Name: spheroid assay to measure tgf-&#946;-induced invasion
Uploaded: 2011-11-16T13:00:00
Description: Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

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&deg;C) for 20 min.</li>
	<li>Add 250 ml of DMEM/F12 (RT) containing 10% horse serum. Mix overnight (4&deg;C).</li>
	<li>Clear the solution by centrifugation (5000g, 2h, 4&deg;C).</li>
	<li>Take the clear highly viscous solution to a new tube (about 90-95% of the stock solution).</li>
	<li>Sto...

<ol>
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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 border="1">
	<tbody>
 <tr>
	<td>Name of the reagent</td>
	<td>Company</td>
	<td>Catalogue number</td>
	<td>Comments (optional)</td>
 </tr>
 <tr>
	<td>Methyl cellulose</td>
	<td>Sigma-Aldrich</td>
	<td>M0512</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
	<td>PureCol</td>
	<td>Advanced Biomatrix</td>
	<td>5005-B</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
	<td>pH indicator strips</td>
	<td>Merck</td>
	<td>9533</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
	<td>96-well suspension plates</td>
	<td>Greiner Bio-one</td>
	<td>650 185</td>
	<td>&nbsp;</td>
 </tr>
 <tr>
	<td>96-well adhesion TC plates</td>
	<td>Greiner Bio-one</td>
	<td>655 180</td>
	<td>&nbsp;</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.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

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

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.

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

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