The authors gratefully acknowledge Dr Vasily Gurchenkov (Institut Curie) for image acquiring and processing in Figure 3 and PICT-IBISA Imaging Facility (Institute Curie). This work was supported by ANR-09-JCJC0023-01, ARC SFI20111203863 and PIC 3D &#8211; Complex in vitro cellular models.

1. TAMRA-collagen I Labeling
<ol>
	<li>Prepare a 10 mg/ml TAMRA solution by adding 2.5 ml DMSO to the supplied 25 mg TAMRA powder. Dissolve it by vortexing until complete dissolution. Store at -20 &#186;C and protect from light.</li>
	<li>Prepare 2 L of Labeling Buffer (0.25 M NaHCO3, 0.4 M NaCl). Adjust pH to 9.5 using 10 M solution of NaOH. Keep at 4 &#186;C. From this point, all operations are carried out at 4 &#186;C unless otherwise stated and fluorescent material is protected from light using aluminum ...

<ol>
	<li>Schiro, J. A., Chan, B. 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=Integrin+alpha+2+beta+1+(VLA-2)+mediates+reorganization+and+contraction+of+collagen+matrices+by+human+cells.">Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells.</a> Cell. 67 (2), 403-410 (1991).</li><li>Klein, C. E., Dressel, D., 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=Integrin+alpha+2+beta+1+is+upregulated+in+fibroblasts+and+highly+aggressive+melanoma+cells+in+three-dimensional+collagen+lattices+and+mediates+the+reorganization+of+collagen+I+fibrils.">Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.</a> J. Cell Biol. 115 (5), 1427-1436 (1991).</li><li>Friedl, P., Maaser, K., Klein, C. E., Niggemann, B., Krohne, G., Z&#228;nker, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+highly+aggressive+MV3+melanoma+cells+in+3-dimensional+collagen+lattices+results+in+local+matrix+reorganization+and+shedding+of+alpha2+and+beta1+integrins+and+CD44.">Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44.</a> Cancer Res. 57 (10), 2061-2070 (1997).</li><li>Wirtz, D., Konstantopoulos, K., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+of+cancer:+the+role+of+physical+interactions+and+mechanical+forces+in+metastasis.">The physics of cancer: the role of physical interactions and mechanical forces in metastasis.</a> Nat. Rev. Cancer. 11 (7), 512-522 (2011).</li><li>Zaman, M. H., Trapani, L. 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=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (29), 10889-10894 (2006).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J. Cell Biol. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., Feng, Y., 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=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat. Cell Biol. 12 (6), 598-604 (2010).</li><li>Harunaga, J. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+adhesions+in+3D.">Cell-matrix adhesions in 3D.</a> Matrix Biol. 30 (7-8), 363-368 (2011).</li><li>Raub, C. B., Suresh, V., 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=Noninvasive+assessment+of+collagen+gel+microstructure+and+mechanics+using+multiphoton+microscopy.">Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.</a> Biophys. J. 92 (6), 2212-2222 (2007).</li><li>Geraldo, S., Simon, A., Elkhatib, N., Louvard, D., Fetler, L., 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=Do+cancer+cells+have+distinct+adhesions+in+3D+collagen+matrices+and+in+vivo?.">Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?.</a> Eur. J. Cell Biol. 91 (11-12), 930-937 (2012).</li><li>Geraldo, S., Gordon-Weeks, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+in+growth-cone+steering.">Cytoskeletal dynamics in growth-cone steering.</a> J. Cell Sci. 122 (Pt 20), 3595-3604 (2009).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol. Bioeng. 83 (2), 173-180 (2003).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J. Vis. Exp. (51), (2011).</li><li>Haji-Karim, M., Carlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+viability+in+cellular+spheroids+of+human+origin.">Proliferation and viability in cellular spheroids of human origin.</a> Cancer Res. 38 (5), 1457-1464 (1978).</li><li>Eyre, D. R., Paz, M. A., Gallop, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+in+collagen+and+elastin.">Cross-linking in collagen and elastin.</a> Ann. Rev. Biochem. 53, 717-748 (1984).</li><li>Baici, A., Cohen, G., Fehr, K., B&#246;ni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+handy+assay+for+collagenase+using+reconstituted+fluorescein-labeled+collagen+fibrils.">A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils.</a> Anal. Biochem. 108 (2), 230-232 (1980).</li><li>Stein, A. M., Vader, D. A., Jawerth, L. M., Weitz, D. A., Sander, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+algorithm+for+extracting+the+network+geometry+of+three-dimensional+collagen+gels.">An algorithm for extracting the network geometry of three-dimensional collagen gels.</a> J. Microsc. 232 (3), 463-475 (2008).</li><li>Sabeh, F., Ota, 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=Tumor+cell+traffic+through+the+extracellular+matrix+is+controlled+by+the+membrane-anchored+collagenase+MT1-MMP.">Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.</a> J. Cell Biol. 167 (4), 769-781 (2004).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Curr. Prot. Cell Biol. Chapter 10, Unit 10.18.1-Unit 10.18.20 (2010).</li><li>Jawerth, L. M., M&#252;nster, S., Vader, D. A., Fabry, B., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blind+spot+in+confocal+reflection+microscopy:+the+dependence+of+fiber+brightness+on+fiber+orientation+in+imaging+biopolymer+networks.">A blind spot in confocal reflection microscopy: the dependence of fiber brightness on fiber orientation in imaging biopolymer networks.</a> Biophys. J. 98 (3), L1-L3 (2010).</li><li>Cicchi, R., Vogler, N., Kapsokalyvas, D., Dietzek, B., Popp, J., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+molecular+structure+to+tissue+architecture:+collagen+organization+probed+by+SHG+microscopy.">From molecular structure to tissue architecture: collagen organization probed by SHG microscopy.</a> J. Biophotonics. 6 (2), 129-142 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The protocol described here to fluorescently label collagen I using TAMRA provides an excellent method to allow easy visualization of the collagen network organization, by using a standard confocal microscope equipped with a 561 nm laser. An advantage of this technique comparing to reflectance confocal microscopy is the ability to image collagen fibers deeper into the 3D matrix. The intensity and contrast of the fibers reflection diminishes substantially with depth due to laser light absorption and scattering. Also, the ...

The field of cell migration has been braving into the brand new third dimensional world. It is intuitive to study cell migration in an environment that most closely resembles the physiological one and, therefore, three-dimensional (3D). However, due to technical limitations, most cell migration studies have been done analyzing cell movement across rigid two-dimension (2D) substrates, either untreated or coated with appropriate extracellular matrix (ECM) proteins.
The first studies dedicated to cell migration in three dimensional collagen lattices go back over 20 years1-3. However, only over the past 5 years it has become clear that migratory cells could substantially differ in their morphology and mode of migration depending on the dimensionality of the substrate. In 2D, cells only contact the substrate with their ventral surface using focal adhesions, resulting in the formation of broad flat protrusions (lamellipodia) with embedded finger-like protrusions (filopodia) at their leading edge. These structures, together with stress fibers that connect the cell front to the trailing edge, are believed to be crucial for cell movement in 2D. In 3D matrices, cells are generally more elongated, with the entire cell surface contacting the ECM, causing considerable changes in the formation and functional relevance of many of these structures. Conversely, other cellular features gain relevance in 3D migration, such as nuclear deformation and structures involved in ECM remodeling4.
Despite these known morphological alterations, as well as differences in migration modes5-7, which can vary depending on the ECM and cell types, structural and functional analysis of cells embedded within 3D matrices still remains unusual. Working with thick and dense 3D matrices carries technical difficulties, such as high-resolution microscopy imaging, and incompatibilities with most standard protocols optimized for 2D cultures, like immunofluorescent labeling of endogenous proteins. Also, because the use of 3D matrices is a relatively new approach, researchers are still investigating the best conditions to closely resemble specific in vivo situations, such as the normal stromal architecture of different tissue organs or the ECM organization around a tumor. Discrepancies in results by different groups concerning, for instance, cancer cell modes of migration or the existence of focal adhesions, have generated some controversy8. A lot of effort has been recently dedicated to reach a consensus in terms of ECM chemical nature, pore size, fiber thickness, and matrix stiffness. Many different types of 3D ECMs are currently used, varying from cell derived matrices to commercially available matrigel, pepsinized bovine collagen I, or nonpepsinized rat tail collagen I. Each of these matrices has specific physical and chemical properties and one needs to relate the matrix of choice to the physiological process being studied. In addition, pore size and fiber thickness can depend on polymerization conditions, such as pH and temperature9,10. Binding to and distance from rigid substrates such as glass, can also change the elastic properties of the matrix10,11.
This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. Methods for making cancer cell spheroids have previously been described, the most popular ones being the hanging drop method12,13 and the agarose-coated plate method14. As an appropriate ECM substrate for cancer cell migration, nonpepsinized rat tail collagen I polymerized at room-temperature is used at 2 mg/ml. Nonpepsinized acid-extracted collagen I from rat tail retains both N- and C-terminal telopeptides, nonhelical portions of the collagen molecule responsible for native collagen intermolecular crosslinking and fibrilar stability15. Together, these conditions allow the formation of collagen networks that most closely resemble the ones observed in vivo10. To allow visualization of the collagen fibers, both in fixed and living cultures, a detailed protocol is &#160;provided to fluorescently label collagen in vitro10 using 5-(and-6)-carboxytetramethylrhodamine (TAMRA), succinimidyl ester. This protocol has been adapted from Baici&#160;et al.16,17, where fluorescein isothiocyanate is used to label soluble collagen molecules. As fluorescein, TAMRA is an amino-reactive fluorescent dye that reacts with nonprotonated aliphatic amino groups of proteins, such as the free amino group at the N-terminus and, more importantly, the side amino group of lysines. This reaction only occurs at basic pH, when the lysine amino group is in the nonprotonated form. In addition to TAMRA being more stable than fluorescein over time, its emission spectra falls on the orange/red range (ex/em = 555/518 nm), which can be usefully combined for live cell imaging of GFP-tagged proteins. Using soluble collagen labeled molecules with amino-reactive dyes does not affect the polymerization process nor the density, pore size and crosslinking status of the collagen matrix10,16,18,19.
This protocol also includes a method for 3D immunofluorescent labeling of endogenous proteins, which has been further optimized to label the cytoskeleton or cytoskeleton associated proteins. The final focus of this protocol is on methods to acquire high-resolution images of 3D cultures using confocal microscopy with reduced contribution from rigid glass coverslips on the collagen matrix tension.

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TAMRA, SE</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:136px;">C-1171</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen High Concentration</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P2141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxol (Paxitel)</td>
			<td>Sigma</td>
			<td>T7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-alpha Tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 633 Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A21052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting media</td>
			<td>Fisher Scientific</td>
			<td>106 226 89 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis Cassette</td>
			<td>Pierce</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris 7.2.3</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Labeling rat-tail collagen I with TAMRA allows an easy preparation and visualization of 3D collagen networks. Slow polymerization at room temperature results in the formation of collagen networks with comparable organization to the ones found in vivo10.
Here we present a protocol for immunofluorescence staining of the cytoskeleton of CT26 cancer cells, both as spheroids and as single cells. To better preserve the cytoskeleton, buffers are supplemented with unlabele...

Watch this Scientific Journal Video about Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D at JoVE.com

Revealing the Cytoskeletal Organization of Invasive Cancer Cells 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 ...

revealing-the-cytoskeletal-organization-of-invasive-cancer-cells-in-3d

Research

JoVE Journal

Medicine

15.6K visualizações.  Institut Curie.  Este artigo apresenta um método para rotular fluorescência de colagénio que pode ser ainda utilizado para ambos e correcção de imagem ao vivo de culturas de células em 3D. Nós também fornecer um protocolo optimizado para visualizar as proteínas do citoesqueleto endógenos de células cultivadas em ambientes em 3D. 