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 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.
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.
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.
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. Please click here to view a larger version of this figure.
2. Cell seeding, time of culture, and fixation
Cell line | Number of cells per coverslip | Time of contact with matrix for linear invadosome quantification | Time of contact with matrix for quantification of linear invadosome degradation activity |
MDA MB 231 | 40,000 | 4 h | 12 h |
HUH7 | 40,000 | 12 h | 24 h |
HEP 3B | 40,000 | 12 h | 24 h |
SNU 398 | 40,000 | 12 h | 24 h |
NIH 3T3 src | 30,000 | 4 h | 12 h |
A431 | 50,000 | 6 h | 24 h |
A549 | 40,000 | 12 h | 24 h |
RAW | 40,000 | 12 h | 12 h |
PAE | 40,000 | 12 h | 12 h |
3. Immunofluorescence
4. Quantifications
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. Please click here to view a larger version of this figure.
5. 3D Collagen Invasion Assay
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 °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. Please click here to view a larger version of this figure.
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 degradation activity can also be determined using the ImageJ software, as describe by Martin et al. and Diaz et al.9,10. The mixed matrix allows for the characterization of linear invadosomes by defining DDR1 as the receptor necessary for linear invadosome formation and functionality (Figure 6).
In order to understand the invasive capacity of cells, we have also developed a 3D type I collagen plug assay (Figures 3 and 7). This assay allows us to determine the number of cells that have invaded into the collagen I plug, as well as the distance traveled, by using z-stack reconstructions.
Figure 4: Comparison of gelatin and gelatin-collagen I mixed matrices using confocal microscopy. Schematic representation showing the organization of fluorescent gelatin coverslips on the top panel. (A) Confocal z-stack reconstruction demonstrates that the fluorescent gelatin matrix forms a thin, uniform layer that covers the entire surface of the coverslip. (B) In the mixed matrix, after the deposition of fluorescent gelatin on the coverslips, type I collagen fibrils polymerize on top. The collagen I fibrils are stained in red. The collagen I matrix is thick and heterogeneous on the coverslip. The distribution of the collagen I fibers is dependent on the polymerization of collagen I α chains. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 5: Impact of gelatin and gelatin-collagen I matrices on invadosome formation and activity. The cells used for these assays are MDA-MB-231 breast cancer cells. (A) A schematic representation shows cells seeded on a fluorescent gelatin coverslip on the top panel. Degradation areas are visualized in black due to the reduction of fluorescence. Tks5 staining was used as an invadosome marker. On gelatin, the invadosomes that form are organized as dots. (B) A schematic representation shows cells seeded on a mixed matrix of gelatin and collagen I. Collagen I is labeled in red; the addition of type I collagen fibrils increases the ability of the cell to degrade gelatin. Interestingly, the invadosome marker Tks5 is reorganized, and the dots are replaced by linear structures representing linear invadosomes. Degradation areas are visualized in black due to the reduction of fluorescence. Scale bar = 5 µm. Please click here to view a larger version of this figure.
Figure 6: Confocal microscopy analysis of the molecular composition and organization of invadosomes in both gelatin and mixed-matrix conditions. (A) In the gelatin condition, invadosomes are organized in dots, and F-actin (red) co-localizes with Tks5 (green, bottom panel), but not with DDR1 (green, top panel). These are classical invadosomes. Scale bars = 5 µm. (B) In the gelatin-collagen I mixed-matrix condition, DDR1 (green, top panel) colocalizes with the collagen I fibrils (red, top panel), Tks5 (green, bottom panel), and F-actin (red, bottom panel). The type I collagen fibrils induce the formation of linear invadosomes. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 7: Cell invasion assay of MDA-MB-231 cells in a collagen I plug. 1 h after seeding the cells, control inserts are fixed and stained. (A) z-stack acquisition of the collagen I plug is performed using confocal microscopy. The cells do not invade the collagen I plug at this time point; rather, they remain on top of the collagen I plug. Thus, this is used as the control time point when no invasion occurs. MMP activation is necessary for the cells to invade a collagen I plug at this density. (B) Three days after seeding, some cells invade the collagen I plug. This assay allows for the observation and quantification of cell invasion. In addition, it can be used to study the impact of various drugs or siRNAs, for example. Scale bar = 20 µm. Please click here to view a larger version of this figure.
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 association between gelatin and fibrillar type I collagen, was used. The use of type I collagen fibrils allows us to highlight a novel class of invadosomes, known as linear invadosomes, which specifically form on collagen I in its physiological architecture7. Labeling the collagen I allows us to observe linear invadosomes along the fibers and to quantify their formation using cellular markers such as Tks5 and cortactin. Using the mixed matrix, we have identified a new receptor, the discoidin domain receptor 1 (DDR1), involved in the formation of linear invadosomes8.
The 2D mixed matrix allows us to quantify the matrix degradation activity of linear invadosomes via the zymography in situ assay. This assay reports the proteolysis activity of MMPs by analyzing the presence of black holes in the fluorescent gelatin layer. Interestingly, the organization of F-actin into linear invadosomes increases the matrix degradation activity compared to gelatin-only7. One limitation of this assay is that the gelatin is a non-physiological matrix, and it has been demonstrated that a more physiological matrix changes the cellular response to the microenvironment. An additional limitation exists in the way in which MMP activity is quantified on the gelatin-collagen I mixed matrices. Only the gelatin degradation that localizes under the collagen I fibril layer can be quantified; therefore, this is an indirect method to quantify collagen I matrix degradation. An alternative method to visualize the degradation activity of linear invadosomes is to label the collagen I fibers with a specific antibody against collagen cleavage sites (Col1-3/4C, immunoglobulin). This allows for the visualization of cleaved fibers by immunofluorescence. However, due to cell migration, this antibody is not very specific in 2D for degradation due to linear invadosome formation. Another way to visualize and quantify the degradation activity of the collagen I fibers is to use multiphoton microscopy and second harmonic generation8. This method allows for the imaging of collagen I fibers without any staining.
Our method of polymerizing type I collagen is different compared to other methods used in the literature12,13. For example, Artym et al. centrifuge coverslips coated with an alpha helix collagen I solution and allow for a brief polymerization of 30 min. This high-density fibrillar collagen (HDFC) is much stiffer and denser than our collagen I fibers but is less polymerized. This difference in matrix structure shifts the invadosome shape from linear to dot-like. The time, pH, concentration, and temperature are parameters that have to be controlled for ECM polymerization. For example, the lower the temperature of polymerization, the larger the fibers will be.
To study the involvement of linear invadosomes in cell invasion, the 3D collagen I plug assay was used. The 3D collagen I plug is already used by the scientific community in order to study invasive structures or the involvement of metalloproteinases in the invasion process14,15,16. These types of 3D matrices have limits regarding their rigidity-for example, the 3D collagen I plug is less rigid than the 2D matrix. Moreover, the type and origin of the collagen I is also important regarding the different organization of collagen fibers in vivo17. Finally, in regards to the microenvironment composition in vivo, only one element of the extracellular matrix, the type I collagen, was focused on. Further study is necessary to determine the relevance of linear invadosomes in vivo.
In addition to the study of linear invadosomes, the mixed matrix, described herein, could be used with other types of matrix components, such as fibronectin, vitronectin, and other types of collagens (e.g., type IV collagen). This protocol could be adapted, depending on the types of matrix elements that are of interest and the processes to be studied. However, it is clear that generating complex and physiological matrices will allow for the identification of new pathways involved in cell adhesion, migration, and invasion.
The authors have nothing to disclose.
J.D.M. was supported by a PhD fellowship from INSERM/Région 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 Ministère de l’Enseignement Supérieur 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 Labellisée Ligue Nationale contre le Cancer 2016” and Institut National du Cancer, INCA_8036, and PLBio2012.
Gelatin solution | Sigma | G1393 | Stock 20 mg/ml used at 1 mg/ml |
Gelatin from pig skin Oregon green 488 conjugate | Molecular probe life technologies | G13186 | 5mg dilute at 1 mg/ml in sterile water |
Microscope cover glasses | Marlenfeld GmbH & Co | 111520 | Ø12mm No.1 |
2.5% Glutaraldehyd in 0.1M sodium cacodylate buffer pH 7.4 | Electron microscopy science | 15960 | Dilute at 0.5 % in sterile water |
1X PBS pH 7.4 | Gibco by life technologies | 10010-015 | Use this PBS in all steps before fixation |
Collagen I Rat tail | Corning | 354236 | |
5 carboxy X rhodamin siccinimidyl ester | Life technologies | C-6125 | |
DPBS 1X + calcium + magnesium | Gibco by life technologies | 14040-091 | |
Paraformaldehyde 16% solution | Electron microscopy science | 15710 | Dilute at 4% in 1X PBS |
Triton X 100 | Sigma | T9284 | |
10X PBS buffer pH 7.4 | Ambion | AM9625 | Dilute at 1X in water Use in steps after fixation |
Tks5 antibody | Santa Cruz | sc-30122 | Invadosome markers Dilution : 1/100 |
Cortactin 4F11 antibody | Millipore | 5180 | Invadosome markers Dilution :1/100 |
DDR1 antibody | Cell signaling | 5583 | Linear invadosome receptor |
Dilution :1/100 | |||
Phalloidin FluoProbes | Interchim | FT-AZ0330 | Fibrillary actin marker Dilution :1/200 |
Hoechst | Sigma | 33258 | Nucleus marker Dilution :1/200 |
Secondary antibodies FluoProbes | Interchim | FP-488 FP-547H or FP-647H | |
Albumin from bovine serum | Sigma | A2153 | Dilute at 4% in 1X PBS |
Fluoromount G mounting medium | Interchim | FP 483331 | |
ImageJ software | Public domain | http://www.macbiophotonics.ca/imagej/ | |
Cell culture insert | Corning | 353097 | 8.0µm pore size / 24 wells |
Sodium hydroxide (NaOH) | Sigma | 221465 |