Name: Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells (Video) | JoVE Journal
Uploaded: 2015-05-20T14:00:00.000+00:00
Duration: 11 min 6 s
Description: Learn the soft agar colony formation assay to assess breast cancer tumorigenicity using BB-Cl-amidine treatment. This method enables quantitative measurement of cell proliferation in semi-solid matrices.

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5 minutes

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>
2. Preparation of the Bottom Layer: 0.6% Agarose Gel
<ol>
	<li>Pre-warm several 5 ml&#160;and 10 ml&#160;pipettes in a 37 &#176;C incubator to prevent the agarose from solidifying in the pipette when handling.</li>
	<li>Partially loosen the bottle lid and microwave the pre-made 3% 2-hydroxyethyl agarose solution for 15 sec. Then, gently swirl the solution and microwave for another 15 sec. CAUTION: Be careful when swirling the agarose solution because the solution rises up when exposed to air and can spill over.</li>
	<li>If there is residual solid gel in the bottle, microwave for a few more seconds.</li>
	<li>Keep the bottle containing the agarose solution in a 45 &#176;C water bath during the next steps to prevent the agarose solution from solidifying prematurely.</li>
	<li>Warm MCF10DCIS media in a 37 &#176;C water bath. Note: MCF10DCIS media consists of DMEM/F12, 5% horse serum, 5% penicillin streptomycin.</li>
	<li>Transfer 3 ml&#160;of the 3% agarose solution using the pre-warmed pipettes into a sterile 50 ml&#160;conical tube.</li>
	<li>Immediately add 12 ml&#160;of warm MCF10DCIS media and gently invert the conical tube to mix the agarose with the media. Try not to form any bubbles as it will interfere with the colony counting later.</li>
	<li>Gently add 2 ml&#160;of this mixture into each well of a 6-well culture plate without forming any air bubbles.</li>
	<li>Incubate the 6-well culture plate horizontally on a flat surface at 4 &#176;C for 1 hr to allow the mixture to solidify.</li>
	<li>After the mixture solidifies, place the plate into a 37 &#176;C incubator for 30 min. The bottom layer is now ready for use.</li>
</ol>
3. Preparation of the Cell-containing Layer: 0.3% Agarose Gel
<ol>
	<li>Trypsinize MCF10DCIS cells and dilute them to a cell concentration of 4 x 104 /ml.</li>
	<li>Take 2 ml&#160;of the 3% agarose using pre-warmed pipettes and transfer into a sterile 50 ml&#160;conical tube.</li>
	<li>Immediately add 8 ml&#160;of MCF10DCIS media to the conical tube and gently invert to mix the agarose with the media. Avoid forming any bubbles.</li>
	<li>Take 2 ml&#160;of the MCF10DCIS cells (4 x 104 /ml) and treat with BB-CLA (0 &#181;M (DMSO) or 1 &#181;M).</li>
	<li>In a 1:1 dilution, mix the cells with the 0.6% agarose.</li>
	<li>Take 1 ml&#160;of the cell-agarose mixture and gently add onto the bottom layer of the 6-well culture plate (2 x 104 cells/ml).</li>
	<li>Place the 6-well culture plate horizontally on a flat surface at 4 &#176;C for at least 15 min to allow the top layer to solidify.</li>
	<li>After the mixture solidifies, place the plate into a 37 &#176;C incubator for a week before adding the feeding layer.</li>
</ol>
4. Preparation of the Feeder Layer: 0.3% Agarose Gel
<ol>
	<li>Microwave the pre-made 3% 2-Hydroxyethyl agarose solution for 15 sec. gently swirl the solution and microwave for another 15 sec.</li>
	<li>Equilibrate the agarose solution bottle in a 45 &#176;C water bath.</li>
	<li>Warm the MCF10DCIS media in a 37 &#176;C water bath.</li>
	<li>Mix 1 ml&#160;of 3% agarose solution with 9 ml&#160;of warm MCF10DCIS media into a 50 ml conical tube and gently invert to mix the agarose with the media. Avoid forming air bubbles.</li>
	<li>Treat the mixture with BB-CLA (0 &#181;M (DMSO) or 1 &#181;M).</li>
	<li>Gently add 1 ml&#160;of this mixture (without forming bubbles) into each well of the 6-well culture plate containing the bottom and soft layers.</li>
	<li>Place the 6-well culture plate horizontally on a flat surface at 4 &#176;C for at least 15 min to allow the mixture to solidify.</li>
	<li>After the feeder layer solidifies, place the plate into a 37 &#176;C incubator.</li>
	<li>Repeat this feeding procedure weekly by overlaying 1 ml&#160;of 0.3% agarose/medium/treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed. Note: Agar in the soft and feeder layers is very soft and, therefore, the added nutrients from the feeder layer will readily diffuse into the cell-containing layer to reach the cells.</li>
</ol>
5. Data Collection
<ol>
	<li>After 2.5 weeks of cell growth in the soft agar, count the number of colonies in each well using a light microscope. To facilitate quantification, print a grid onto a transparency and attach the grid to the 6-well plate to help locate where the cells are during counting. Since colony size (as quantified by the diameter of each colony) will vary, predefine a reference colony size to determine which colonies will be scored. For example, include colony sizes of 70 &#181;m or larger in the data analysis.</li>
	<li>Store the samples at 4 &#176;C to prevent further colony formation and for future counting. Seal the 6-well culture plate with Parafilm to prevent the gels from drying out.</li>
</ol>

<ol><li>Hamburger, A. W., Salmon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+bioassay+of+human+tumor+stem+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Molecular+signaling+regulating+anchorage-independent+growth+of+cancer+cells%5BTitle%5D)+AND+%22Mt+Sinai+J+Med%22%5BJournal%5D)">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/pubmed?term=((PAD%2C+a+growing+family+of+citrullinating+enzymes%3A+genes%2C+features+and+involvement+in+disease%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">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/pubmed?term=((Role+for+peptidylarginine+deiminase+enzymes+in+disease+and+female+reproduction%5BTitle%5D)+AND+%22J+Reprod+Dev%22%5BJournal%5D)">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/pubmed?term=((Potential+role+of+peptidylarginine+deiminase+enzymes+and+protein+citrullination+in+cancer+pathogenesis%5BTitle%5D)+AND+%22Biochem+Res+Int%22%5BJournal%5D)">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/pubmed?term=((Identification+of+PADI2+as+a+potential+breast+cancer+biomarker+and+therapeutic+target%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">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/pubmed?term=((Peptidylarginine+deiminase+inhibition+disrupts+NET+formation+and+protects+against+kidney%2C+skin+and+vascular+disease+in+lupus-prone+MRL%2Flpr+mice%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">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/pubmed?term=((MCF10DCIS.com+xenograft+model+of+human+comedo+ductal+carcinoma+in+situ%5BTitle%5D)+AND+%22J+Natl+cancer+Inst%22%5BJournal%5D)">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/pubmed?term=((Cooperative+evaluation+of+human+tumor+chemosensitivity+in+the+soft-agar+assay+and+its+clinical+correlations%5BTitle%5D)+AND+%22J+Cancer+Res+Clin+Oncol%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+human+tumor+colony+growth+in+soft+agar+by+serum%5BTitle%5D)+AND+%22Int+J+Cell+Cloning%22%5BJournal%5D)">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/pubmed?term=((A+high-throughput+soft+agar+assay+for+identification+of+anticancer+compound%5BTitle%5D)+AND+%22J+Biomol+Screen%22%5BJournal%5D)">A high-throughput soft agar assay for identification of anticancer compound.</a> J Biomol Screen. 12, 938-945 (2007).</li>
</ol>

The authors have nothing to disclose.

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><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

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...

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 ...

The overall goal of this procedure is to quantitatively determine the effect of treatment on the ability of cells to proliferate in semi-solid matrices as enhanced ability is a hallmark of cell tumor genicity using the soft agar assay tumor genicity is measured through a cell's ability to form colonies within a semi-solid AROS gel. Since non transformed cells are unable to rapidly propagate in this environment, the semi-solid AROS gel is constructed by first making a 0.6%AROS bottom layer. Next, a cell containing 0.3%AROS gel layer is added above the bottom layer.In this assay, the experimental cells are treated with an inhibitor of peptidyl, arginine, dase, or pad enzyme developed by Dr.Subramanian. Every week, an additional feeding layer is added, which is a 0.3%aros gel with or without treatment. The final step is to count the number of colonies larger than 70 micrometers for analysis.Ultimately, inverted microscopy is used to show the difference in the colony number between the control and the treatment group. Hi, my name is Achi Huta. I am a graduate student in Dr.Scott Kros lab at the Baker Institute for Animal Health.At Cornell University, soft tiger colony formation assay can help answer key questions in cancer biology field, such as assessing the tumor genicity of a wide range of cancer cells with regards to their sensitivity to drugs, hormones, and a multitude of other treatment conditions. Begin this procedure by preparing 3%two hydroxyethyl aros into a clean, dry 100 milliliter glass bottle. Add 0.9 grams of two hydroxyethyl aros, followed by 30 milliliters of distilled water.Microwave the mixture for 15 seconds and gently swirl. Repeat this step until the AROS powder fully dissolves. Next, autoclave the solution containing bottle for 15 minutes.Allow the agros solution to cool down to room temperature. Before further, use Prewarm several five milliliter and 10 milliliter pipettes in a 37 degrees Celsius incubator to prevent the aros from solidifying in the pipette. When handling partially, loosen the bottle lid and microwave the pre-made 3%two HYDROXYETHYL aro solution for 15 seconds.Then gently swirl the solution and microwave for another 15 seconds. Be careful when swirling the ARO solution because the solution rises up when exposed to air and can spill over if there is residual solid gel in the bottle microwave. For a few more seconds, keep the bottle containing the ARO solution in a 45 degree Celsius water bath during the next steps to prevent the agro solution from solidifying prematurely.Next, transfer 12 milliliters of warmed media using the Prewarm pipettes into a sterile 50 milliliter conical tube. Immediately add three milliliters of the 3%aros solution and gently invert the conical tube to mix the aros with the media. Then gently add two milliliters of this mixture into each well of a six well culture plate without forming any air bubbles.Incubate the six well culture plate horizontally on a flat surface at four degrees Celsius for one hour to allow the mixture to solidify. After the mixture solidifies, place the plate into a 37 degree Celsius incubator for 30 minutes. The bottom layer is now ready for use.A difficult aspect of this procedure is to make sure that all cells are distributed equally and that the melted agra shell do not solidify before addition to each well to ensure that the seltz are mixed in the media before adding the liquid agri gel. Additionally, the pipes are prewarm beforehand to avoid the solutions from solidifying while handling. To prepare the cell containing layer first trypsin eyes MCF 10 DCIS cells and dilute them to a cell concentration of 40, 000 cells per milliliter, then take eight milliliters of media using prewarm pipettes and transfer into a sterile 50 milliliter conical tube.Immediately add two milliliters of 3%aros to the conical tube and gently invert to mix the aros with the media. Avoid forming any bubbles. Next, take two milliliters of the cells and treat them with two micromolar BB chloramine in DMSO or DMSO alone as the control following treatment, mix the cells with the 0.6%aros in a one-to-one dilution to make a final concentration of one micromolar BB chloramine.Then take one milliliter of the cell aros mixture and gently add onto the bottom layer of the six well culture plate. Place the six well culture plate horizontally on a flat surface at four degrees Celsius for at least 15 minutes to allow the top layer to solidify. After the mixture solidifies, place the plate into a 37 degrees Celsius incubator for a week before adding the feeding layer.Begin feeding layer preparation by microwaving the pre-made 3%two hydroxyethyl aro solution as before and equilibrating the agro solution bottle in a 45 degree Celsius water bath mix one milliliter of 3%agro solution with nine milliliters of warm media into a 50 milliliter conical tube and gently invert to mix the aros with the media. Avoid forming air bubbles. After treating the mixture with BB chloramine gently add one milliliter of the mixture into each well of the six well culture plate containing the bottom and soft layers.Then place the six well culture plate horizontally on a flat surface at four degrees Celsius for at least 15 minutes to allow the mixture to solidify. After the feeder layer solidifies, place the plate into a 37 degrees Celsius incubator. Bader, repeat this feeding procedure weekly by overlaying one milliliter of 0.3%agros medium treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed.After two and a half weeks of cell growth in the soft agar, the colony number in each well is counted. Using a light microscope to facilitate quantification, print a grid onto a transparency and attach the grid to the six well plate to help locate where the cells are during counting. Colony size is quantified by the diameter of each colony and will vary predefine a reference colony size to determine which colonies will be scored.For example, here, colony sizes of 70 micrometers or larger are included in the data analysis. After counting seal the six well culture plate with paraform to prevent the gels from drying out. Store the samples at four degrees Celsius to prevent further colony formation and for future counting.The results demonstrate that BB chloramine significantly inhibits the formation of MC 10 DCIS cell derived colonies in the presence of a pad inhibitor. There was a reduction in both colony formation and colony size when compared to the DMSO control. The size of colonies for BB chloramine treated MCF 10 DCIS cells was predominantly within the range of 20 to 100 micrometers.While the size of colonies for the DMSO control exhibited a greater range of 70 to 150 micrometers after two and a half weeks of growth colonies larger than 70 micrometers were counted and analyzed. There was an average of about 3, 500 colonies in the DMSO control, whereas only approximately 2000 colonies were seen in the BB chloramine treated group. After two and a half weeks of soft agriculture, this represents a 44%decrease in the average colony formation in the presence of one micromolar BB Chloramine indicating a significant tumorogenic inhibition of breast cancer cells by the pad inhibitor.While attempting this procedure, it is important to keep in mind to prewarm all the reagents and equipments beforehand to avoid the solutions from solidifying when handling, and thanks for watching.

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