2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Our group works on the interaction between cell and extracellular matrix. Cells can produce but also can degrade the extracellular matrix element. Degradation of the matrix can occur during physiological but also in pathological processes, such as neurodegensis and the invasion of cell tumor.

At the cellular level we can notice the presence of actin-based structure involved in degradation of the matrix. And these structures are named invadosomes. Invadosome, it's a general term including podosomes in normal cells and invadopodia in cancer cells.

The degradation of this invadosome can be self-organized in different shapes, dots, aggregates and rosettes. And the activity of this invadosome is driven by metalloproteinases, such as MMP2 and MT1-MMP. This is not for a long time that the couple, MMP2, MT1-MMP is activated by the type 1 collagen fibrils.

To study the activity of invadosomes, a classical zymography in-situ assay was used. But to study the role and impact of type 1 collagen fibrils in invadosomes, we decided to create a new assay. To do this, we created a mixed matrix composed of gelatin and type 1 collagen fibrils.

In this movie in the first part, Eloide Henriet will show you how we generate this kind of matrix. In the second part, Zakaria Ezzoukhry will show you how we are able to measure the impact of type 1 collagen fibrils in the morphology, formation and activation of the invadosomes. And in the last part, Julie Di Marino will show you how we adapt a 3D collagen invasion assay.

I will show you how to prepare a mixed matrix composed of gelatin and type 1 collagen fibrils. I will do two types of mixed matrix. One to do the zymography in-situ assay with fluorescein gelatin and non-fluorescein type 1 collagen.

And another one allowing us visualize the collagen fibrils with non-fluorescein gelatin and labeled collagen fibrils. To prepare the gelatin matrix, aliquot 20 microliter droplets of gelatin on a piece of parafilm. Cover each droplet with an autoclaved glass coverslip.

Incubate at room temperature for 20 minutes and protect from light. For gelatin fixation, aliquot 40 microliter droplets of 0.5%glutaraldehyde in PBS. Using forceps, transfer gelatin-coated coverslips on to each glutaraldehyde droplet.

Incubate at room temperature for 40 minutes and protect from light. Take a multiwell cell culture plate and fill each well with 500 microliters of sterile 1X PBS. At the end of the incubation period, place each coverslip, gelatin side up into each well.

Wash coverslips twice in 1X PBS for five minutes each. At this step, coverslips can be used for experimentation or stored for later use. Pipette 0.4 mgs per ml collagen one into a tube.

Add the fluorescent dye for a final concentration of 10 micrograms per ml. Mix the solution in the tube and incubate for five minutes at room temperature protected from light. Dilute to the final volume with 1x DPBS and add 500 microliters of the solution on top of gelatin-coated coverslips in a multiwell plate.

Incubate for four hours at 37 degrees Celsius for collagen polymerization. After incubation remove the excess collagen. Add cells for a final volume of 500 microliters and incubate the cells at 37 degrees Celsius before fixation.

This type of matrices could be modified with other matrix elements such as fibronectin, vitronectin or type IV collagen. For my part, I will show you how to perform immunofluorescence by using Tks5, a marker of invadosomes, and how to quantify linear invadosomes on collagen type 1 coated coverslips. We can present the results by number of cells present in linear invadosomes, but also we can present the number of structures in each cell.

Fix the coverslips for 10 minutes in 500 microliters of 4%paraformaldehyde in PBS. Wash twice with 1x PBS and permeabilize the cells with 0.2%Triton X-100 in 1x PBS for 10 minutes. Aliquot 50 microliter droplets of primary antibody solution in 4%BSA in 1x PBS on a piece of parafilm.

Place each coverslip on a droplet. Incubate for 40 minutes at room temperature protected from the light. Wash coverslips twice with 1x PBS and incubate coverslips in secondary antibody, colloidin and Hoechst dye for 30 minutes protected from light.

After the incubation, wash twice with 1x PBS, followed by distilled water to remove salts. Mount coverslips on a glass slide with polymerizing mounting medium. Let the slide dry overnight at room temperature before imaging.

For the image analysis acquire an image of actin and Tks5 sequentially using a confocal microscope. To quantify the size and number of linear invadosomes per cell, use the ImageJ software and the provided macro. First run the macro in ImageJ and open the actin image.

Afterwards, open the Tks5 image. Set the threshold and apply the macro. Analyzed results will appear in two tables.

The number of linear invadosomes per cell and other information such as the percentage area used by linear invadosomes in the cell will be in the summary table. The size of each linear invadosome will be in the results table. After the quantification of the images, we can present the results by showing the number of structures per cell.

So in my part, we adapt a method to demonstrate the presence of linear invadosomes in 3D. We realized the gel only composed of collagen one fibrils and this assay is used to evaluate the cell invasion in the collagen one context. Prepare type 1 collagen solution on ice at a final concentration of two mgs per ml.

Add the fluorescent dye at a final concentration of one microgram per milliliter. Mix the solution and incubate on ice for five minutes protected from light. Afterwards add 50 microliters of ice-cold 10x PBS.

Then add 6.25 microliters of ice-cold one molar sodium hydroxide. Add sterile water to obtain the correct concentration and mix the solution. Place Boyden chamber inserts into a multiwell cell culture dish.

Add 100 microliters of collagen one solution to each insert. To polymerize the collagen gel, incubate the dish for one hour at 37 degrees Celsius. Add serum-enriched cell culture medium to each well and place inserts in media.

Seed 30, 000 cells in serum-free media on top of the collagen gel. To fix the cells, add 4%paraformaldehyde solution into an empty well. Remove the cell medium from the insert without touching the collagen gel.

Place the insert in wells with the 4%paraformaldehyde solution and add additional 4%paraformaldehyde solution on top of the collagen gel. Incubate at room temperature for 30 minutes. After fixation, remove the 4%paraformaldehyde solution and wash with 1x PBS.

After washing, the collagen gel should become transparent. Now the inserts are ready for immunofluorescence. First drain the inserts.

Then place the inserts into new wells of the multiwell plate. To permeabilize the cells, add a solution of 0.2%Triton X-100 in 1X PBS on top of the collagen gel for 30 minutes. Drain the inserts.

Wash with 1X PBS and add primary antibody solution on top of the collagen gel for one hour protected from light. Remove the primary antibody solution, wash with 1X PBS and incubate the collagen gel with the secondary antibody solution for 1.5 hours protected from light. Wash the secondary antibody solution with 1X PBS.

To prepare the collagen gel for mounting, cut the insert membrane with a scalpel. Place the collagen gel attached to the membrane in a glass bottom dish. It is important for the top of the gel to be in contact with the bottom of the dish.

Add mounting media and a coverslip on top for microscopy observation. With this assay, we demonstrated the key role of the DDR1 receptor in the cell invasion in collagen one gel. This assay can be also used to evaluate the effect of drugs on cell invasion.

In our different study, we demonstrated that just adding a new element of extracellular matrix in its physiological confirmation, here type 1 collagen fibrils, can modify the morphology, the molecular composition and the activity of invadosomes. So using the different assays described in this movie, we demonstrated the existence of new structures, a new organization of invadosome that we named linear invadosomes. Generating complex and more physiological matrices, we allow identification of new pathways involved cell addition, migration and invasion.