vested and analyzed. Haematoxylin & Eosin staining of histological sections of a Py2T tumor and a late stage MMTV-PyMT tumor revealed that MMTV-PyMT tumors were mainly well differentiated with some less well-differentiated areas 
and necrosis towards the tumor center. The tumor borders were passively 5 Py2T EMT Model 6 Py2T EMT Model invading the fat pad by proliferation. In contrast, Py2T tumors were characterized by streams of elongated cells that were actively invading the surrounding fat tissue. Of note, Py2T tumors lacked excessive necrosis, possibly because they were well vascularized as determined by staining for the blood vessel marker CD31. Furthermore, Py2T tumors contained a high stromal component intermixed with tumor cells. To exclude the possibility that immune cell infiltration was due to a possible re-expression of the PyMT transgene, tumor tissue sections were stained with an antibody against the PyMT protein. As expected, PyMT expression could be detected in MMTVPyMT tumors, but not in Py2T tumors. When Py2T cells were orthotopically implanted into immuno-deficient nude mice, all mice developed tumors with a indoleamine-2,3-dioxygenase inhibitor INCB024360 substantial infiltration of CD45-positive stromal cells, with a high content of macrophages. The spindle-like appearance of cells in the Py2T tumors suggested that Py2T cells might have undergone an EMT in these tumors. We thus compared lysates from mainly epithelial MMTVPyMT tumors with lysates from mainly invasive Py2T tumors for expression of EMT markers. Indeed, expression of E-cadherin in MMTV-PyMT tumors was 22880633 readily detectable as expected, however, very little if any E-cadherin expression was detectable in lysates of Py2T tumors, supporting the hypothesis that Py2T cells had undergone EMT-like changes in vivo. Expression of the mesenchymal markers fibronectin and Ncadherin was also higher in some but not all Py2T tumors as compared to MMTV-PyMT tumors. Collectively, these results demonstrate that Py2T cells are tumorigenic, despite the absence of PyMT expression, and that they undergo oncogenic EMT-like changes in vivo. Notably, neither FVB/N nor immuno-deficient mice bearing Py2T tumors developed apparent metastasis, as determined by histological sectioning of various organs. TGFb-dependent EMT of Py2T Tumors We next assessed whether the EMT occurring during Py2T tumor growth in the mammary fat pad of mice could be attributed to stimulation by host-derived TGFb. First, we generated Py2T cell lines that stably express GFP for their distinction from host stromal cells. Next, we superinfected these cells with a lentiviral construct encoding a dominant-negative form of TGFb receptor II or empty vector as control. Cultured Py2T TBRDN-expressing cells did not show any apparent changes in phenotype as compared to control cells in the absence of TGFb, but were resistant against TGFb-induced EMT. In a next step, we transplanted Py2T control and Py2T TBRDN into fat pads of immuno-deficient nude mice to evaluate their ability to 24952596 undergo EMT in vivo. All mice developed tumors, and tumor growth was not significantly different between the two experimental groups, although TBRDN tumors tended to grow more slowly with increasing size in comparison to Py2T control tumors. H&E staining of Py2T control tumors revealed the same stream-like cellular growth pattern as observed in Py2T tumors in FVB/N mice, with cells displaying a spindle-like morphology. Interestingly, tumors formed by Py2T TBRDN contained patches of more differen
