J.D.M. was supported by a PhD fellowship from INSERM/R&#233;gion Aquitaine and is now supported by a post-doctoral ARC fellowship and the Tisch Cancer Institute at the Mount Sinai School of Medicine. E.H. is supported by a PhD from the Minist&#232;re de l'Enseignement Sup&#233;rieur et de la Recherche. Z.E. is supported by a post-doctoral fellowship from Agence Nationale de la Recherche (ANR). C.M. is supported by the Tisch Cancer Institute at the Mount Sinai School of Medicine and J.J.B.C. is supported by the NIH/NCI grant K22CA196750, the TCI Young Scientist Cancer Research Award JJR Fund, and the Tisch Cancer Institute at Mount Sinai School of Medicine. This work was supported by a grant from ANR-13-JJC-JSV1-0005. F.S. is supported by the "Ligue nationale contre le cancer." V.M. and F.S. are supported by funding from "Equipe Labellis&#233;e Ligue Nationale contre le Cancer 2016" and Institut National du Cancer, INCA_8036, and PLBio2012.

NOTE: Prior to fixation, all steps are completed in a sterile laminar flow hood.
1. 2D matrix
NOTE: 2D matrices are used to determine the percentage of cells forming invadosomes and the matrix degradation area per cell.
<img alt="Figure 1" src="/files/ftp_upload/54911/54911fig1.jpg" /> 
Figure 1: Protocol. Summary of the protocol used to prepare gelatin and collagen I matrices. It is possible to make the gelatin matrix only or a gelatin and collagen I mixed matrix. <a href="http://cloudfront.jove.com/files/ftp_upload/54911/54911fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Gelatin matrix 
	NOTE: Fluorescent gelatin matrices are used to quantify matrix degradation, and non-fluorescent gelatin is used to promote the adhesion of collagen I fibers. Fluorescent gelatin matrices are also used for podosome and invadopodia studies (see Figure 1).
	<ol>
		<li>Under a sterile laminar flow hood, place a piece of parafilm (20 cm x 15 cm) on the work area and disinfect it with 70% ethanol.</li>
		<li>Aliquot 20 &#181;L droplets of 1 mg/mL gelatin in a line, spaced approximately 1 cm apart; refer to Figure 1 for droplet organization.</li>
		<li>Cover each 20 &#181;L gelatin droplet with an autoclaved glass coverslip (12 mm in diameter).</li>
		<li>Incubate for 20 min at room temperature. If the gelatin is fluorescent, protect it from light.</li>
		<li>Aliquot 40 &#181;L droplets of 0.5% glutaraldehyde in 1x phosphate-buffered saline (PBS) in a line, each spaced approximately 1 cm below the line of coverslips from step 1.1.3. 
		CAUTION: Glutaraldehyde is a toxic fixative agent. Use gloves and do not inhale.</li>
		<li>Using jeweled forceps, transfer each gelatin-coated coverslip to a glutaraldehyde droplet. 
		NOTE: It is normal that a small gelatin droplet remains on the parafilm. Do not reuse this for another experiment.</li>
		<li>Incubate for 40 min at room temperature. For fluorescent coverslips, protect them from light.</li>
		<li>Take a 24-well cell culture plate and fill each well with 500 &#181;L of sterile 1x PBS. Place the coverslips, gelatin side up, into each well.</li>
		<li>Wash twice in 1x PBS for 5 min each. 
		NOTE: At this step, the coverslips can be used for experimentation or stored in 1x PBS at 4 &#176;C. Non-fluorescent coverslips can be stored for one week, and fluorescent coverslips can be stored for 48 h.</li>
	</ol>
	</li>
	<li>Fibrillar collagen type I matrix 
	NOTE: Fibrillar collagen type I matrix is used to study linear invadosome formation and the associated matrix degradation. A combination of fluorescent collagen I and non-fluorescent gelatin coverslips is used to colocalize invadosome markers with collagen fibers (e.g., F-actin in the far-red channel, collagen I in the red channel, Tks5 in the green channel, and Hoechst dye for a nuclear stain). On the other hand, a combination of non-fluorescent collagen I with non-fluorescent gelatin coverslips is used to maximize the number of invadosome markers stained (e.g., F-actin in the far-red channel, cortactin in the red channel, Tks5 in the green channel, and Hoechst dye for a nuclear stain). Finally, a combination of non-fluorescent collagen I with fluorescent gelatin coverslips is used to quantify the gelatin-associated degradation (e.g., F-actin in the far-red channel, Tks5 in the red channel, in the green channel, and Hoechst dye for a nuclear stain) (see Figure 1). Hoechst dye is used to stain the nucleus, and it also checks for mycoplasma contamination, in addition to a PCR test done regularly on the cell lines used.
	<ol>
		<li>Fluorescent collagen I
		<ol>
			<li>Use commercial collagen I extracted from rat-tail tendon in 0.02 N acetic acid. Withdraw the quantity of collagen I necessary to prepare 500 &#181;L of solution per coverslip at a final concentration of 0.4 mg/mL of collagen I.</li>
			<li>Add 5-carboxy x rhodamine succinimidyl ester at a final concentration of 10 &#181;g/mL, calculated for the final volume of collagen I solution (n x 500 &#181;L).</li>
			<li>Mix the solution by pipetting up and down and incubate for 5 min at room temperature, protected from light.</li>
			<li>Dilute to the final volume with 1X Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) containing calcium and magnesium, and use directly.</li>
		</ol>
		</li>
		<li>Fibrillar collagen I coverslip
		<ol>
			<li>Use the non-fluorescent or fluorescent gelatin coverslips previously prepared in step 1.1.</li>
			<li>Prepare a solution of fluorescent or non-fluorescent collagen I at a final concentration of 0.4 mg/mL in 1X DPBS containing calcium and magnesium.</li>
			<li>Add 500 &#181;L of fluorescent or non-fluorescent collagen I solution per coverslip in a 24-well cell culture plate.</li>
			<li>Incubate for 4 h at 37 &#176;C for collagen I polymerization.</li>
			<li>After incubation, remove the excess collagen I by tilting the plate and pipetting on the side of the well (not directly on the coverslip) to avoid damaging the matrix. 
			NOTE: This matrix cannot be stored. Seed the cells immediately after removing excess collagen I.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Cell seeding, time of culture, and fixation
<ol>
	<li>Depending on the cell type, prepare the cells in the appropriate culture medium and add cells for a final volume of 500 &#181;L per well.</li>
	<li>To characterize the ability of a cell line to form linear invadosomes and to degrade the matrix, plate the cells for 4 h, 12 h, and 24 h on collagen I matrix to determine the kinetics of linear invadosome formation and the associated degradation activity. 
	NOTE: Table 1 contains examples of cell lines tested in the laboratory.
	<table border="1">
		<tbody>
			<tr>
				<td>Cell line</td>
				<td>Number of cells per coverslip</td>
				<td>Time of contact with matrix for&#160; linear invadosome quantification</td>
				<td>Time of contact with matrix for&#160; quantification of&#160; linear invadosome degradation activity</td>
			</tr>
			<tr>
				<td>MDA MB 231</td>
				<td>40,000</td>
				<td>4 h</td>
				<td>12 h</td>
			</tr>
			<tr>
				<td>HUH7</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>24 h&#160;</td>
			</tr>
			<tr>
				<td>HEP 3B</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>24 h&#160;</td>
			</tr>
			<tr>
				<td>SNU 398</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>24 h&#160;</td>
			</tr>
			<tr>
				<td>NIH 3T3 src</td>
				<td>30,000</td>
				<td>4 h</td>
				<td>12 h</td>
			</tr>
			<tr>
				<td>A431</td>
				<td>50,000</td>
				<td>6 h</td>
				<td>24 h&#160;</td>
			</tr>
			<tr>
				<td>A549</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>24 h&#160;</td>
			</tr>
			<tr>
				<td>RAW</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>12 h</td>
			</tr>
			<tr>
				<td>PAE</td>
				<td>40,000</td>
				<td>12 h</td>
				<td>12 h</td>
			</tr>
		</tbody>
	</table>
	Table 1: Cell seeding and incubation time reference. This table presents a non-exhaustive list of cell lines used to study linear invadosomes. It indicates the number of cells to seed on the matrix, the time necessary to observe linear invadosomes, and the time necessary to observe the associated matrix degradation. Linear invadosomes have a short lifetime. To be able to observe linear invadosomes in the maximum number of cells, the cells have to be in contact with the collagen I matrix for a short period of time. On the other hand, to be able to visualize the reported gelatin degradation, the cells must be in contact with the collagen I matrix for a longer period of time than those listed above.</li>
	<li>After the removal of the cell culture medium, fix the coverslips for 10 min in 500 &#181;L of 4% paraformaldehyde in 1X PBS.</li>
	<li>Wash twice with 1x PBS.</li>
	<li>Store at 4 &#176;C, protected from light.</li>
</ol>
3. Immunofluorescence
<ol>
	<li>Permeabilize the cells for 10 min with a freshly prepared solution of 0.2% Triton X-100 in 1x PBS. Do not reuse this solution.</li>
	<li>Wash the coverslips twice with 1x PBS.</li>
	<li>Place a piece of parafilm (20 cm x 15 cm) on the bench.</li>
	<li>Dilute primary antibody in 4% bovine serum albumin (BSA) in 1x PBS, as described in the Materials Table.</li>
	<li>Aliquot 50 &#181;L droplets of primary antibody solution in a line, each spaced approximately 1 cm apart.</li>
	<li>Place each coverslip on a droplet.</li>
	<li>Incubate for 40 min at room temperature, protected from light.</li>
	<li>After the incubation period, wash twice with 1x PBS.</li>
	<li>Dilute the secondary antibody, phalloidin, and Hoechst dye in 4% BSA in 1x PBS.</li>
	<li>Aliquot 50 &#181;L droplets of the mix, each spaced approximately 1 cm below the first line of coverslips, as done previously.</li>
	<li>Place each coverslip on a droplet.</li>
	<li>Incubate for 30 min at room temperature, protected from light.</li>
	<li>After incubation, wash twice with 1x PBS.</li>
	<li>Wash once with distilled water to remove the salts.</li>
	<li>Mount the coverslips on a glass slide with polymerizing mounting medium.</li>
	<li>Let the coverslips dry overnight at room temperature, protected from light, before microscopy.</li>
</ol>
4. Quantifications
<img alt="Figure 2" src="/files/ftp_upload/54911/54911fig2.jpg" /> 
Figure 2: Linear invadosome quantification. The macro requires confocal images of F-actin and Tks5 staining (format: 1,024 x 1,024). (A) First, open the F-actin picture and make a mask. (B) Then, open the Tks5 picture and threshold it. Apply the macro. (C) 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. <a href="http://cloudfront.jove.com/files/ftp_upload/54911/54911fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Percentage of cells forming linear invadosomes
	<ol>
		<li>Count the cells forming linear invadosomes, approximately 100 cells per coverslip (use technical triplicates). Quantify at least 300 cells per n, with n = 3.</li>
		<li>Calculate the percentage of cells forming linear invadosomes by taking the average of three independent experiments, resulting in 900 quantified cells.</li>
	</ol>
	</li>
	<li>Linear invadosome size and number per cell 
	NOTE: Use a confocal microscope to take sequential images of the F-actin and Tks5 stains. The macro is adapted to a 1,024 x 1,024 image format.
	<ol>
		<li>Use ImageJ with the supplementary macro.</li>
	</ol>
	</li>
	<li>Degradation per cell
	<ol>
		<li>Use three coverslips per condition.</li>
		<li>Take 30 images per coverslip of fluorescent gelatin and Hoechst-stained nuclei.</li>
		<li>Use the ImageJ software as described in Martin KH et al.9.</li>
	</ol>
	</li>
</ol>
5. 3D Collagen Invasion Assay
<img alt="Figure 3" src="/files/ftp_upload/54911/54911fig3.jpg" /> 
Figure 3: Invasion assay schema. Summary of the invasion assay protocol. Collagen I is cross-linked with succinimidyl ester dye and polymerized for 1 h at 37 &#176;C. Cells are added in serum-free medium on top of the collagen I plug. The insert is placed in medium with 10% FBS. Collagen I plugs are fixed 1 h after cell seeding in the control and 3 days after cell seeding in the experimental condition to determine the invasion capacity of the cells. <a href="http://cloudfront.jove.com/files/ftp_upload/54911/54911fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Collagen I plug preparation
	<ol>
		<li>Make a time course of 4 h, 12 h, 24 h, 48 h, and 72 h for the first experiment to determine the kinetics of invasion into the gel according to the cell type. For each experiment, add a 1 h time point as a reference time point when no invasion occurs.</li>
		<li>Prepare the collagen I solution on ice under a sterile laminar flow hood. Prepare 500 &#181;L of solution for 3 Boyden chamber inserts.</li>
		<li>Aspirate 1 mg of commercial rat-tail collagen I to prepare a solution with a final concentration of 2 mg/mL collagen I.</li>
		<li>Add 5-carboxy x Rhodamine succinimidyl ester at a final concentration of 1 &#181;g/mL, calculated for the final volume of collagen I solution (in this case, 500 &#181;L).</li>
		<li>Mix well and incubate for 5 min on ice under the sterile laminar flow hood, protected from light.</li>
		<li>Add 50 &#181;L of ice-cold, filtered 10X PBS and 6.25 &#181;L of ice-cold 1 M sodium hydroxide (NaOH).</li>
		<li>Add sterile water according to the formula 443.75 - x &#181;L of collagen I.</li>
		<li>Add 100 &#181;L of solution per Boyden chamber insert.</li>
		<li>Incubate for 1 h at 37 &#176;C to allow for polymerization.</li>
		<li>Add cell culture medium enriched with fetal bovine serum (FBS) into new wells; place inserts into these wells.</li>
		<li>Seed 30,000 cells on top of the collagen I plug in FBS-free medium.</li>
		<li>After culturing the cells at 37 &#176;C, fix the collagen I plug for 30 min in 4% paraformaldehyde in 1x PBS.</li>
		<li>Complete immunofluorescence as in step 3, with the exception of the following steps: permeabilize with 0.2% Triton X-100 in 1x PBS for 30 min, not 10 min; incubate with primary and secondary antibody solutions for 1.5 h each, rather than 40 min and 30 min, respectively. Stain for F-actin or nuclei to localize the cells.</li>
		<li>After immunofluorescence, use a scalpel to cut around the membrane of the insert and carefully transfer the collagen I plug to a glass-bottom dish. Make sure that the top of the collagen I plug is in contact with the glass. Keep the insert membrane attached to the bottom of the collagen I plug in order to maintain the integrity of the plug. 
		NOTE: The collagen I plug remains intact in the glass-bottom dish. The depth between the glass bottom and the plastic surrounding the glass bottom is equivalent to the thickness of the plug.</li>
		<li>Place a coverslip on the collagen I plug and mount with polymerizing mounting medium to prevent the plug from drying out.</li>
		<li>Use an inverted confocal microscope to image the collagen I plug; the top of the plug is in contact with the glass bottom of the dish.
		<ol>
			<li>Acquire a z-stack from the top to the bottom of the collagen I plug to visualize cell invasion. Determine the thickness of the z-stack by examining how deeply the cells invade the imaging field. Perform acquisitions with a 40X oil objective in a 512 x 512 format with a z-step size of 0.25 &#956;m. 
			NOTE: For reference, the average z-stack for MDA-MB-231 cells 3 days after seeding is 30 &#181;m.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Classically, invadosomes are studied in vitro without regard to the microenvironment and the matrix on which the cells are plated. Several types of matrices are currently used, including gelatin, fibronectin, vitronectin, or high-density fibrillar collagen (HDFC)7,11; however, these are often not representative of the microenvironment in which cells reside and are not physiologically relevant. Here, a novel type of matrix, which consists of an associatio...

Extracellular matrix (ECM) remodeling occurs during physiological and pathological processes, such as angiogenesis and tumor cell invasion. In physiological conditions, many cell types, mostly from the hematopoietic lineage, are able to degrade ECM elements. For example, macrophages are able to cross anatomical barriers to reach tissues, and osteoclasts degrade bone matrix to ensure calcium homeostasis. More globally, all matrices in the body renew to maintain their physical and chemical properties. In cancer tissues, the tumor microenvironment composition is altered. For example, in breast and lung cancer, type I collagen is overexpressed and accumulates around the tumor. Moreover, this accumulation is associated with an increased risk of developing metastasis1,2. Cancer metastasis is dependent on the ability of cancer cells to degrade the ECM and invade adjacent tissues.
The invasive activity of cells is attributed to specialized actin-rich structures known as invadosomes. This term includes podosomes and invadopodia, which are present, respectively, in normal and cancer cells (e.g., macrophages, endothelial cells, and cancer cells such as the MDA-MB-231 breast cancer cell line). In vitro, invadosomes can organize into different shapes: dots, aggregates, or rosettes3. Classically, invadosomes are composed of an F-actin core containing several proteins, such as the scaffold protein Tks5 and cortactin, surrounded by adhesion plaque molecules, such as integrins and vinculin4. Recently, it was shown that Tks5 and the RhoGTPase Cdc42 can be used as a minimum molecular signature for functional invadosomes5. These structures are able to degrade the ECM via the recruitment and activation of specific metalloprotease proteins, such as MT1-MMP and MMP-26. In a previous study, we reported that the interaction between type I collagen fibrils and the discoidin domain receptor 1 (DDR1), a specific receptor of type I collagen fibrils, leads to the formation of a new class of invadosomes, named linear invadosomes. Linear invadosomes are formed along type I collagen fibrils7. These structures are able to degrade type I collagen fibrils via the recruitment and activation of MT1-MMP/MMP2. Moreover, their formation is dependent on Tks5 and Cdc425. In vitro, we demonstrated the existence of linear invadosomes and the involvement of DDR1 in their formation and activity8 using different strategies consisting of a combination of various matrix elements, including: i) fluorescent gelatin-coated coverslips, ii) fluorescent type I collagen fibril-coated coverslips, and iii) 3D type I collagen plugs. Due to the use of different matrices, we were able to study and characterize linear invadosome formation and their degradation activity7,8.
Finally, to better understand the biology of invasive cells, a combination of ECM components in both 2D and 3D culture systems were used, mimicking the complexity of the tumor microenvironment composition. Below are protocols for coating coverslips with gelatin/type I collagen in 2D and 3D culture systems.

<table><tbody><tr><td>Gelatin solution</td><td>Sigma</td><td>G1393</td><td>Stock 20 mg/ml used at 1 mg/ml</td></tr><tr><td>Gelatin from pig skin Oregon green 488 conjugate</td><td>Molecular probe life technologies</td><td>G13186</td><td>5mg dilute at 1 mg/ml in sterile water</td></tr><tr><td>Microscope cover glasses</td><td>Marlenfeld GmbH &amp;amp; Co</td><td>111520</td><td>&amp;Oslash;12mm No.1</td></tr><tr><td>2.5% Glutaraldehyd in 0.1M sodium cacodylate buffer pH 7.4</td><td>Electron microscopy science</td><td>15960</td><td>Dilute at 0.5 % in sterile water</td></tr><tr><td>1x PBS pH 7.4</td><td>Gibco by life technologies</td><td>10010-015</td><td>Use this PBS in all steps before fixation</td></tr><tr><td>Collagen I Rat tail</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>5 carboxy X rhodamin siccinimidyl ester</td><td>Life technologies</td><td>C-6125</td><td></td></tr><tr><td>DPBS 1x + calcium + magnesium</td><td>Gibco by life technologies</td><td>14040-091</td><td></td></tr><tr><td>Paraformaldehyde 16% solution</td><td>Electron microscopy science</td><td>15710</td><td>Dilute at 4% in 1X PBS</td></tr><tr><td>Triton X 100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>10x PBS buffer pH 7.4</td><td>Ambion</td><td>AM9625</td><td>Dilute at 1X in water Use in steps after fixation</td></tr><tr><td>Tks5 antibody</td><td>Santa Cruz</td><td>sc-30122</td><td>Invadosome markers Dilution : 1/100</td></tr><tr><td>Cortactin 4F11 antibody</td><td>Millipore</td><td>5180</td><td>Invadosome markers Dilution :1/100</td></tr><tr><td>DDR1 antibody</td><td>Cell signaling</td><td>5583</td><td>Linear invadosome receptor</td></tr><tr><td>Dilution :1/100</td><td></td><td></td><td></td></tr><tr><td>Phalloidin FluoProbes</td><td>Interchim</td><td>FT-AZ0330</td><td>Fibrillary actin marker Dilution :1/200</td></tr><tr><td>Hoechst</td><td>Sigma</td><td>33258</td><td>Nucleus marker Dilution :1/200</td></tr><tr><td>Secondary antibodies FluoProbes</td><td>Interchim</td><td>FP-488 FP-547H or FP-647H</td><td></td></tr><tr><td>Albumin from bovine serum</td><td>Sigma</td><td>A2153</td><td>Dilute at 4% in 1X PBS</td></tr><tr><td>Fluoromount G mounting medium</td><td>Interchim</td><td>FP 483331</td><td></td></tr><tr><td>ImageJ software</td><td>Public domain</td><td></td><td>http://www.macbiophotonics.ca/imagej/</td></tr><tr><td>Cell culture insert</td><td>Corning</td><td>353097</td><td>8.0&amp;micro;m pore size / 24 wells</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma</td><td>221465</td><td></td></tr></tbody></table>

2d and 3d matrices to study linear invadosome formation and activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

Using a combination of two types of matrices, gelatin and type I collagen (Figures 1 and 4), we have highlighted a new type of invadosome, known as linear invadosomes. The labelling of these matrices allows for the observation of linear invadosome formation along collagen I fibers and of their degradation capabilities (Figure 5). The number of invasive structures can then be quantified by the macro described previously (step 4.2), and the...

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

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

2d-and-3d-matrices-to-study-linear-invadosome-formation-and-activity

Research

JoVE Journal

Bioengineering

9.9K Views.  INSERM U1053. This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines. 

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

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Cancer Research

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Medicine

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Biology

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

Immunology and Infection

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain. This method includes a MATLAB script for analyzing TrackMate output files to calculate displacements, motility rates, and trajectory angles in single cells and in bulk for an imaging site. This motility analysis script allows for quick, straightforward, and scalable analysis of cell motility rates from TrackMate data and could be broadly used to identify and study the regulation of motility in epithelial cells. We also provide a MATLAB script for reorganizing microscopy videos collected on a microscope and converting them to TIF stacks, which can be analyzed using the ImageJ TrackMate plugin in bulk. Using this methodology to explore the roles of adherens junctions and actin cytoskeletal dynamics in regulating cell motility in HaCaT keratinocytes, we demonstrate evidence that Arp2/3 activity is required for the elevated motility seen after &#945;-catenin depletion in HaCaT keratinocytes.

This protocol for high-throughput measurement of cell motility in HaCaT keratinocytes describes methods for collecting and processing images of cell nuclei and performing particle tracking using the ImageJ plugin TrackMate.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To ...

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

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Przeglądaj więcej filmów

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking