This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA1385282522717 to RW.

1. Preparation of Materials and Reagents
<ol>
	<li>Label each well of a tissue culture treated 6-well plate appropriately for each cell line or condition being investigated.</li>
	<li>Prepare 2x cell culture medium by dissolving 1 g of powder medium and 0.2 g of sodium bicarbonate in de-ionized water to a final volume of 50 ml.</li>
	<li>Pass this medium through a 0.2 &#956;m filter to sterilize.</li>
	<li>Add additional components needed for normal culture of the cell line of interest. For example, grow CMT 167 cell l...

<ol>
	<li>Puck, T. T., Marcus, P. I., Cieciura, 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=Clonal+growth+of+mammalian+cells+in+vitro;+growth+characteristics+of+colonies+from+single+HeLa+cells+with+and+without+a+feeder+layer.">Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer.</a> J Exp Med. 103, 273-283 (1956).</li><li>Taddei, M. L., Giannoni, E., Fiaschi, T., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis:+an+emerging+hallmark+in+health+and+diseases.">Anoikis: an emerging hallmark in health and diseases.</a> The Journal of pathology. 226, 380-393 (2012).</li><li>Wend, P., Holland, J. D., Ziebold, U., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+stem+and+cancer+stem+cells.">Wnt signaling in stem and cancer stem cells.</a> Semin Cell Dev Biol. 21, 855-863 (2010).</li><li>Reya, T., 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+role+for+Wnt+signalling+in+self-renewal+of+haematopoietic+stem+cells.">A role for Wnt signalling in self-renewal of haematopoietic stem cells.</a> Nature. 423, 409-414 (2003).</li><li>Klaus, A., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+and+its+impact+on+development+and+cancer.">Wnt signalling and its impact on development and cancer.</a> Nat Rev Cancer. 8, 387-398 (2008).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+development+and+disease.">Wnt/beta-catenin signaling in development and disease.</a> Cell. 127, 469-480 (2006).</li><li>Takahashi-Yanaga, F., Kahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Wnt+signaling:+can+we+safely+eradicate+cancer+stem+cells.">Targeting Wnt signaling: can we safely eradicate cancer stem cells.</a> Clin Cancer Res. 16, 3153-3162 (2010).</li><li>De, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/Ca2++signaling+pathway:+a+brief+overview.">Wnt/Ca2+ signaling pathway: a brief overview.</a> Acta Biochim Biophys Sin (Shanghai). 43, 745-756 (2011).</li><li>Tennis, M. A., Vanscoyk, M. M., Wilson, L. A., Kelley, N., Winn, 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=Methylation+of+Wnt7a+is+modulated+by+DNMT1+and+cigarette+smoke+condensate+in+non-small+cell+lung+cancer).">Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer).</a> PLoS One. 7, (2012).</li><li>Winn, R. 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=Restoration+of+Wnt-7a+expression+reverses+non-small+cell+lung+cancer+cellular+transformation+through+frizzled-9-mediated+growth+inhibition+and+promotion+of+cell+differentiation.">Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation.</a> J Biol Chem. 280, 19625-19634 (2005).</li><li>Winn, R. 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=Antitumorigenic+effect+of+Wnt+7a+and+Fzd+9+in+non-small+cell+lung+cancer+cells+is+mediated+through+ERK-5-dependent+activation+of+peroxisome+proliferator-activated+receptor+gamma.">Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma.</a> J Biol Chem. 281, 26943-26950 (2006).</li><li>Tennis, M. 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=Sprouty-4+inhibits+transformed+cell+growth,+migration+and+invasion,+and+epithelial-mesenchymal+transition,+and+is+regulated+by+Wnt7A+through+PPARgamma+in+non-small+cell+lung+cancer.">Sprouty-4 inhibits transformed cell growth, migration and invasion, and epithelial-mesenchymal transition, and is regulated by Wnt7A through PPARgamma in non-small cell lung cancer.</a> Mol Cancer Res. 8, 833-843 (2010).</li><li>Steele, J. G., Rowlatt, C., Sandall, J. K., Franks, 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=Cell+surface+properties+of+high-+and+low-metastatic+cell+lines+selected+from+a+spontaneous+mouse+lung+carcinoma.">Cell surface properties of high- and low-metastatic cell lines selected from a spontaneous mouse lung carcinoma.</a> Int J Cancer. 32, 769-779 (1983).</li></ol>

The authors have nothing to disclose.

In vitro confirmation of tumor suppressive function of signaling proteins is difficult. One of the most rigorous assays available to investigate this property is the soft agar colony formation assay. Here, we have illustrated the soft agar colony formation assay using a murine lung carcinoma cell line stably overexpressing Wnt7a and Fzd9 compared to its parental CMT167 cell line.
There are several important points to consider regarding the soft agar assay. The most critical step in th...

The soft agar colony formation assay is a technique widely used to evaluate cellular transformation in vitro. Historically, another assay, the clonogenic assay, described by Puck et al. in 1956 was used to evaluate the ability of cells to form colonies1. In this technique, cells were dispersed onto a culture plate and grown in the presence of &#39;feeder&#39; cells or conditioned medium to provide necessary growth factors. The limitation of this technique was that it only provided information regarding colony formation. Normal cells are prevented from anchorage-independent growth, due to a particular type of apoptotic death, called anoikis2. However, transformed cells have the capability to grow and divide without binding to a substrate. To capitalize on this concept, researchers developed the soft agar colony formation assay. The soft agar colony formation assay has since been modified, in more recent years, to address specific needs. One variation involves incorporation of fluorometric dye to allow for high-throughput colony counting. Another variation involves the use of specialized agar solution to allow for retrieval of viable cells after colony formation when protein or DNA samples are needed.
In the traditional soft agar colony formation assay, cells are grown in a layer of soft agar mixed with cell culture medium that rests on another layer of soft agar, also mixed with cell culture medium, but containing a higher concentration of agar. This prevents cells from adhering to the culture plate, yet allows transformed cells to form visible colonies. The rationale behind this technique is that normal cells depend on cell to extracellular matrix contact to be able to grow and divide. Conversely, transformed cells have the ability to grow and divide irrespective of their surrounding environment. Therefore, cells able to form colonies in an anchorage-independent manner were considered to be transformed and carcinogenic. The overall goal of this method is to measure this capability in cells in a semi-quantitative and stringent manner.
The Wnt signaling pathway is critical in embryogenesis and often de-regulated in tumorigenesis3-6. There are multiple pathways associated with Wnt signaling. The canonical pathway involves Wnt signaling and regulation of downstream gene transcription through its effects on the transcriptional coactivator beta-catenin. Wnts also signal through several non-canonical pathways, for example, the planar cell polarity pathway, which regulates elements involved in cytoskeletal structure7, and the Wnt-calcium pathway, which regulates release of calcium from the endoplasmic reticulum8. Wnt ligands exert their activity through binding Frizzled receptors. Although several Wnts have been shown to be upregulated in lung cancer, Wnt7a has been shown to be down-regulated in non-small cell lung cancer through promoter methylation9. Wnt7a binds Fzd9 and acts as a tumor suppressor through a non-canonical pathway. Restoration of Wnt-7a and Fzd-9 inhibits the growth of non-small cell lung cancer cells10. The effects of Wnt7a/Fzd9 are mediated through the activation of ERK-5, which in turn, activates peroxisome proliferator-activated receptor &#947; (PPAR&#947;)11,12. Here, we show that overexpression of Wnt7a and Fzd9 results in the suppression of anchorage-independent growth of a murine lung carcinoma cell line. Murine CMT167 cells were derived from a lung carcinoma in C57BL/lcrf mice13 and were stably transfected with Wnt7A and Fzd9. Overexpression of Wnt7A and Fzd9 were confirmed by quantitative-PCR (Q-PCR) and the functionality of Wnt7A and Fzd9 overexpression was confirmed through downstream activation of PPAR&#947;.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cancer Cell Line of Interest</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">10032302</td>
			<td>CMT-167 Cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Powdered RPMI 1640 Medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-089</td>
			<td style="width:167px;">Used to prepare 2X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Liquid RPMI 1650 Medium</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:167px;">Referred to as 1X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">HyClone</td>
			<td style="width:119px;">SH30910.03</td>
			<td style="width:167px;">Used to supplement cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">CellGro</td>
			<td style="width:119px;">30-002-Cl</td>
			<td style="width:167px;">Used in cell culture medium.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Difco Noble Agar</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">214230</td>
			<td style="width:167px;">Used to prepare 1.0% and 0.6% Agar.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP-328-1</td>
			<td style="width:167px;">Used in 2X cell culture medium.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Trypsin</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">25-050-Cl</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sterile Bottle-Top Filters</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">09-761-126</td>
			<td style="width:167px;">Used to sterile filter 2X medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Lipofectamine Reagent</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">18324-020</td>
			<td style="width:167px;">Used in PPAR-RE luciferase assay.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well Plates Tissue-culture Treated</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">37 degree C/5% CO2 Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Chemi-Doc Imager</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to take pictures of colonies.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Quantity One Software</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to count cell colonies.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">50mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">250mL Erlenmeyer Flasks</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microwave</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">5mL Serological Pipettes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Aid</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micropipette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Hemacytometer w/ cover slip</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Tips</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Light Microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Heat-Resistant Gloves</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Saran Wrap</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice Bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the soft agar colony formation assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The expression of Wnt7A and Fzd9 in CMT167 cells is effective in tumor suppression as illustrated by our soft agar colony formation assay. Preliminarily, we used Q-PCR to show that Wnt7A and Fzd9 mRNA are expressed in low levels in CMT167 cells. CMT167 cells showed low levels of endogenous Wnt7A and Fzd9 when compared to MLE-12 cells, an SV40-transformed murine lung epithelial cell line (Figure 1). We then transfected CMT167 cells with two retroviral overexpression vectors expressing human constructs of ...

Watch this Scientific Journal Video about The Soft Agar Colony Formation Assay at JoVE.com

The Soft Agar Colony Formation Assay

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

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Biology

110.3K vistas.  University of Illinois at Chicago. The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.