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 line in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin solution. Warm medium to 37 &#176;C in hot water bath prior to use.</li>
	<li>Prepare 1x cell culture medium separately as you would for normal cell culture of the cell line of interest.</li>
	<li>Prepare 1% noble agar by adding 1 g of noble agar to 100 ml of deionized water. 
	NOTE: Noble agar will not dissolve completely with agitation alone.</li>
	<li>Prepare 0.6% noble agar by adding 0.6 g of noble agar to 100 ml of de-ionized water. Both agar solutions can be made in 100 ml glass bottles with closable lids for long-term storage.</li>
	<li>Autoclave the noble agar mixtures to sterilize. These mixtures can be made in advance and stored at 4 &#176;C but should be heated again at the time of the experiment until agar has completely dissolved.</li>
	<li>Prepare nitroblue tetrazolium chloride solution by making a 1 mg/ml stock solution in 1x PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in H2O to final volume of 1,000 ml). This will be used at the end of the experiment to stain the colonies.</li>
</ol>
2. Plating of Bottom Layer of Agar
<ol>
	<li>Loosen the cap on the bottle of 1% noble agar and microwave for about 1-2 min. While heating in a microwave, monitor the solution closely to avoid boiling over. Continue heating, while mixing intermittently, until agar is completely dissolved and the solution is clear. 
	NOTE: Use heat-resistant gloves to handle flask after heating. Failing to do so may cause burn or serious injury.</li>
	<li>Place melted agar solution and pre-warmed 2x culture medium in an ice bucket filled with hot tap water (42 &#176;C). Also place a 50 ml conical tube in a tube holder in the ice bucket with hot water. Transfer bucket to cell culture hood for subsequent steps.</li>
	<li>For the bottom layer of agar, you will need 1.5 ml of a mix of agar and medium per well of a 6-well plate.</li>
	<li>To ensure an adequate amount of the mixture, prepare a total of 12 ml for each 6-well plate.</li>
	<li>Start by adding 6 ml of culture medium to the 50 ml conical tube and then 6 ml of the 1% noble agar solution.</li>
	<li>Invert the conical tube several times to mix. Working at a brisk pace will prevent premature hardening of the soft agar.</li>
	<li>Draw up approximately 5.5 ml of mixture into a 5 ml serological pipette.</li>
	<li>Allow the air bubbles to rise to top of pipette column before depositing 1.5 ml of this mixture into each well. Use caution to avoid deposition of any air bubbles into the plate wells.</li>
	<li>Cover the plates and allow agar mixture to solidify at room temperature, in cell culture hood, for 30 min.</li>
</ol>
3. Plating the Upper Layer of Agar Containing Cells
<ol>
	<li>Once the lower layer of agar has solidified, begin preparation of the upper layer.</li>
	<li>First, harvest cells by trypsinization and dilute them 1:5 in culture medium (e.g. for 1 ml of trypsin, add 4 ml of medium) into a 15 ml conical tube.</li>
	<li>Count cells and calculate the number of cells needed per well to prepare a final cell suspension at this time. This number will vary depending on cell type. Use 5,000 cells/well as a starting point and adjust as needed. For this cell number, you would prepare a cell suspension of 6,667 cells/ml (i.e. each well will receive 0.75 ml of this suspension and 0.75 ml of agar for a total volume of 1.5 ml; the concentration of cells will also be diluted 1:2 for a final total cell count of 5,000).</li>
	<li>The volume of cell suspension needed per well of a 6-well plate will again be 1.5 ml. Prepare additional cell suspension totaling 12 ml per 6-well plate.</li>
	<li>Melt 0.6% agar solution in a microwave as above and place into ice bucket containing hot water along with a 50 ml conical tube in a tube holder and the final cell suspension from Step 3.3.</li>
	<li>Transfer the ice bucket with melted 0.6% agar to cell culture hood for subsequent steps.</li>
	<li>Mix 0.6% agar and cell suspension in a 1:1 ratio, preparing a total volume of 12 ml per 6-well plate. 1.5 ml will be required per well but extra should be made as above.</li>
	<li>Pipette 6 ml of cell suspension into the 50 ml conical tube.</li>
	<li>Then, add 6 ml of 0.6% agar to the tube. 
	NOTE: The temperature of this mixture must be kept around 42 &#176;C to avoid premature hardening and to maximize cell survival.</li>
	<li>Working quickly, pipette this mixture 2-3x to distribute cells, then draw up 5.5 ml of mixture into a 5 ml serological pipette.</li>
	<li>Allow any air bubbles to rise to top of pipette column before depositing 1.5 ml of this mixture into each well. Use caution to avoid deposition of any air bubbles into the plate wells.</li>
	<li>Allow cell/agar mixture to solidify at room temperature, in cell culture hood, for 30 min before placing into a 37 &#176;C humidified cell culture incubator.</li>
	<li>The time required for adequate colony formation varies for each cell line, typically around 21 days.</li>
	<li>A layer of growth medium should be maintained over the upper layer of agar to prevent desiccation. 100 &#956;l of medium added twice weekly is sufficient for this purpose.</li>
</ol>
4. Staining the Plates and Counting Colonies
<ol>
	<li>Stain cells by adding 200 &#956;l of nitroblue tetrazolium chloride solution per well and incubating plates overnight at 37 &#176;C.</li>
	<li>Once colonies are stained, take photographs of wells using an imager and count colonies using image analysis software.</li>
</ol>

<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 this assay is plating of the cells. Cell counts must be accurate, and the agar solution must not be too hot. If no colonies are formed, the cells may have been damaged due to heat stress. In this situation, the assay should be repeated, taking precautions to keep agar temperature as close to 42 &#176;C as possible. Alternatively, a greater number of cells can be plated.
There are a few limitations to the soft agar assay. One such limitation is that it takes two to three weeks for completion. Another is that it does not allow for cell retrieval upon completion. Modifications of this technique employ specialized agar solutions to allow for cells to be harvested for DNA or protein upon assay completion. Alternative methods also utilize fluorometric dye to allow for high throughput assays. Nevertheless, the traditional soft agar colony formation assay remains as one of the most rigorous tests for confirmation of anchorage-independent cell growth.

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><tbody><tr><td>Cancer Cell Line of Interest</td><td>Sigma-Aldrich</td><td>10032302</td><td>CMT-167 Cells</td></tr><tr><td>Powdered RPMI 1640 Medium</td><td>Gibco</td><td>31800-089</td><td>Used to prepare 2x cell culture medium.</td></tr><tr><td>Liquid RPMI 1650 Medium</td><td>Cellgro</td><td>10-040-CV</td><td>Referred to as 1x cell culture medium.</td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30910.03</td><td>Used to supplement cell culture medium.</td></tr><tr><td>Penicillin/Streptomycin</td><td>CellGro</td><td>30-002-Cl</td><td>Used in cell culture medium.</td></tr><tr><td>Difco Noble Agar</td><td>BD Biosciences</td><td>214230</td><td>Used to prepare 1.0% and 0.6% agar.</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher</td><td>BP-328-1</td><td>Used in 2x cell culture medium.</td></tr><tr><td>Trypsin</td><td>Cellgro</td><td>25-050-Cl</td><td></td></tr><tr><td>Sterile Bottle-Top Filters</td><td>Fisher</td><td>09-761-126</td><td>Used to sterile filter 2x medium.</td></tr><tr><td>Lipofectamine Reagent</td><td>Invitrogen</td><td>18324-020</td><td>Used in PPAR-RE luciferase assay.</td></tr><tr><td>6-well Plates Tissue-culture Treated</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176;C/5% CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td></td><td></td><td></td></tr><tr><td>Chemi-Doc Imager</td><td>Bio-Rad</td><td></td><td>Used to take pictures of colonies.</td></tr><tr><td>Quantity One Software</td><td>Bio-Rad</td><td></td><td>Used to count cell colonies.</td></tr><tr><td>15 ml Conical Tubes</td><td></td><td></td><td></td></tr><tr><td>50 ml Conical Tubes</td><td></td><td></td><td></td></tr><tr><td>250 ml Erlenmeyer Flasks</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>5 ml Serological Pipettes</td><td></td><td></td><td></td></tr><tr><td>Pipette Aid</td><td></td><td></td><td></td></tr><tr><td>Micropipette</td><td></td><td></td><td></td></tr><tr><td>Hemacytometer w/ cover slip</td><td></td><td></td><td></td></tr><tr><td>Pipette Tips</td><td></td><td></td><td></td></tr><tr><td>Inverted Light Microscope</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Heat-Resistant Gloves</td><td></td><td></td><td></td></tr><tr><td>Saran Wrap</td><td></td><td></td><td></td></tr><tr><td>Ice Bucket</td><td></td><td></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 Wnt7A (LNCX-Wnt7A) and Fzd9 (LPCX-Fzd9) to create a stable cell line that expresses both Wnt7A and Fzd9 (CMT LL Wnt7a/Fzd9). We confirmed expression of Wnt7A and Fzd9 by Q-PCR in this cell line (Figure 2). As our previous work has shown, PPARγ is a downstream effector of Wnt7A/Fzd9 signaling. Wnt signaling pathways are diverse. Therefore, proper expression and activation of a particular Wnt and its Fzd receptor must be assessed using a downstream effector that is known to be activated by the Wnt/Fzd pair of interest. For Wnt7A and Fzd9, PPARγ was chosen for this reason. We transfected CMT167 cells overexpressing empty vectors (LNCX/LPCX) or Wnt7A/Fzd9 with a Luciferase reporter construct containing a PPARγ response element. We showed that our CMT167 LL Wnt7A/Fzd9 cell line had almost six-fold increased PPAR-RE activity (Figure 3). Finally, as mentioned previously, to confirm the tumor suppressor activity of Wnt7A and Fzd9 in vitro, we performed a soft agar colony formation assay. CMT167 vector expressing and Wnt7A/Fzd9 expressing stable cell lines were plated onto soft agar plates and allowed to form colonies for two to three weeks. CMT167 LL Wnt7A/Fzd9 cells formed significantly less colonies when compared to vector-transfected cells, illustrating the tumor suppressor function of Wnt7A and Fzd9 (Figure 4). Pictures were taken of all plates, and colonies were counted using an imager and imaging software.
<img alt="Bar chart comparing Wnt7a/GAPDH and Fzd9/GAPDH mRNA expression in MLE12 vs CMT167 cells." src="/files/ftp_upload/51998/51998fig1highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Decreased levels of Wnt7A and Fzd9 mRNA in CMT167 cells compared to MLE-12 cells. Q-PCR analysis was performed to determine mRNA levels of Wnt7a and Fzd9 normalized to GAPDH. Levels in CME167 murine lung carcinoma cells were compared to those in MLE-12 SV40-transformed murine lung epithelial cells, which were normalized to 1.0. CMT167 cells showed lower levels of Wnt7a (A) and Fzd9 (B) mRNA when compared to MLE-12 cells.
<img alt="Bar graphs of Wnt7A and Fzd9 expression ratios relative to GAPDH; CMT cell line comparison results." src="/files/ftp_upload/51998/51998fig2highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Overexpression of Wnt7a/Fzd9 in CMT167 LL Wnt7a/Fzd9 stable cell line. Wnt7A/Fzd9 stable overexpression human constructs were made using an LNCX retroviral vector. Q-PCR was performed for Wnt7A and Fzd9, and mRNA levels were normalized to GAPDH in empty-vector transfected cells and in Wnt7A/Fzd9 overexpressing cells. CMT167 LL Wnt7a/Fzd9 cells expressed high levels of Wnt7a (A) and Fzd9 (B) mRNA.
<img alt="Bar chart showing PPAR-RE promoter activity data; LL Wnt7a/Fzd9 increases activity versus LNCX/LPCX." src="/files/ftp_upload/51998/51998fig3highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: CMT167 Wnt7a/Fzd9 stable overexpressing cell line exhibits increased PPAR-RE promoter activity. CMT167 LL Wnt7a/Fzd9 or empty vector CMT167 LNCX/LPCX stable overexpressing cells were transfected with a PPAR response element promoter luciferase plasmid using Lipofectamine reagent. Promoter activity was quantified compared to vector-transfected cells, where promoter activity was normalized to 1.0. CMT167 LL Wnt7a/Fzd9 stable overexpressing cells showed an approximately six-fold increase in PPAR promoter activity.
<img alt="Bar graph and Petri dishes showing cloning efficiency, LNCX/LPCX vs. LL Wnt7a/Fzd9 experiment." src="/files/ftp_upload/51998/51998fig4highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Soft Agar Colony Formation Assay of vector-expressing CMT167 LNCX/LPCX cells and CMT167 LL Wnt7a/Fzd9 overexpressing cells. Vector-expressing CMT167 LNCX/LPCX cells and CMT167 LL Wnt7A/Fzd9 overexpressing cells were plated in a soft agar colony formation assay per protocol described above. CMT167 LL Wnt7A/Fzd9 cells showed a marked decrease in colony formation. Representative wells showing colonies for each cell line. Picture of wells are representative of three independent experiments.

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 ...

Nanyang Technological University

Research

JoVE Journal

Biology

111.4K Views.  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.

Video: The Soft Agar Colony Formation Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

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.

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 ...

Medicine

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Immunology and Infection

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Keratinocyte Colony Formation Assay: An In Vitro Assay to Assess the Ability of Keratinocytes to Grow into Colonies

Source: Morris, R. J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/58701"> Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture</a>. J. Vis. Exp. (2019)
In this video, we perform a clonogenic assay of keratinocytes harvested from adult mice to determine the ability of individual cells to proliferate and form colonies. This keratinocyte harvesting technique is useful for studying other downstream aspects of cutaneous chemical carcinogenesis and tumor promotion.

Source: Morris, R. J. et al.  Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture. J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

Cancer Research

Skin Cancer

Assays and techniques

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

Breast Cancer

In vitro studies

Colony Formation Assay: Assessing the Efficacy of Anticancer Agents on Colony-Forming Lung Cancer Cells

Source: Lee, J. Y.et al, <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp.(2018).
This video describes the colony formation assay, which helps determine lung cancer cells&#39; ability to proliferate indefinitely and form individual colonies. We can analyze the effect of an anticancer agent on the colony-forming cells by analyzing the changes in the number and/or size of the resulting colonies.

Source: Lee, J. Y.et al, Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...

Lung Cancer

In vitro study

Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells

To carry out the CFU assay, prepare 10 milliliters of Giemsa solution per dish, by using sterile water to dilute the stock solution 1 to 10. Remove the medium from the cell culture dish, and use PBS to carefully wash the cells.
With pure methanol, fix the cells at room temperature for 5 minutes, then, discard the methanol and add the Giemsa solution. Incubate in a humidified chamber at 37 degrees Celsius for 60 minutes. After the incubation, use PBS to wash the cells twice. Then, air-dry the plate head-first on a paper towel. With a marker on the back of the plate, manually count the colonies.

The Soft Agar Colony Formation Assay

Samenvatting

Meer video's verkennen