Morphotypic plasticity in vitro and in nude mice of epithelial mouse mammary cells (NMuMG) displaying an epithelioid (e) or a fibroblastic (f) morphotype in culture

Abstract

Transition from an epithelioid (e-) to a fibroblastic (f-) morphotype marks invasiveness in clinical and experimental cancer. To understand better the factors influencing such transitions, we have subcloned and manipulated mouse mammary gland (NMuMG) cell cultures and compared the invasive phenotype of multiple subelones in vitro and in vivo. Cell lines with an e-morphotype expressed E-cadberin homogeneously and were not invasive in vitro. Cells with an f-morphotype were E-cadherin-negative and became fully invasive in vitro upon expression of the ras oncogene. Invasive tumors were produced in nude mice after subcutaneous injection of e-type or f-type cells. These tumors showed cystic, glandular and undifferentiated structures. Tumors from f-type cells were E-cadherin-negativewhereas e-type tumors stained heterogeneously in immunohistochemical preparations. Our observations demonstrate the impact of the micro-ecosystem on the invasive phenotype, with in vivo downregulation of E-cadherin and stimulation of the e-to f-morphotype transition.

Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition

“…The heterogeneity in parental NMuMG cells might contribute to some of the inconsistencies we see in the literature concerning EMT in this cell line. Van den Broecke et al characterized subclones of NMuMG cells and reported that the epithelioid clones expressed E-cadherin homogenously and were not invasive whereas the fibroblastic clones were E-cadherin-negative and more invasive than the epithelioid clones (Van den Broecke et al, 1996). NMuMG/E9 cells and NMuMG/E11 cells are similar to the clones in this earlier report.…”

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo

“…Both bTC and 4T1 cells stimulated migration of BPMECs (Figure 7g,j, arrowheads indicate endothelial cells; Figure 7l,m). The induction of migration appeared to be specific to tumor cells, as non-malignant NMuMg (Owens et al, 1974;Van den Broecke et al, 1996) cells did not induce BPMEC migration (Figure 7i,m). Addition of EphA2-Fc inhibited migration of BPMECs in response to both 4T1 and bTC (P50.01, Figure 7k,h,l,m), demonstrating that Eph A class receptor/ligand function is necessary for endothelial cell migration in response to tumor cells.…”

“…We selected the 4T1 model due to the ability of transplanted 4T1 cells to rapidly and reproducibly generate mammary adenocarcinoma in syngeneic animals in vivo (Aslakson and Miller, 1992;Lin et al, 1998). Expression of ephrin-A1 and EphA2 in EMT6 cells was also analysed, as these tumor cells also produce malignant adenocarcinomas in vivo, though with a slower progression than 4T1 cells (Rockwell, 1977(Rockwell, , 1978(Rockwell, , 1981. As shown in Figure 3a, ephrin-A1 was barely detectable in normal primary mouse mammary epithelial cell (PMEC) lysates, but was expressed at high levels in 4T1 and EMT6 tumor cells.…”

“…Immunoblot and immunoprecipitation analyses bTC pancreatic carcinoma (Radvanyi et al, 1993), 4T1, and EMT6 (Rockwell, 1977(Rockwell, , 1978(Rockwell, , 1981 mouse mammary adenocarcinoma cell lines were maintained in DMEM with 10% FCS. Primary mouse mammary epithelial cells were isolated and maintained as described previously Muraoka et al, 2001).…”

“…For co-culture experiments, transwells were coated with growth factor-reduced Matrigel (1 : 20 dilution in DMEM) and 2610 5 4T1, bTC, or NMuMg (Owens et al, 1974;Van den Broecke et al, 1996) were plated on the lower surface of the transwell filter. Tumor cells on the lower filter surface were labeled with FITC ovalbumin (Molecular Probes; 0.5 mg/ml in DMEM).…”