mice. Second, the loss of PyMT expression together with the fact that these cells are nevertheless tumorigenic suggests that outgrowing Py2T cells that have undergone EMT have escaped oncogene addiction. In support of this hypothesis, we observed that mesenchymal Py2T LT cells formed significantly more colonies when grown under anchorage-independent conditions in soft agar. Intriguingly, in several other mouse models of breast cancer, discontinued oncogene expression is followed by the appearance of tumors that display Paritaprevir web EMT-like features. For example, after turning off Her2/ neu expression in tumors induced by this oncogene in the mammary gland, tumors regress, yet regrow as spindle cell ”EMT�� tumors that are strikingly similar if not identical in phenotype to the tumors we describe here. In agreement with our study, these tumors have not been observed to metastasize. It is likely that our model recapitulates these events, whose underlying mechanisms have yet to be determined. If so, the Py2T model system could be instrumental in elucidating mechanisms of tumor recurrence and of therapy resistance development, which has been previously attributed to EMT. Finally, in light of the recent findings that EMT confers stem cell-like traits to cancer cells, Py2T cells also offer a unique 18849973 system to study these events in vitro and in vivo. Reagents: recombinant human TGFb1, recombinant mouse EGF, rmIGF-1, rmHGF, rmbasicFGF, rhPDGF-BB, Dexamethasone, Matrigel, growth factor reduced, SB431542, SB203580, SP600125, PD98059. Cells and Cell Lines A subclone of NMuMG cells was a kind gift of Dr. M. J. Wheelock and has been previously described. NMuMG cells were originally obtained from the American Type Culture Collection. Py2T cells were isolated from a breast tumor of an MMTVPyMT female mouse with an FVB/N background. Isolation of this cell line was done with approval, and according to the rules and guidelines of, the Swiss Federal Veterinary Office and the local ethics committee;. NMuMG and Py2T cells were cultured in DMEM supplemented with glutamine, penicillin, streptomycin, and 10% FBS. Mouse Strains MMTV-PyMT 21804608 were received from N. Hynes. BALB/c nude mice were purchased from JANVIER SAS. Primers Primers used for quantitative RT-PCR are listed in Conclusions We have established and functionally characterized a novel cellular model of murine breast cancer EMT. While Py2T cells undergo EMT in response to TGFb stimulation in vitro, orthotopic transplantation into mice results in tumors displaying oncogenic, TGFb-dependent EMT. Py2T cells thus represent a versatile model to investigate the molecular mechanisms underlying EMT and to delineate how EMT contributes to therapy resistance, loss of oncogene addiction and tumor recurrence. Genotyping To extract DNA, cells from 
a confluent 10 cm dish were trypsinized, washed in PBS and pelleted. To the pellet, 450 mL tail tip buffer and 180 mL 6 M NaCl were added, the samples were mixed and spun at full speed in a tabletop centrifuge. Supernatant was added to 600 mL isopropanol, vortexed and spun for 5 min at full speed. Supernatant was discarded and 500 mL of 70% EtOH was added, vortexed and spun for 3 min at full speed. Supernatant was discarded and the pellet was dried and resuspended in TE buffer. Samples were analyzed using standard PCR procedure. Materials and Methods Antibodies and Reagents Antibodies: PyMT, Actin, E-cadherin, E-cadherin, Ncadherin, fibronectin, GAPDH, cytokeratin 14, cytokeratin 8/18, v
