o confluent sheets of necrotic tumor cells, as well as prominence of residual stromal cells. In concert with the lack of histologic distinctions between control and cyclopamine treated animals, no differences were found in the immunohistochemical expression of the Hh ligand Shh HIF-1 Alpha and the Hh receptor Ptch, with abundant expression observed in both arms. Nestin immunohistochemistry, which was used to highlight tumor neovasculature of murine origin, also revealed no significant differences between cyclopamine and control arms. A variety of pharmacodynamic parameters were then examined in the primary xenografts from the four treatment arms. Murine nestin is expressed in the tumor neovasculature of xenografts, whereas the human nestin assay is expected to detect any nestin expression within the neoplastic cells themselves.
Quantitative AZD8330 MEK inhibitor RT PCR analysis showed significant down regulation of murine nestin mRNA in the treatment groups receiving gemcitabine, with or without addition of cyclopamine, compared with the control arm, which is consistent with reduction in tumor neovasculature, whereas no significant differences were noted between control and cyclopamine only groups. Human nestin levels showed a trend towards decreased expression in both treatment groups that received cyclopamine, however, due to the small sample number and considerable variation between the respective samples, these differences did not reach statistical significance. Murine Ptch levels, reflecting Hh expression in the stromal components, showed a trend towards decreased expression in the treatment groups, however, because this decrement was also observed in the gemcitabine arm, the mechanism based significance is uncertain.
We saw no significant differences or trends in the expression of human Ptch, similarly, there were no significant differences or trends in murine or human Gli1 or Gli2 mRNA. No significant differences in snail mRNA levels could be observed, again possibly because the sample numbers studied were too small. Hh signaling Oxaliplatin in pancreatic cancer cells significantly inhibits invasion, and, conversely, ectopic Hh activation in immortalized human pancreatic ductal cells renders them profoundly invasive in modified Boyden chamber assays. It has been previously reported that an active Hh pathway induces epithelial to mesenchymal transition through Gli1 dependent transcriptional down regulation of the cell adhesion molecule E cadherin, which, in turn, confers a greater invasive ability in cancer cells.
Our data confirm that similar mechanisms are active in the pancreatic epithelium, with profound loss of E cadherin expression in HPDE Gli1 cells and the reciprocal up regulation of E cadherin in E3LZ10.7 cells treated with cyclopamine. The second issue that we address in this study pertains to minor cellular subpopulations that may be preferentially affected by Hh blockade in pancreatic cancer. Specifically, we show an 3 fold reduction in the proportion of ALDH expressing cells by cyclopamine therapy in E3LZ10.7 cells. Our finding of Gli overexpression in ALDH bright cells might suggest that this subpopulation could be particularly Hh dependent and, therefore, preferentially sensitive to Hh inhibition with cyclopamine. Expression of elevated ALDH levels in human hematop