Name: characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics
Uploaded: 2018-03-26T14:30:00
Description: Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

The Ohse Grant Foundation funded this work.

1. Cell Extension Characteristics
<ol>
	<li>Maintain SW-13, SW-Neo, and SW-DKK3 cells in a standard humidified incubator at 37.0 &#176;C and 5% CO2 in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% fetal bovine serum and 10,000 U/mL penicillin and streptomycin (designated as &#39;growth medium&#39;).
	<ol>
		<li>Count cells using a hemocytometer, and plate 5,000 cells per well for SW-13, SW-Neo, and SW-DKK3 lines into separate 6-well plates and grow overnight in growth medium on sterile glass cov...

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, 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=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., 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=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, 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=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. 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+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, 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=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

Here we describe an in vitro quantitative method to characterize cell extensions with ease, few pit-falls, and reliable reproducibility that can be applied for various test conditions. Moreover, simple, quantifiable adhesion and/or motility assays can be performed simultaneously to correlate potential functional significance of observed alterations in cell membrane extensions.
However, these methods may present some potential limitations. First, the criteria specified here to identify...

Dysregulated WNT signaling plays a critical role in adrenocortical malignancies1. The methods used in this study investigate whether silencing of DKK3, a negative regulator of WNT signaling, represents a dedifferentiation event in the adrenal cortex and promotes tumor formation in the context of cell-extension repertoire changes. DKK3 is a 38 kDa secreted glycoprotein with an N-terminal signal peptide and previous studies have demonstrated that its enforced expression resulted in cell cycle arrest, inhibited aggressive malignant behavior, and reversed epithelial-mesenchymal transition2.
The malignant behavior of adrenocortical carcinoma (ACC) and other cancers is, in part, influenced by the ability of tumor cells to interface with the surrounding surfaces, including the extracellular matrix, which in turn facilitates tumor cell invasion and migration3. The role of specific cell membrane extensions in cancer progression is being increasingly demonstrated in various contexts, primarily via the formation of filopodia. For example, overexpression of L-type calcium channels has been found to induce filopodia formation and promote tumor cell invasion4. Similarly, Fascin, an actin binding protein minimally expressed in normal tissue, is also overexpressed in cancer cells in association with filopodia formation5. Lobopodia formation enables non-malignant fibroblasts to migrate effectively through the extra-cellular matrix, however, it has been shown that fibrosarcoma cells rely on metalloproteinase activity in lieu of lobopodia to facilitate cell migration and invasion6. We have shown that tumor suppressors, including Ras association domain family 1 isoform A (RASSF1A) and DKK3, can function to alter cytoskeletal elements and promote lamellipodia formation and stymie invasive properties7,8.
As such, it is critical to characterize the effects of genes involved in carcinogenesis and their relationship to cell-membrane extension alterations, specifically assessing filopodia, lobopodia, and lamellipodia formation under test conditions. Current state-of-the art techniques include the use of increasingly sophisticated microscopy methods, fluorescent labeling, and/or complex computer algorithms for data acquisition and interpretation. While these methods provide new and powerful analytic tools, their complexity limits their widespread use and adaptability in cell biology experiments. Furthermore, the precise quantification and observation of changes in cell extension morphology is not typically measured9,10. In contrast, we introduce a technique here that accurately quantifies cell extension alterations using standard microscopic techniques and readily adaptable in vitro methods. These methods also quantify each cell extension type simultaneously for each cell analyzed and determine overall changes in the cell membrane extension repertoire. We also show how these changes can relate to cell adhesion properties.
As an experimental example, we will use a previously created cell line of SW-13, designated SW-DDK3, which has been stably transfected with pCMV6-Entry/DKK3 plasmid vectors and constitutively overexpresses DKK3, a differentiating factor in the adrenal gland. Non-transfected SW-13 cells and SW-13 cells stably transfected with empty vector (pCMV6-Entry), designated SW-Neo, will serve as experimental controls.

<table width="653">
	<tbody>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:115px;">ThermoFisher</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:203px;">Supplemented with fetal bovine serum and penicllin and streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Deulbeco&#39;s Phosphate Buffered Saline</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>1X solution, used directly</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">3.7% formaldehyde&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">0.05% crystal violet&#160;</td>
			<td style="width:115px;">Harleco</td>
			<td style="width:119px;">192-12</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">De-ionized water</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Microscope with 400X magnification&#160;</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td>NA</td>
		</tr>
		<tr height="43">
			<td height="43" style="width:216px;height:43px;">Cell dissociation solution non-enzymatic 1X</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5914&#160;</td>
			<td>1X solution, used directly</td>
		</tr>
	</tbody>
</table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

Using the above assays, the effects of DKK3 overexpression on cell extension morphology and cell adhesion properties were tested in the established ACC cell line SW-13, in vitro. Cells overexpressing DKK3 were generated by stably transfecting SW-13 cells with Myc-DDK tagged pCMV6-Entry/DKK3 plasmid vectors and designated as SW-DKK3. Similarly, cells stably transfected with empty vector pCMV6-Entry were created as a transfection and passaging control and defined as SW-Neo. SW-13 c...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

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