This work was supported by the National Institutes of Health (NIMHD-G12MD007581) through the RCMI-Center for Environmental Health at Jackson State University and the U.S. Department of Defense, Idea Award 11-1-0205.

NOTE: This protocol is described in McArdle et al.11. A timeline is provided in Figure 1.
1. Preparation of the culture plate
<ol>
	<li>Wash the 35-mm culture dish containing a 10-mm glass-bottom well with 1 mL of 1 M hydrochloric acid (HCl) for 15 min to lower the hydrophobicity of the glass-bottom.</li>
	<li>Wash the culture plate twice with 1 mL of phosphate buffer saline (PBS) to get rid of all the HCl.</li>
	<li>Wash the cul...

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Fusion:+Mechanisms+Slowly+Unravel.">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <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+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, 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=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

Here, we describe a simple inverted vertical invasion assay which is convenient for a variety of cell types including cells that are rare and/or dependent on their microenvironment. In this 3D invasion assay, cells remain in their original microenvironment and are covered by the collagen gel containing a chemoattractant. The cells then migrate and invade in the collagen. In this assay, the cells can be stained and easily monitored within the gel. The simplicity and reproducibility of this assay makes it a convenient tool...

Cell motility is a normal developmental and physiological process of cells. However, changes in the pattern and the ability of cells to migrate and invade are associated with pathological conditions including inflammatory diseases and cancer metastasis1,2,3. Metastasis is arguably the most poorly understood aspect in cancer. To metastasize, cancer cells must degrade the extra cellular matrix (ECM), invade the local tissue and intravasate. Once in circulation, the cancer cells will disseminate to the distant site, extravasate and form secondary tumors. Therefore, the ability of cells to degrade the ECM, to migrate, and to invade is related to their metastatic potential. Different approaches have been developed to analyze and quantify the migration and invasion capabilities of cells. The most used approaches include the wound healing assay, time-lapse microscopy, and the Boyden chamber assay. The wound healing assay4 and time lapse microscopy are simple and convenient assays that would give preliminary insight into cell migration. However, both are 2D assays that do not reflect the 3D environment in which cells exist in vivo. Studies have shown that cells respond differently to a 3D environment compared to a 2D one5,6. Moreover, wound healing assays are difficult to reproduce and to yield quantitative results7,8,9. The Cell Exclusion Zone assay, which is a 3D modification of the wound healing assay, is a more physiologically relevant assay but it is not suitable for cells low in number such as hybrid cells resulting from cell fusion events10,11.
The Boyden chamber assay is a 3-dimensional assay that provides relevant microenvironments for cellular studies. However, it requires cells to be removed from their original microenvironment and to be deposited into the upper chamber of the Boyden assay system to migrate and invade downwards toward the chemoattractant containing medium. This 3D approach is not appropriate in analyzing the migration and invasion capability of cells vulnerable to their microenvironment and/or limited in number10,11,12. A newer approach to analyze cell migration and invasion is the microfluidic gradient chamber assay13. Although suitable and reliable in migration and invasion studies, this assay is more expensive and could be technically challenging for a typical laboratory. We describe here a simple inverted vertical invasion assay that is compatible with many cell types including cells sensitive to their microenvironment and/or restricted in number. This assay was adapted from the protocol proposed by Hooper et al.14. In this assay, the cells remain in their original microenvironment and their migration and invasion is monitored as they move upwards in the collagen gel containing a chemoattractant11. This assay is reproducible and has been optimized and validated in the 35-mm culture dish. It could be adapted to different assay conditions including different cell culture sizes, different matrices or strength of adhesion, and different chemoattractants. This assay could serve as an in vitro platform for initial screening in the drug discovery process.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">MatTek P35G-1.5-10C</td>
			<td style="width:269px;">MatTek Corporation, Ashland, MA</td>
			<td style="width:151px;">P35G-1.5-10-C</td>
			<td style="width:232px;">mattek glass bottom dishes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I</td>
			<td>Corning, Corning, NY, USA</td>
			<td align="right">354236</td>
			<td>Collagen gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>H1758</td>
			<td>HCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">10010023</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>D2429</td>
			<td>10X DMEM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Fluorescent microscope</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>Olympus IX81</td>
			<td>Confocal&#160; microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td>
			<td>SH30070.03</td>
			<td>FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ti:Sapphire multi-photon laser scanning microscope</td>
			<td>Prarie Technologies, Sioux Falls, SD, USA</td>
			<td>40x NA 0.8 objective</td>
			<td>MPLSM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris software</td>
			<td>Bitplane Inc. Zurich, CH</td>
			<td>Imaris 7.5.2</td>
			<td>Imaris software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td style="width:269px;">Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">50980487</td>
			<td>PFA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-minimum essential medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MDA-MB-231</td>
			<td>ATCC; Manassas, VA, USA</td>
			<td>HBT-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MSC</td>
			<td>Trivedi and Hematti, 2007</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20X lens UPLAPO</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>36-064</td>
			<td>Olympus UPLSAPO 20X Objective</td>
		</tr>
	</tbody>
</table>

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).

We have used a 35-mm culture dish with a glass bottom and rat tail collagen I to develop and optimize an in vitro 3D invasion assay protocol. Using this assay, we showed that breast cancer cells MCF7 and MSC cells could invade into the collagen gel to an average distance of 0.04 &#177; 1.8 &#181;m, and 41.3 &#177; 76.0 &#181;m respectively after 48 h when IGF1 (18.6 ng/mL) was used as chemoattractant (Figure 2). More importantly, we showed that fusio...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

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.

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. ...

The overall goal of this invasion assay is to quantify the migratory and invasive capacities of rare and sensitive cells in a 3D setting without dislodging the cells from their micro-environment. This method can help answer key questions in the cancer research field about the invasion capability of sensitive and oray events, such as cancer cell fusion product. The main advantage of this technique is that the cells are analyzed in their original micro-environment using a 3D setting.The implications of this technique extend toward therapy of cancer because it can be used to screen pharmacological agents capable of inhibiting cancer cell invasion and metastasis. Visual demonstration of this technique is critical. Aspiration in the temperature and pH of the collagen mixture affect its polymerization and rough handling causes the gel to detach.Begin by treating a 35 millimeter culture dish with a 10 millimeter glass bottom well with one milliliter of one molar hydrochloric acid to lower the hydrophobicity of the glass. After 15 minutes, wash the plate two times with one milliliter of PBS per wash, followed by one wash with one milliliter of 70%ethanol. Next, rinse the plate with one milliliter of culture medium specific to the cells of interest to be used.And seed the plate with the cells of interest. Then place the plate in a cell culture incubator. When the cells reach 100%confluency combine 114.5 microliters of freshly prepared rat-tail collagen, 16.6 microliters of 10 X DMEM medium, and 33.1 microliters of culture medium supplemented with chemo attractant of interest in a microcentrifuge tube on ice.Neutralize the collagen mixture with 14 microliters of sodium bicarbonate and overlay the cells with 80 microliters of gel. Allow the collagen to polymerize in a humidified 37 degree Celsius and five percent carbon dioxide incubator for 30 minutes. Then layer two milliliters of reduced two percent serum cell culture medium over the gel and carefully return the cells to the incubator for the appropriate experimental time period without dislodging the collagen.At the end of the incubation, gently remove the medium and carefully rinse the gel with one milliliter of PBS. Fix the cells with one milliliter of four percent paraformaldehyde for 15 minutes followed by two washes in one milliliter of PBS per wash. Then label the cells with DAPI solution for 20 minutes at room temperature and image the in vitro three dimensional collagen invasion by confocal microscopy.Here, quantification of the invasion of the collagen gel by a breast cancer cell line and breast cancer cell fusion products in response to a chemo attractant over a 48-hour migration period, is shown. Notably, the migration of rare breast cancer cell fusion products into the collagen gel in response to the chemo attractant was also observed. The cells remain healthy throughout the collagen gel experiment, as evidenced by their maintenance of robust cell numbers and their morphology at 48 hours after gel polymerization.This vertical collagen gel assay also allows the kinetics of cell migration at invasion, to be analyzed by imaging the cell migration in Z-series as well as the morphology of the migrating cells to be captured in situ. When attempting this procedure, it is important to remember to use a culture dish that is compatible with your microscope. Following this procedure, other variables like modifying the strength of collagen or using different chemo attractants can be introduced to answer additional questions about the effects of ECM engagement on cell invasion or the of different chemo attractant on cell invasion, respectively.After its development, this technique paved the way for researchers in the field of biology and cancer to screen for pharmacological inhibitors of metastasis as well as to explore wound healing in all inflammatory responses in vivo. After watching this video, you should have a good understanding of how to assess the migratory and invasive capability of rare and sensitive cells, in a 3D setting, without distorting the cells on the micro involvement. Don't forget that walking with human cancer cells, hydrochloric acid, paraformaldehyde, and DAPI can be extremely hazardous and that precautions, such as using a number two bio septic cabinet and a fume hold should always be taken while performing this procedure.

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

Research

JoVE Journal

Cancer Research

5.9K Views.  University of Minnesota - Twin Cities. The overall goal of this invasion assay is to quantify the migratory and invasive capacities of rare and sensitive cells in a 3D setting without dislodging the cells from their micro-environment. This method can help answer key questions in the cancer research field about the invasion capability of sensitive and oray events, such as cancer cell fusion product. The main advantage of this technique is that the cells are analyzed in their original micro-environment using a 3D setting.The ...