In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total downregulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of ''primary'' tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced ''ALDH-high'' cells by f3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce ''bulk'' tumor size. [Cancer Res 2007;67(5):2187-96]

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials.The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

Background Pancreatic cancer is a near uniformly lethal disease and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. The Axl receptor tyrosine kinase is implicated in cellular transformation and tumor progression, although its role in pancreatic cancer has not been previously documented. Results Axl labeling was present in 54 of 99 (55%), and was absent in 45 of 99 (45%) cases, respectively. Axl expression in pancreatic cancer was significantly associated with lymph node metastases (p < 0.01), and a shorter median survival (12 versus 18 months, p < 0.01), than in tumors with negative labeling. Stable knockdown of Axl resulted in significant reduction in cell viability (p < 0.001), anchorage independent growth (p = 0.0031), as well as attenuation of migratory (p < 0.001) and invasive properties (p < 0.005), compared to vector-transfected cells. Profound inhibition of p42/p44 MAP kinase and PI-3kinase/Akt effector pathways was observed in MIAPaCa-2 cells with loss of Axl function. The reduction in invasion and migration upon Axl knockdown was mirrored by a decrease in the amounts of activated (GTP-bound) GTPase proteins Rho and Rac, significant downregulation in transcript levels of the epithelial mesenchymal transition (EMT)-associated transcription factors slug, snail and twist, and significant decrease in matrix metalloproteinase MMP-9 mRNA levels. Materials The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Endogenous Axl expression was stably downregulated by lentiviral short hairpin shRNA directed against AXL mRNA in MIAPaCa-2 cells, and the effects on cell viability, anchorage independent growth, invasion, migration and intracellular effector pathways was assessed, by comparing to lentiviral vector-transfected cells. Conclusion Expression of Axl tyrosine kinase in pancreatic cancers confers an adverse prognostic influence, and represents a new therapeutic target in this malignancy.