Transforming growth factor β treatment is sufficient to induce postsurgical recurrence and pulmonary metastasis. (A) Normal mammary epithelial (NME) cells were left untreated (before transforming growth factor treatment (pre-TGF)) or treated with TGF-β1 (post-TGF) and engrafted (2 × 106 cells/mouse) onto the mammary fat pad, at which point primary tumor growth was monitored by bioluminescence. Five weeks after engraftment of pre- and post-TGF NME cells, the primary tumors were surgically removed and tumor recurrence was similarly monitored by bioluminescence imaging. Shown are two representative mice from each experimental cohort, both before and 3 weeks after surgical resection of the primary tumor. (B) NME cells were treated with TGF-β1 (Post) as described in (A) and monitored for expression of the mesenchymal marker fibronectin (FN) and the marker epithelial cadherin (E-cad). Estrogen receptor α (ERα) was potently downregulated in NME cells following TGF-β treatment. (C) Photograph showing ex vivo pre- and post-TGF primary tumors following surgical resection demonstrating complete and intact primary tumor excision. (D) The average weight (±SE) of pre- and post-TGF primary tumors shown in (C) and the indicated P value. (E) Pre- and post-TGF primary tumors were analyzed by immunohistochemistry for the in vitro markers described in Panel B. All images were taken at 100x magnification and insets were taken at 400x magnification. (F) Growth of pre- and post-TGF NME primary and recurrent tumors were monitored using digital caliper measurements (n=5 mice/group). Vertical line indicates time of primary tumor resection. Data are the mean (±SE) tumor size for each group . *P < 0.05. (G) Photograph of a representative mouse 3 weeks after resection of a post-TGF NME tumor. Yellow outlines show widespread recurrent tumor formation. As shown in (A), mice bearing pre-TGF NME tumors failed to form recurrent tumors. (H) Pulmonary metastasis of pre- and post-TGF NME tumors was quantified by bioluminescence. Line indicates time of primary tumor resection. Inset: Bioluminescent images of ex vivo lungs from representative mice bearing pre- and post-TGF NME tumors. Data are the mean (±SE) area flux values for each group. *P < 0.05 (n = 5 mice/group).