Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSClow) and highly aggressive CSC-like cells (CSChigh) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSChigh cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSClow cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSChigh cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSChigh cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.

Recruitment of activated leukocytes from peripheral blood into the tumor tissue is a crucial step of the immune response, which is controlled by the interaction between specific adhesion molecules such as endothelial ICAM-1 and leukocyte b 2 -integrins. Although attenuated expression of adhesion molecules on tumor endothelium has been proposed to represent a mechanism, which suppresses the intratumoral leukocyte infiltration, the relevance of adhesion molecules for leukocyte recruitment in tumor tissue is poorly understood. The present study is the first investigation of the role of ICAM-1 and b 2 -integrins in leukocyte recruitment in pancreatic and hepatocellular cancer in vivo, which was studied using knockout mice, intravital time-lapse microscopy and immunohistochemistry. We found that tumor tissue of both pancreatic and hepatocellular cancer was infiltrated with numerous active lymphoid and myeloid leukocytes, although the leukocyte extravasation rate in tumor blood vessels was very low. The knockout of LFA-1 (also known as a L b 2 integrin) strongly suppressed recruitment of CD8 1 T cells whereas no significant differences of leukocyte adhesion and infiltration were found in ICAM-1 2/2 and Mac-1mice. Analysis of the interstitial leukocyte migration demonstrated that intratumoral leukocytes used haptokinetic type of migration, however, no significant differences of leukocyte migration between any knockout strains were found. We concluded that leukocyte recruitment in pancreatic and hepatocellular cancer is a slow-going process whose dynamics clearly contrasts to a high-speed leukocyte recruitment during acute inflammation. In contrast to acute inflammatory reaction, only LFA-1 controls recruitment of CD8 1 T-cells in both pancreatic and hepatocellular cancer, whereas ICAM-1 and Mac-1 are dispensable.Pancreatic and hepatocellular cancer remain an extremely aggressive malignancies with a poor prognosis. 1 The mean 5-year survival is 6% and 14% for pancreatic and liver cancer, respectively.

No abstract

No abstract

Purpose: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA.Experimental Design: Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells.Results: Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced.Conclusions: This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.