This work was supported by endowment funds from the West Virginia University Mary Babb Randolph Cancer Center. We thank Susette Mueller (Georgetown University) and Laura Kelley for early advice and assistance. The use of the West Virginia University Microscopy Imaging Facility (supported by the Mary Babb Randolph Cancer Center and, NIH grants P20 RR16440, P30 RR032138 and P30 GM103488) is gratefully acknowledged.

1. Production of Oregon Green 488-gelatin Coated Coverslips 
<ol>
 <li>Prepare an unlabeled 5% (w/w) stock gelatin/sucrose solution by adding 1.25 g gelatin and 1.25 g sucrose in PBS to a final volume of 50 ml. Warm the stock gelatin solution to 37 &deg;C and ensure it is entirely melted before use. Store the final mixture at 4 &deg;C. </li>
 <li>Clean 13 mm diameter #1 glass coverslips by placing an individual coverslip into each well of a 24 well plastic tissue culture plate. Add 500 &mu;l of 20% nitric ac...

<ol>
	<li>Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+at+the+leading+edge.">Life at the leading edge.</a> Cell. 145, 1012-1022 (2011).</li><li>Murphy, D. A., Courtneidge, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'ins'+and+'outs'+of+podosomes+and+invadopodia:+characteristics,+formation+and+function.">The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function.</a> Nat. Rev. Mol. Cell Biol. 12, 413-426 (2011).</li><li>Linder, S., Wiesner, C., Himmel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degrading+Devices:+Invadosomes+in+Proteolytic+Cell+Invasion.">Degrading Devices: Invadosomes in Proteolytic Cell Invasion.</a> Annu. Rev. Cell. Dev. Biol. , (2010).</li><li>Chen, W. T., Chen, J. M., Parsons, S. J., Parsons, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+degradation+of+fibronectin+at+sites+of+expression+of+the+transforming+gene+product+pp60src.">Local degradation of fibronectin at sites of expression of the transforming gene product pp60src.</a> Nature. 316, 156-158 (1985).</li><li>Artym, V. V., Zhang, Y., Seillier-Moiseiwitsch, F., Yamada, K. M., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interactions+of+cortactin+and+membrane+type+1+matrix+metalloproteinase+at+invadopodia:+defining+the+stages+of+invadopodia+formation+and+function.">Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.</a> Cancer Res. 66, 3034-3043 (2006).</li><li>Ayala, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+inputs+converge+on+cortactin+to+control+invadopodia+biogenesis+and+extracellular+matrix+degradation.">Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation.</a> J. Cell Sci. , (2008).</li><li>Ammer, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saracatinib+impairs+head+and+neck+squamous+cell+carcinoma+invasion+by+disrupting+invadopodia+function.">Saracatinib impairs head and neck squamous cell carcinoma invasion by disrupting invadopodia function.</a> J. Cancer Sci. Ther. 1, 52-61 (2009).</li><li>Seals, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptor+protein+Tks5/Fish+is+required+for+podosome+formation+and+function,+and+for+the+protease-driven+invasion+of+cancer+cells.">The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.</a> Cancer Cell. 7, 155-165 (2005).</li><li>Yamaguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+invadopodium+formation:+the+role+of+the+N-WASP-Arp2/3+complex+pathway+and+cofilin.">Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin.</a> J. Cell Biol. 168, 441-452 (2005).</li><li>Kopp, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinesin+KIF1C+and+microtubule+plus+ends+regulate+podosome+dynamics+in+macrophages.">The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages.</a> Mol. Biol. Cell. 17, 2811-2823 (2006).</li><li>Oser, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortactin+regulates+cofilin+and+N-WASp+activities+to+control+the+stages+of+invadopodium+assembly+and+maturation.">Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation.</a> J. Cell. Biol. 186, 571-587 (2009).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36-42 (2004).</li><li>Kelley, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+Src+requires+a+wild-type+counterpart+to+regulate+invadopodia+maturation.">Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation.</a> J. Cell Sci. 123, 3923-3932 (2010).</li><li>Chen, W. T., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+is+not+present+in+the+focal+adhesions+formed+between+normal+cultured+fibroblasts+and+their+substrata.">Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata.</a> Proc. Natl. Acad. Sci. U.S.A. 77, 7318-7322 (1980).</li><li>Chen, W. T., Olden, K., Bernard, B. A., Chu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transformation-associated+protease(s)+that+degrade+fibronectin+at+cell+contact+sites.">Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites.</a> J. Cell Biol. 98, 1546-1555 (1984).</li><li>Bharti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src-dependent+phosphorylation+of+ASAP1+regulates+podosomes.">Src-dependent phosphorylation of ASAP1 regulates podosomes.</a> Mol. Cell Biol. 27, 8271-8283 (2007).</li><li>Albrechtsen, R., Stautz, D., Sanjay, A., Kveiborg, M., Wewer, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+engagement+of+ADAM12+induces+clusters+of+invadopodia+with+localized+ectodomain+shedding+activity.">Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity.</a> Exp. Cell Res. 317 (10), 195-209 (2011).</li><li>Scott, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell Biol. 191, 169-185 (2010).</li><li>Mueller, S. C., Yeh, Y., Chen, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+phosphorylation+of+membrane+proteins+mediates+cellular+invasion+by+transformed+cells.">Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells.</a> J. Cell. Biol. 119, 1309-1325 (1992).</li><li>Bowden, E. T., Coopman, P. J., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia:+unique+methods+for+measurement+of+extracellular+matrix+degradation+in+vitro.">Invadopodia: unique methods for measurement of extracellular matrix degradation in vitro.</a> Methods Cell Biol. 63, 613-627 (2001).</li><li>Baldassarre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin+participates+in+focal+extracellular+matrix+degradation+by+invasive+cells.">Dynamin participates in focal extracellular matrix degradation by invasive cells.</a> Mol. Biol. Cell. 14, 1074-1084 (2003).</li><li>Alexander, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+promotes+invadopodia+activity.">Extracellular matrix rigidity promotes invadopodia activity.</a> Curr. Biol. 18, 1295-1299 (2008).</li><li>Artym, V. V., Yamada, K. M., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+degradation+assays+for+analyzing+local+cell+invasion.">ECM degradation assays for analyzing local cell invasion.</a> Methods Mol. Biol. 522, 211-219 (2009).</li><li>Schoumacher, M., Goldman, R. D., Louvard, D., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin,+microtubules,+and+vimentin+intermediate+filaments+cooperate+for+elongation+of+invadopodia.">Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia.</a> J. Cell Biol. 189, 541-556 (2010).</li><li>Clark, E. S., Whigham, A. S., Yarbrough, W. G., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortactin+is+an+essential+regulator+of+matrix+metalloproteinase+secretion+and+extracellular+matrix+degradation+in+invadopodia.">Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia.</a> Cancer Res. 67, 4227-4235 (2007).</li><li>Yamaguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase+signaling+pathway+mediated+by+p110alpha+regulates+invadopodia+formation.">Phosphoinositide 3-kinase signaling pathway mediated by p110alpha regulates invadopodia formation.</a> J. Cell Biol. 193, 1275-1288 (2011).</li><li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-bundling+protein+fascin+stabilizes+actin+in+invadopodia+and+potentiates+protrusive+invasion.">The actin-bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion.</a> Curr. Biol. 20, 339-345 (2010).</li><li>Yamaguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+4,5-bisphosphate+and+PIP5-kinase+Ialpha+are+required+for+invadopodia+formation+in+human+breast+cancer+cells.">Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Ialpha are required for invadopodia formation in human breast cancer cells.</a> Cancer Sci. , (2010).</li></ol>

No conflicts of interest declared.

The ability to visualize cells degrading the extracellular matrix has aided in discovering the molecular mechanisms employed in the early steps of cell invasion. Pioneered by Wen-Tien Chen in the early 1980's4,14,15, coating fluorescently labeled extracellular proteins on glass coverslips for subsequent microscopic analysis has emerged as the primary technique in evaluating invadopodia function across a wide range of cell types. The prescribed protocol demonstrates the basic method used for preparing ge...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Instrument</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>gelatin</td>
 <td>Sigma</td>
 <td>G1890</td>
 <td>Porcine skin</td>
 </tr>
 <tr>
 <td>sucrose</td>
 <td>Fisher</td>
 <td>BP220</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 mm coverslips</td>
 <td>Fisher</td>
 <td>50-121-5159</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>24 well plates</td>
 <td>Fisher</td>
 <td>08-772-1</td>
 <td>BD Falcon</td>
 </tr>
 <tr>
 <td>nitric acid</td>
 <td>Ricca </td>
 <td>R5326000</td>
 <td>20% solution</td>
 </tr>
 <tr>
 <td>poly-L-lysine</td>
 <td>Electron Microscopy Sciences</td>
 <td>19320-B</td>
 <td>0.1% solution</td>
 </tr>
 <tr>
 <td>glutaraldehyde </td>
 <td>Sigma </td>
 <td>G7526</td>
 <td>8% solution</td>
 </tr>
 <tr>
 <td>Oregon Green 488-conjugated gelatin </td>
 <td>Invitrogen</td>
 <td>G-13186</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>sodium borohydride </td>
 <td>Sigma</td>
 <td>213462</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Fisher</td>
 <td>BP151</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>rhodamine-phalloidin</td>
 <td>Invitrogen</td>
 <td>R415</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>anti-cortactin (clone 4F11)</td>
 <td>Millipore </td>
 <td>05-180 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-FLAG antibody</td>
 <td>Millipore</td>
 <td>MAB3118</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor 647 Goat anti-mouse IgG</td>
 <td>Invitrogen</td>
 <td>A21235</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ProLong Gold antifade </td>
 <td>Invitrogen</td>
 <td>P36930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSM 510 Confocal Microscope </td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ImageJ software</td>
 <td>Public domain</td>
 <td>&nbsp;</td>
 <td><a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a></td>
 </tr>
 <tr>
 <td>AutoQuant X2.2 software</td>
 <td>Media Cybernetics</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NIS Elements software</td>
 <td>Nikon</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

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 ...

quantitative-measurement-invadopodia-mediated-extracellular-matrix

Research

JoVE Journal

Biology

19.1K Views.  West Virginia University. Descriviamo il metodo di prototipo per la produzione di vetrini al microscopio a fluorescenza rivestite con gelatina per la visualizzazione invadopodia-mediata degradazione della matrice. Tecniche di calcolo utilizzando il software disponibile sono presentati per quantificare i livelli risultanti di proteolisi matrice di singole cellule all'interno di una popolazione mista e per gruppi multicellulari che comprende interi campi microscopici.