Name: modeling and imaging 3-dimensional collective cell invasion
Uploaded: 2011-12-07T13:00:00
Description: Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Funding for this research is from Cancer Research UK.

1. Retroviral labeling of cells with fluorescent proteins
<ol>
	<li>Plate retroviral packaging cells (e.g. Phoenix) out at 0.25 x 106 cells per well of a 6-well dish in 10% fetal bovine serum (FBS)/DMEM.</li>
	<li>Transfect cells with retroviral DNA 48 hours later cells using Effectene according to the manufacturers' instructions.</li>
	<li>Rinse wells twice with medium 24 hours later, then add 1.5 ml of 10% FBS/DMEM per well.</li>
	<li>Collect packaged virus in tissue culture mediu...

<ol>
	<li>Olson, M. 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=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. 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+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. 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+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</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. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>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=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Matrigel invasion assays have traditionally been set up with cells placed onto a layer of extracellular matrix protein with chemoattractant-induced motility towards and through a filter at the bottom. Invasiveness was scored as a function of how many cells could be counted on the underside of the filter. Although practically there is little difference with the &ldquo;inverse&rdquo; invasion assay described above, there is considerably more information about the process of invasion that can be determined by visualizing in...

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
</tr>
<tr>
<td>DMEM (Dulbecco&rsquo;s Modified Eagle&rsquo;s Medium)</td>
<td>GIBCO</td>
<td>21969</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>PAA</td>
<td>A15-101</td>
</tr>
<tr>
<td>Penicillin Streptomycin</td>
<td>GIBCO</td>
<td>15140</td>
</tr>
<tr>
<td>200 mM L-Glutamine (100x)</td>
<td>GIBCO</td>
<td>25050-032</td>
</tr>
<tr>
<td>Puromycin</td>
<td>Sigma-Aldrich</td>
<td>P8833</td>
</tr>
<tr>
<td>0.05&#37; Trypsin EDTA</td>
<td>GIBCO</td>
<td>25300</td>
</tr>
<tr>
<td>Polybrene</td>
<td>Sigma-Aldrich</td>
<td>AL-118</td>
</tr>
<tr>
<td>Lipofectamine 2000 Reagent</td>
<td>Invitrogen</td>
<td>11668019</td>
</tr>
<tr>
<td>6.5mm Transwells, 8.0 &micro;m pore size </td>
<td>Corning</td>
<td>3422</td>
</tr>
<tr>
<td>Complete Matrigel</td>
<td>BD Biosciences</td>
<td>354234</td>
</tr>
<tr>
<td>Calcein AM</td>
<td>Invitrogen</td>
<td>C1430</td>
</tr>
<tr>
<td>RNase</td>
<td>Qiagen</td>
<td>19101</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
</tr>
<tr>
<td>Confocal microcope</td>
<td>Leica</td>
<td>SP2MP</td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

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 ...

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