These signaling molecules were evaluated before and after imatinib treatment. See the Supporting Materials for details. Results are shown as mean of “x” experiments ± standard deviation. Statistical comparisons

were made using Student t tests or Wilcoxon-Mann-Whitney’s two-sample rank-sum test. In Wilcoxon-Mann-Whitney’s two-sample rank-sum test, the P value was obtained from the exact permutation null distribution. Statistical analysis was performed selleck screening library using SPSS 16.0 software (SPSS, Inc., Bologna, Italy); P values <0.05 were considered as significant. The amount of tumor reactive stroma, measured as the percentage of the α-SMA-positive area present within the boundaries of the neoplastic area, was homogeneously represented in all CCA samples

(11.11% ± 4.70%) (Table 1; Supporting Fig. 1). Several phenotypic features of EMT were present in CCA bile ducts, but morphologic criteria supporting a complete transition toward a mesenchymal phenotype (coexpression of K7 and α-SMA) were never met. No EMT phenotype differences were observed find more between intra- (n = 10) and extrahepatic (n = 5) CCA (Table 1; Supporting Fig. 1). EGI-1 cells were xenotransplanted in SCID male mice after transduction with lentiviral vectors encoding firefly luciferase and EGFP to detect tumor engraftment in the liver in vivo (Fig. 1). Nine of ten xenotransplanted SCID mice developed a luminescent signal over the liver area 30-150 days postxenotransplantation. One animal died at day 55 before developing a detectable luciferase signal. Once the bioluminescent signal intensity in the liver reached a value >1 × 105 p/sec/cm2/sr, tumor-bearing mice were sacrificed at a median of 71 days after xenotransplantation (range, 50-155). Fig. 1A,B shows the correspondence between the bioluminescent signal and the macroscopic presence of liver tumors. Liver tumors were analyzed by dual IF for EGFP (expressed by transplanted EGI-1 cells) and α-SMA (myofibroblast/CAF marker). Xenotransplanted cancer cells that underwent

a complete EMT would be expected to coexpress EGFP and α-SMA. EGFP-positive, EGI-1-derived tumors were found embedded in abundant stroma, rich in α-SMA-positive Ponatinib cells strictly adjacent to tumor cells (Fig. 1C,D). However, coincident labeling between EGFP and α-SMA was never observed (Fig. 1D). In selected mice, a FISH analysis was performed using both human and mouse Y-probes for their coexpression with CAFs to confirm the above-mentioned results. Preliminary studies in mouse (n = 2) and human liver specimens (n = 2) indicated that both Y-probes were highly specific and did not cross-react between the two species. Consistent with the EGFP data, α-SMA-positive cells expressed the mouse, but not the human, Y-probe, which was instead normally expressed by infiltrating EGI-1 cells (Fig. 1E,F). These data demonstrate that CAF-infiltrating liver metastases are not generated through an EMT of xenografted EGI-1 cells.