We are grateful to Ken Iwata (OSI Pharmaceuticals, New York, USA) for reagents and Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) for the cell lines.

1. Preparation of the methocel solution (500 mL)
<ol>
	<li>Autoclave 6 g of methylcellulose in a 500 ml bottle containing a magnetic stirrer.</li>
	<li>Dissolve the methylcellulose in 250 ml of preheated DMEM/F12 without serum (60°C) for 20 min.</li>
	<li>Add 250 ml of DMEM/F12 (RT) containing 10% horse serum. Mix overnight (4°C).</li>
	<li>Clear the solution by centrifugation (5000g, 2h, 4°C).</li>
	<li>Take the clear highly viscous solution to a new tube (about 90-95% of the stock solution).</li>
	<li>Store at 4°C until use.</li>
</ol>
2. Spheroid culture
<ol>
	<li>Prepare a 20% methocel solution (10 ml methocel and 40 ml growth medium).</li>
	<li>Wash cells twice with PBS, add 0.1% trypsin and incubate cells at 37 °C</li>
	<li>Resuspend cells in growth medium and count the cells.</li>
	<li>Prepare a suspension of 10 000 cells per ml in 20% methocel.</li>
	<li>Add the cell suspension to the 96 well round bottom suspension plates (100 μl per well).</li>
	<li>Next day, check the spheroid formation in the suspension plates. Spheroids will be ready for use after 1-3 days.</li>
</ol>
3. Preparation of collagen
<ol>
	<li>Prepare on ice a solution of 8 ml collagen, 1 ml 10x PBS, 1 ml 0.1 N NaOH and 3 drops 0.1 N HCl. Check if pH is 7.4 by spotting some of the solution on pH indicator strips. Adjust pH if out of range.</li>
	<li>Add 50 μl of neutralized collagen solution in the wells of a 96 well plate</li>
	<li>Incubate at 37°C until the collagen is solidified (90 min).</li>
	<li>Prepare methocel-collagen-ligand solutions on ice: for each ligand pipette 1 part of of collagen solution. Add 20 ng per ml of collagen TGF-β. After dilution with methocel and diffusion over the different layers, the final concentration of TGF-β will be 5 ng/ml Mix by vortexing. Add 1 part of methocel- solution. Mix by vortexing.</li>
</ol>
4. Spheroid embedding
<ol>
	<li>Place a 200 μl tip which has approximately 5 mm of the tip cut off, on a 200 μl pipette. Suck up one spheroid with the pipette and carefully dispense the medium into an empty plate. Watch if the spheroid stays inside the tip. The spheroid is visible as a white dot. Without releasing the plunger, suck up 100 μl collagen-methocel mixture and dispense the spheroid in the collagen mixture into a collagen-coated 96 well. Do this for 12 spheroids for each condition.</li>
	<li>Let collagen solidify at least 30 minutes at 37°C</li>
	<li>Remove air bubbles with a 200 μl tip.</li>
	<li>Add 50 μl of medium with 1.6 % serum and inhibitors dissolved in DMSO. For SB-431542 add 4 μl of stock solution (10 mM) to 1 ml of medium. After diffusion, the final concentration of the inhibitor will be 10 μM. TGF-β and inhibitors will stay the course of the experiment. Take pictures of the spheroids under the microscope using 40X magnification.</li>
	<li>After 48h of stimulation with TGF-β and/or inhibitors, take pictures of the spheroids under the microscope using 40X magnification.</li>
	<li>Measure the area of the invading spheroids after 2 days and subtract the starting area. Measuring the area of a spheroid: Open the picture from the spheroid in Adobe Photoshop Extended (figure 2A) and select the Quick Selection tool (figure 2B). The mouse pointer changes into a circle with a 'plus' (+) sign (figure 2C). While dragging the cursor over the spheroid, Photoshop will recognize the borders of the spheroid. If an area outside the spheroid is selected, this region can be excluded from the selection by pressing the Alt key or by using the negative selection tool which is indicated by a (-) sign in the toolbar. The cursor changes into a circle with a 'minus' (-) sign and by dragging the cursor over the wrongly selected area, this area is removed from the selection (figure 2D). If necessary, multiple regions can be selected by pressing the Shift button. To work more accurately, the size of the mouse pointer can be adjusted by changing the brush diameter in the toolbar (figure 2E). When the whole spheroid is selected, the area of the selection is calculated via ANALYSIS-MEASURE in the menu bar or by pressing Ctrl + Shift + M (figure 2F). The measurement recording window will appear showing the file name and the area of the selection in pixels, as well as other data (figure 2G). If multiple selections are measured, the first line shows the sum of all the areas. The measurements of the individual selections are presented in the lines below. The data of all the measurements can be exported into a text file and opened in Excel.</li>
</ol>
5. Representative results:
An example of the spheroid assay with MCF10A1 (M1) normal breast epithelial cells, H-RAS transformed MCF10AneoT (M2) cells and M2-derived MCF10CA1a (M4) is given in Figure 3. M1 cells showed only weak invasion without stimulation, but invaded significantly better upon stimulation by TGF-β. In contrast, the RAS-transformed M1-derivative M2 invaded already efficiently without addition of stimuli, and this was further increased 4-5 fold upon TGF-β treatment. M4 cells showed the strongest invasion, both with and without TGF-β addition.
Next, we examined whether we could interfere with TGF-β-induced invasion in this spheroid model using chemical inhibitors. For this purpose we used SB-431542, an ATP analogue and selective inhibitor of the kinase activity of TβRI, as well as activin type IB receptor (ActRIB) and activin receptor-like kinase (ALK)712. As expected, SB-431542 potently inhibited TGF-β-induced invasion of M1, M2 and M4 cells (Fig 4), indicating that TGF-β-induced invasion is TβRI-kinase dependent. In addition, the basal invasion of the spheroids was also strongly inhibited, suggesting that basal invasion is also dependent on TGF-β.
<img alt="Spheroid formation and invasion diagram using collagen; cellular process illustration for research." src="/files/ftp_upload/3337/3337fig1.jpg" data-altupdatedat="1761809329000" data-alt="Figure 1"> 
Figure 1. Flow chart of the 3D spheroid assay. First, cells are plated under non-adherent conditions in u-shaped suspensions plates. Cells aggregrate into multcellular spheroids and can be embedded into a collagen matrix. Into this collagen matrix, they are able to invade.
<img alt="Digital analysis workflow using graphic software; tools and menu selections illustrated in steps." src="/files/ftp_upload/3337/3337fig2.jpg" data-altupdatedat="1761809329000" data-alt="Figure 2"> 
Figure 2. Quantification of the area of the spheroids using Adobe Photoshop Extended. (A) The picture of the spheroid is opened in Adobe Photoshop Extendend (B) Selection of the Quick Selection tool (C) Dragging the cursor over the spheroid to select the area of the spheroid (D) Removal of a wongly included area using the negative Quick Selection tool (E) Adjustment of the brush size of the Quick Selection tool to more accurately select the area (F) Selection of the measurement command to record (G) Example of a measurement record.
<img alt="TGF-β effects on cell line M1, M2, M4 over 2 days; includes invasion assay graph; comparative analysis." src="/files/ftp_upload/3337/3337fig3.jpg" data-altupdatedat="1761809329000" data-alt="Figure 3"> 
Figure 3. TGF-β-induced invasion of spheroids of spontaneously immortalised MCF10A1 (M1) (A), RAS-transformed MCF10AneoT (M2 (B) and its metastatic derivative MCF10CA1a (M4) (C). M1, M2 and M4 spheroids embedded into collagen were treated with 5ng/ml of TGF-β for 48h as indicated. A-C Representative pictures taken at the day of embedding and two days later. D Relative invasion was quantified as area difference on day 2 minus day 0. The results are expressed as mean ± s.d. Adapted from13.
<img alt="Cell migration assay results; TGF-β influence on cell formation; microscopy images; DMSO, SB-431542 comparison." src="/files/ftp_upload/3337/3337fig4a.jpg" data-altupdatedat="1761809329000" data-alt="Figure 4a"> 
<img alt="Bar chart comparing cell invasion under control and SB-431542 conditions in invasion assay results." src="/files/ftp_upload/3337/3337fig4b.jpg" data-altupdatedat="1761809329000" data-alt="Figure 4b"> 
Figure 4. Basal and TGF-β-induced spheroid invasion is inhibited by SB-431542. M1, M2 and M4 spheroids were embedded into collagen and treated with 5ng/ml TGF-β and/or 10 μM SB-431542 as indicated. Representative pictures taken after 2 days (A). Relative invasion was quantified as area difference on day 2 minus day 0 (B). The results are expressed as mean ± s.d. Adapted from13.

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<li>Wiercinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+TGF-%CE%B2%2FSmad+pathway+induces+breast+cancer+cell+invasion+through+the+up-regulation+of+matrix+metalloproteinase+2+and+9+in+a+spheroid+invasion+model+system%5BTitle%5D)+AND+%22Breast+Cancer+Res.+Treat%22%5BJournal%5D)">The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system.</a> Breast Cancer Res. Treat. 128, 657-657 (2011).</li>
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<li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collagen+density+promotes+mammary+tumor+initiation+and+progression%5BTitle%5D)+AND+%22BMC.+Med%22%5BJournal%5D)">Collagen density promotes mammary tumor initiation and progression.</a> BMC. Med. 6, 11-11 (2008).</li>
<li>Ramaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+signature+of+metastasis+in+primary+solid+tumors%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">A molecular signature of metastasis in primary solid tumors.</a> Nat. Genet. 33, 49-49 (2003).</li>
<li>Debnath, J., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modelling+glandular+epithelial+cancers+in+three-dimensional+cultures%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">Modelling glandular epithelial cancers in three-dimensional cultures.</a> Nat. Rev. Cancer. 5, 675-675 (2005).</li>
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No conflicts of interest.

We established a spheroid model in which MCF10A1 cells and its malignant derivatives invade into a collagen matrix in a TGF-&beta;&#45;dependent manner. First, spheroids are formed in 96-wells round bottom suspension plates in the presence of methylcellulose. Of course, also U-shaped poly&#45;HEMA coated plates can be used for this purpose. Methylcellulose is absolutely required in this step, since cells will die in the absence of methylcellulose. TGF&#45;&beta; is added directly to the collagen-methocel mixture since we...

<table><tbody><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td></td></tr><tr><td>PureCol</td><td>Advanced Biomatrix</td><td>5005-B</td><td></td></tr><tr><td>pH indicator strips</td><td>Merck &amp;amp; Co., Inc.</td><td>9533</td><td></td></tr><tr><td>96-well suspension plates</td><td>Greiner Bio-One</td><td>650 185</td><td></td></tr><tr><td>96-well adhesion TC plates</td><td>Greiner Bio-One</td><td>655 180</td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

The overall goal of the following experiment is to use a measure the effect of potential regulators of TGF beta induced invasion in a three-dimensional environment. This is achieved by first making spheroids of the cells. The PHE are then embedded in collagen, which provides a 3D environment.Next, the invasive area is quantified. In order to determine the effect of treatment on TGF beta induced invasion, the results show that modulators of the TGF-beta pathway affect the invasive property of the steroids in a 3D collagen matrix. The main advantage of this technique over existing methods like the scratch ass essay, is that it better recapitulates the 3D environment.This method can help to answer key questions in the T meter fields, such as the identification of novel genes involved in teacher beta induced invasion. To prepare methyl cell solution for the TGF beta induced invasion assay begin by autoclaving six grams of Methylcellulose in a 500 milliliter bottle containing a magnetic stir. Dissolve the Methylcellulose in 250 milliliters of preheated D-M-E-M-F 12 without serum for 20 minutes, add 250 milliliters of room temperature D-M-E-M-F 12 containing 10%horse serum mix overnight at four degrees Celsius the next day, clear the solution by centrifugation at 5, 000 G for two hours at four degrees Celsius.Transfer the clear, highly viscous solution to a new tube store at four degrees Celsius until use to culture phe. Prepare a 20%methyl cell solution using 10 milliliters of the prepared methyl cell stock and 40 milliliters of growth. Medium wash MCF 10 A cells twice with PBS.Add 0.1%trypsin and incubate the cells at 37 degrees Celsius until all cells are detached. Resuspend the cells in 10 milliliters, growth medium, and count the cells using a cell counter. Prepare a suspension of 10, 000 cells per milliliter in 20%methyl cell.For each test condition, add 100 microliters of the cell suspension to 12 wells of a 96 well round bottom suspension plate. Incubate the cells overnight in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. The next day check for spheroid formation.Spheroids will be ready for use within one to three days. Prepare on ice a solution of eight milliliters of collagen, one milliliter of 10 XPBS, one milliliter of 0.1 normal sodium hydroxide, and three drops of 0.1 normal hydrochloric acid. Check that the solution's pH is 7.4 by spotting it on pH indicator strips.Adjust the pH if it is out of range. Add 50 microliters of neutralized collagen solution to the wells of a 96 Well plate incubate at 37 degrees Celsius for about 90 minutes or until the collagen is solidified. For each ligand to be tested, prepare a methyl cell collagen ligand solution on ice.Begin with one milliliter of collagen solution per treatment, and add 20 nanograms of TGF beta for a final concentration of five nanograms per milliliter mixed by vortexing. Then add one milliliter of methyl cell solution for a final volume of two milliliters and vortex. Again to embed a steroid place a 200 microliter pipette tip, which had five millimeters or more of the tip cut off on a 200 microliter pipette.Then take up one s spheroid with the pipette. Carefully dispense the medium into an empty plate while ensuring that the S spheroid stays inside the tip. Do not release the plunger.Take up 100 microliters of the collagen methyl cell mixture and dispense the S spheroid collagen mixture into a collagen coated well of a 96. Well plate fill 12 wells for each condition. Let the collagen solidify at least 30 minutes at 37 degrees Celsius and remove the air bubbles with a 200 microliter tip.Prepare a 40 micromolar solution of the TGF beta inhibitor SB 4 31 542 by adding four microliters of SB 4 31 542 stock solution to one milliliter of D-M-E-M-F 12 containing 1.6%horse serum. Add 50 microliters of medium with inhibitor to each well after diffusion. The final concentration of the inhibitor will be 10 micromolar.Take pictures of the steroid under the microscope using 40 x magnification, then incubate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. After a two day incubation, take pictures again and measure the area of the invading steroid by opening a spheroid picture in Adobe Photoshop. Extended and select the quick selection tool.The mouse pointer will change into a circle with a plus sign while dragging the cursor over the spheroid. The software will recognize the borders of the spheroid. If an area outside the steroid is selected, exclude the area by pressing the alt key or by using the negative selection tool, which is a negative sign in the toolbar.Drag the cursor over the wrongly selected area and the software will remove it from the selection. If an area within the spheroid is deselected, the area can be included again by dragging while pressing the shift bar. To work more accurately, adjust the size of the mouse pointer by changing the brush diameter in the toolbar.To calculate the area of all active selections, choose analysis measure In the menu bar. A measurement recording window will appear showing the file name and the area of selection in pixels. If multiple selections are measured within one spheroid, the first line will show the sum of all the areas and the lines below.It will list the values of the individual selections. Export the data into a text file and open it in excel. An example of the S spheroid assay with MCF 10 A one, normal breast epithelial cells, HAS transformed MCF 10 A neo T cells and M two derived MCF 10 CA one A is shown here.M1 cells showed only weak invasion without stimulation, but invaded significantly better after stimulation by TGF beta. In contrast, the RAs transformed M1 derivative M two invaded efficiently without the addition of stimuli, and this was further increased four to fivefold. After TGF beta treatment, M four cells showed the strongest invasion both without and with TGF Beta Edition.A graphical representation of the results for this experiment was obtained using Photoshop and is shown here in this example, SB 4 31 5 42 an A TP analog and selective inhibitor of the kinase activity of TGF beta receptor one, as well as active in type one B receptor and active in receptor like kinase seven was added to the cell cultures SB 4 31 5 42, potently inhibited, TGF beta induced invasion of M1, M two, and M four cells, indicating that TGF-beta induced invasion is TGF-beta receptor one kinase dependent. In addition, the basal invasion of the PHE was strongly inhibited. A graphical representation of the results for this experiment was again created using Photoshop and is shown here.Following this procedure, other methods like RNA isolation or protein isolation can be performed to answer additional questions like which genes are expressed or which proteins are expressed. After watching this video, you should have a good understanding of how to find new modulators of tavita induced invasion.

spheroid-assay-to-measure-tgf-induced-invasion

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