We are thankful to Dr. Richard Cerione, Dr. Marc Antonyak, and Kelly Sullivan, Cornell University, for providing technical advice, and to Dr. Gerlinde Van de Walle, Cornell University, for sharing their Olympus CKX41 inverted microscope.

1. Preparation of 3% 2-Hydroxyethyl Agarose
<ol>
	<li>Into a clean, dry 100 ml&#160;glass bottle, add 0.9 g of 2-hydroxyethyl agarose (Agarose VII) followed by 30 ml&#160;of distilled water.</li>
	<li>Microwave the mixture for 15 sec and gently swirl. Repeat this step at least three more times until the agarose powder fully dissolves.</li>
	<li>Autoclave the solution-containing bottle for 15 min.</li>
	<li>Allow the agarose solution to cool down to RT before further use. Store the solution at RT.</li>
</ol>
<p class="j...

<ol>
	<li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+bioassay+of+human+tumor+stem+cells.">Primary bioassay of human tumor stem cells.</a> Science. 197, 461-463 (1977).</li><li>Wang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells.">Molecular signaling regulating anchorage-independent growth of cancer cells.</a> Mt Sinai J Med. 71 (6), 361-367 (2004).</li><li>Vossenaar, E. R., Zendman, A. J., van Venrooij, W. J., Pruijn, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD,+a+growing+family+of+citrullinating+enzymes:+genes,+features+and+involvement+in+disease.">PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.</a> Bioessays. 25 (11), 1106-1118 (2003).</li><li>Horibata, S., Coonrod, S. A., Cherrington, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction.">Role for peptidylarginine deiminase enzymes in disease and female reproduction.</a> J Reprod Dev. 58 (3), 274-282 (2012).</li><li>Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R., Coonrod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis.">Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis.</a> Biochem Res Int. , 895343 (2012).</li><li>McElwee, J. L., 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=Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target.">Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.</a> BMC Cancer. 12, 500 (2012).</li><li>Knight, J. S., 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=Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney,+skin+and+vascular+disease+in+lupus-prone+MRL/lpr+mice.">Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice.</a> Ann Rheum Dis. , 1-8 (2014).</li><li>Miller, F. R., Santner, S. J., Tait, L., Dawson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ.">MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ.</a> J Natl cancer Inst. 92, 1185-1186 (2000).</li><li>Fan, D., Morgan, L. R., Schneider, C., Blank, H., Fan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations.">Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.</a> J Cancer Res Clin Oncol. 109, 23-28 (2000).</li><li>Hamburger, A. W., White, C. P., Dunn, F. E., Citron, M. L., Hummel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum.">Modulation of human tumor colony growth in soft agar by serum.</a> Int J Cell Cloning. 1 (4), 216-229 (1983).</li><li>Anderson, S. N., Towne, D. L., Burns, D. J., Warrior, U. <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+soft+agar+assay+for+identification+of+anticancer+compound.">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li></ol>

The rate of colony formation in soft agar varies depending on the cell type9. Therefore, the number of cells to start with should be optimized and adjusted accordingly. A suggested starting range is between 5 x 102 to 1 x 104 cells per well using a 6-well plate. In addition, colony size varies depending on the growth rate of each cell. Therefore, a predefined a cut-off for colony size is needed to annotate individual colonies for downstream quantitative analyses. Here, colonies larger tha...

Both non-transformed (normal) and transformed cells can readily proliferate in a 2D monolayer culture. This form of adherent cell growth is quite dissimilar from that which occurs in vivo where, in the absence of mitogenic stimulation, cells do not often rapidly divide within their microenvironment. The soft agar assay on the other hand is distinct from 2D culture systems because it quantifies tumorigenicity by measuring a cell&#8217;s ability to proliferate and form colonies in suspension within a semi-solid agarose gel1. In this setting, non-transformed cells are unable to rapidly propagate in the absence of anchorage to the extracellular matrix (ECM) and undergo apoptosis, a process known as anoikis. In contrast, cells that have undergone malignant transformation lose their anchorage dependence due to activation of signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and Rac/Cdc42/PAK. Therefore, these cells are able to grow and form colonies within the semi-solid soft agar matrix2.
A common use of the soft agar assay is to test whether specific compounds, such as PADI inhibitors, are able to suppress tumor growth in vitro. In general, colony count or colony sizes are quantitative read-outs from the assay that can be compared between control and treatment groups to assess differences in cellular tumorigenicity. Therefore, if one finds that colony formation is inversely correlated with increasing drug concentration, then a conclusion could be drawn that the drug is an effective inhibitor of tumorigenicity in vitro. On the other hand, if the drug does not affect colony formation, the drug is either not at the appropriate dosage or it is not an effective tumorigenic inhibitor. Aside from using a soft agar assay to test the anti-tumor effect of a drug, this assay can also be used to probe the relationship between a specific gene and tumorigenesis. For example, the effect of suppressing PADI2 expression on tumorigenicity can be addressed by PADI2-specific siRNA treatment.
PADIs are calcium-dependent enzymes that post-translationally modify proteins by converting positively charged arginine residues into neutrally charged citrulline in a process known as citrullination or deimination3-5. We have recently found that peptidylarginine deiminase 2 (PADI2) may function as a novel breast cancer biomarker and that PADI inhibitors represent candidate therapies for early stage breast cancers6. For example, we have previously demonstrated that a &#8220;pan-PADI&#8221; inhibitor, Cl-amidine, suppresses the proliferation of breast cancer cells using 2D monolayers and that the inhibitor suppressed the growth of 3D tumor spheroids6. In this report, we extend these studies, and highlight the utility of the soft agar assay, by testing the efficacy of a new PADI inhibitor, BB-CLA, in suppressing the growth of MCF10DCIS breast cancer colonies7. We note that we used MCF10DCIS cells for this experiment because they are oncogenic derivatives of non-transformed human MCF10A cells and because they contain high steady state levels of PADI2 protein8. We hypothesize that PADI2 enzymatic activity plays a key role in the tumorigenicity of this cell line and that BB-CLA-mediated inhibition of PADI2 activity will suppress cancer progression.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zeiss Axiopot</td>
			<td>Carl Zeiss Microscopy</td>
			<td>1021859251</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Lonza BioWhittaker</td>
			<td>12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Donor Equine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH30074.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxyethylagarose: Type VII, low gelling temperature</td>
			<td>Sigma-Aldrich</td>
			<td>39346-81-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of the soft agar colony formation assay to identify inhibitors of tumorigenicity in breast cancer cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

The soft agar colony formation assay can be used for a broad range of applications documenting the tumorigenicity of cancer cells. A major advantage of this technique is that the semi-solid matrix selectively favors the growth of cells that can proliferate in an anchorage-independent manner. This trait is mainly exhibited by cancer cells but not by normal cells. We primarily use this technique to test the efficacy of tumor growth inhibition by drugs and to test for the effect of overexpression or depletion of our genes o...

Watch this Scientific Journal Video about Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells at JoVE.com

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

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28.1K Views.  Cornell University. Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro. 