Holger Kalthoff

Additional file 1: of Cytokeratin 20 positive circulating tumor cells are a marker for response after neoadjuvant chemoradiation but not for prognosis in patients with rectal cancer

The figure shows a sample results of CK20 RT-PCR from 16 patients, including two positive controls (colon cancer cell line HT29). (TIF 2824 kb)

Die Expression von Foxp3 in Pankreascarzinomzelllinien wird durch TGF-β2 funktionell reguliert und vermittelt einen anti-proliferativen Effekt auf naïve T-Zellen

Foxp3 is a member of the forkhead/ winged helix transcription factor family which is highly expressed in CD4+CD25+ regulatory T cells and was recently identified as a key player in T cells with regulatory potential. We describe the expression and function of Foxp3 in pancreatic ductal adenocarcinoma cell lines and cancer tissue resulting in an inhibition of proliferation of naive T cells. The expression of Foxp3 in pancreatic cancer cell lines is regulated by TGF-β2 which resembles the mechanisms in regulatory T cells. We detected Foxp3 mRNA and protein expression in different pancreatic cancer cell lines. In 25/39 pancreatic cancer tissues Foxp3 expression was detectable with immunohistochemistry in the tumor cells, but we found no correlation between Foxp3 expression in the tumor cells and tumor stage and overall-survival. In all pancreatic cancer cell lines Foxp3 positive, tumorinfiltrating lymphocytes were detected. Treatment of pancreatic cancer cell lines with TGF-β2 but not TGF-β1 led to an up regulation of Foxp3 mRNA and protein expression. In line with these findings we found that anti-TGF-β2 treatment resulted in Foxp3 down regulation. In co-culture assays of Panc89 cells with naive T-cells a strong anti-proliferative effect on the proliferation of naive T-cells was observed. Interestingly, this effect could be partially reverted after inhibition of FoxP3 expression in Panc89 cells, indicating that this suppressive effect is dependent on FoxP3 expression. This demonstrates that regulation of Foxp3 expression in pancreatic cancer cells may be similar to the known pathways in Treg and that pancreatic cancer cells might mimic functions of Treg through Foxp3-dependent suppression of T-cell proliferation.

Identifizierung und Validierung von differentiell exprimierten Genen im Stroma von duktalen Pankreaskarzinomen und chronischer Pankreatitis

Background: Pancreatic ductal adenocarcinoma is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesised that the gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. Methods: We investigated the gene expression of eleven stromal tissue from pancreatic ductal adenocarcinoma, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified, and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. Results: We found 255 genes to be over-expressed and 61 genes to be under-expressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway. Therefore, we confirmed the differential expression of SFRP1 and WNT5a using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during co-cultivation with a pancreatic carcinoma cell line. Conclusion: The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The over-expression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

Invasion statt Apoptose — eine neue Funktion des Todesrezeptors CD95 bei Pankreaskarzinomzellen

Pancreatic adenocarcinoma is one of the most aggressive cancers with an extremely poor prognosis. Pancreatic tumor cells are highly invasive and metastatic, and mostly apoptosis-resistant. TRAF2 is an adaptor protein known to interact with death receptors, promoting activation of NF-κB. In this study we found TRAF2 strongly overexpressed in pancreatic tumor tissues and tumor cell lines. To investigate the role of TRAF2 in pancreatic tumor pathophysiology we transfected low TRAF2-expressing Colo357 cells with a TRAF2 expression vector, and analyzed the apoptosis sensitivity and invasive potential of these cells. We found Colo357/TRAF2 cells nearly completely resistant against treatment with either an agonistic anti-CD95 antibody or with CD95-ligand. Conversely, downregulation of TRAF2 by siRNA rendered CD95-resistant Panc89 cells sensitive. Interestingly, Colo357/ TRAF2 cells showed an enhanced constitutive activity of NF-κB and AP-1, and secreted high amounts of MMP-2, MMP-9, uPA, and IL-8. Consequently, an in vitro invasion assay demonstrated that TRAF2-overexpressing cells grew much more invasively than control cells. Treatment with CD95L led to an additional activation of NF-κB and AP-1, followed by strong induction of uPA and IL-8, and resulted in a highly potentiated invasive growth instead of apoptosis. In conclusion, TRAF2 plays a substantial role in the pathophysiology of pancreatic adenocarcinoma by converting the apoptotic signaling into a survival- and invasiveness-promoting phenotype.

CpG-Oligonukleotide hemmen das Wachstum orthotoper Pankreaskarzinome auch im immundefizienten Mausmodell

Background/Aim: Significant inhibitory effects on tumor growth have been demonstrated for modified oligodeoxynucleotides (ODN) in preclinical systems. This was also shown for CpG-ODN in an immune competent subcutaneous tumor model. The object was now to investigate whether these effects were also reproducible for pancreatic cancer in an orthotopic immune deficient model. Methodology: For in vitro analysis in PancTul cells, DNA synthesis was assessed by [3H]-thymidine-incorporation, a colorimetric cell viability assay (easy4you™-Assay) was employed, and DNA fragmentation as an indicator for cell death was analysed (JAM-[3H]-thymidine-incorporation assay). Cell cycle analysis was carried out by PI-FACS-analysis. In vivo testing was done in an orthotopic pancreatic xenotransplantation model using SCID beige as well as nude mice. ODN employed were CpG-1826 and a random control ODN which were applied intraperito-neally. Results: In vitro, CpG-1826 and control-ODN induced an inhibition of DNA synthesis by about 40%, but did not alter cell viability in Panc-Tul cells. Incubation of tumor cells with conditioned medium from human dermal fibroblasts, treated with CpG-ODN lead to a further reduction of DNA-synthesis. In vivo CpG-1826 proved to have a significant growth inhibitory effect (up to 50%) when compared to controls treated by saline or control-ODN. This was accompanied with a seven fold splenomegaly and hepatomegaly of 50%. The reduction of tumor weight by CpG-1826 was only slightly more pronounced in nude compared to SCID beige mice. Conclusion: CpG-1826 induced significant growth inhibitory effects on orthotopic xenotransplanted pancreatic tumours in immune deficient mice. Since in vitro results indicate an effect of conditioned media of fibroblasts on tumor cells and since relevant immunological effector cells are lacking in the animal models employed, our data suggest that tumor-stroma-interactions might be involved in CpG-induced therapeutic effects on solid tumours in immune deficient mice.

Humane duktale Pankreas-Adenokarzinome zeigen eine hohe Expression von Proteinkinase Cμ, einem starken Induktor von Apoptose-Resistenz und Zellproliferation

Aim of the study nRecently, we have shown that CD95-resistant pancreatic adenocarcinoma cells express high levels of Protein Kinase Cμ (PKCμ). In this study we investigated the role of PKCμ in the regulation of proliferation and apoptosis. Additionally we tested the expression of PKCμ in pancreatic tumour tissues.