Spheroid Assay to Measure TGF-β-induced Invasion

Summary

An assay to quantitatively measure Transforming Growth Factor (TGF)-β-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.

Abstract

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage^1,2. Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis ^3,4. The mechanisms by which TGF-β induces invasion are not well understood.

TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI 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-β 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-β-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-β signaling on the invasive properties of breast cells in different stages of malignancy.

Protocol

1. Preparation of the methocel solution (500 mL)

1. Autoclave 6 g of methylcellulose in a 500 ml bottle containing a magnetic stirrer.
2. Dissolve the methylcellulose in 250 ml of preheated DMEM/F12 without serum (60°C) for 20 min.
3. Add 250 ml of DMEM/F12 (RT) containing 10% horse serum. Mix overnight (4°C).
4. Clear the solution by centrifugation (5000g, 2h, 4°C).
5. Take the clear highly viscous solution to a new tube (about 90-95% of the stock solution).
6. Store at 4°C until use.

2. Spheroid culture

1. Prepare a 20% methocel solution (10 ml methocel and 40 ml growth medium).<....

Discussion

We established a spheroid model in which MCF10A1 cells and its malignant derivatives invade into a collagen matrix in a TGF-β-dependent manner. First, spheroids are formed in 96-wells round bottom suspension plates in the presence of methylcellulose. Of course, also U-shaped poly-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-β is added directly to the collagen-methocel mixture since we.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Methyl cellulose	Sigma-Aldrich	M0512
PureCol	Advanced Biomatrix	5005-B
pH indicator strips	Merck & Co., Inc.	9533
96-well suspension plates	Greiner Bio-One	650 185
96-well adhesion TC plates	Greiner Bio-One	655 180