Spheroid assay to measure TGF-ß-induced invasion.

ABSTRACT

TGF-ß 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-ß is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-ß induces invasion are not well understood. TGF-ß elicits its cellular responses via TGF-ß type II (TßRII) and type I (TßRI) receptors. Upon TGF-ß-induced heteromeric complex formation, TßRII phosphorylates the TßRI. The activated TßRI 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 genes(5). 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) pathways(6). To study the role of TGF-ß in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). 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-ß-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 spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-ß signaling on the invasive properties of breast cells in different stages of malignancy.