto fluorescein-gelatin matrices and simultaneously treated with focal adhesion kinase (FAK) inhibitor II or a DMSO control (Figure 4). Over 100 cells per condition were included in analysis of “percent degradation area of total cell area”. FAK inhibition, which has previously been shown to enhance invadopodia formation in certain cell types12, was indeed observed to increase MDA-MB-231 degradation over the course of 24-hour treatment. The non-invasive phenotype of SK-MEL-28 cells was not altered by addition of FAK inhibitor II, but surprisingly, IC-21 degradation was decreased by treatment with the compound. Such opposite effects as those seen between the MDA-MB-231 and IC-21 cells emphasise variations in proteolytic behaviour between cell types, and may be due to differences in degradation signalling pathways between cell types in general, or between cancerous (e.g., MDA-MB-231) and normal (e.g., IC-21) cell phenotypes.

References

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The Authors Janet Anderl, Luke Armstrong and Jun Ma Janet is an R&D research scientist for Cell Based Assay Development; Luke is a senior R&D Manager for Cell Based Assay Development and Jun is a product manager at EMD Millipore

Contact e: jun.ma@merckgroup.com