Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1b)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILb-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anticancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for antiapoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.

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