Grimm et al. BMC Cancer 2010, 10:82 http://www.biomedcentral.com/1471-2407/10/82 immunogenic effects are minor. The causes of a weak immune response to malignant cells are multifarious and subsumed in the term “tumor immune escape”. Important single mechanisms of the immune escape are down regulation of MHC-class I complex, loss of co-sti- mulatory surface antigens, decreased expression of apoptosis inducing death receptors (e.g. Fas/TRAIL receptor) on malignant cell, and loss of tumor infiltrat- ing cytotoxic T cells by tumor induced apoptosis [5]. An additional, very important aspect of the “tumor immune escape” during carcinogenesis is a significant disturbed cellular immune response [6]. Regarding an effective cel- lular immune response the presence or absence of effec- tor and regulatory T-cells is essential. According to the cytokine profile CD4+ T-cells can be divided in Th1 (T- helper 1)-cells, characterised by the secretion of IFN-g, in Th2 (T-helper 2)-cells, characterised by the secretion of IL-4/IL-5 and in so called Tr1 cells, characterized by the secretion of IL-10. Furthermore TGF-b secretion is the main characteristic of Th3 cells [7,8]. Basically, Th1- cells activate cytotoxic CD8+ T-cells thus causing an antitumoral effect whereas Th2/Th3/Tr1-cells boost the tumor progression by secretion of the above mentioned cytokines. The change of a Th-1 cell mediated cytokine profile to a profile typical for Th2/Th3/Tr1-cells is called “Th-1/Th-2 shift” and is considered a major cause of an ineffective cellular immune response during carci- nogenesis [9]. Furthermore, regulatory CD4+CD25+ T- cells (Tregs) which express the transcriptional repressor gene Foxp3 and emerge as mature T cells from the thy- mus, are increasingly under consideration in regard of the ineffectiveness of the cellular immune response to malignant cells [10-12]. Tregs play a critical role in maintaining tolerance to self antigen and in preventing autoimmunity [13]. Inducible Tregs are generated in the periphery in response to pathogens and self antigens [7,14]. However, the induction or activation of Tregs by pathogens may be one strategy to subvert protective immunity whereas depletion of CD4+CD25+ Tregs enhances survival during certain infections [15]. Under certain conditions, typically following exposure to IL-10 or TGF-b, dendritic cells within the tumor microenvir- onment and cancer cells can initiate the development of Tregs limiting effector responses by inhibition of cyto- toxic T-cells thus impairing anti tumor immunity [16,17]. In conclusion, TGF-b and IL-10 expression is intimately implicated in tumor development and contri- butes to many features of tumor cell biology. In this study we used an intraportal liver metastases animal model with CT26.WT colon cancer cells for the time-dependent analysis of immunological tumor escape mechanisms during carcinogenesis considering the above mentioned cytokine and cellular interactions. Page 2 of 11 Methods Animals Female Balb/c mice, 6-8 weeks of age, were purchased from Harlan-Winkelmann (Borchem, Germany). Animals were maintained under conventional housing conditions and under specific pathogen-free conditions. Regular screenings for the presence of murine pathogens (such as Mycoplasma pulmonis, Streptococcus pneumoniae, and Helicobacter spp.) were performed. All mice were fed a commercial diet, given water ad libitum, and subjected to an equal 12-hour light/dark cycle in accordance with institutional guidelines. Housing and all procedures invol- ving animals were performed according to protocols approved by the University’s animal care committee and in compliance with the guidelines on animal welfare of the National Committee for Animal Experiments. Cell line CT26 is an N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated murine colon carcinoma cell line, which was cloned to generate the cell line designated CT26.WT (LGC promochem, Germany). This cell line is tumorigenic and induces colon carcinoma in rodents [18]. CT26.WT cells were propagated and subcultured according to the distributor’s protocol. The cultures were routinely tested for mycoplasma contamination to ensure that only negative cells were used. Surgical procedure The surgical procedure of intraportal tumor cell injec- tion has already been described extensively [19]. 1 × 105 tumor cells in 100 μl PBS were injected into the portal vein using a 32G needle. In all control group animals a laparotomy and an intraportal injection of 100 μl PBS solution without tumor cells was performed (n = 3). Mice were sacrificed at 5, 10, 15, and 20 days after injection (n = 7 mice per group) or earlier if tumor related cachexy or hepatic failure with tumor related ascites occurred. Control group animals were sacrificed on day 5 following PBS injection. Liver and lung tissues of all animals were collected and subjected to histologi- cal examination. Liver metastases were further examined by immunohistochemistry, Real Time PCR, and TUNEL assay. All surgical procedures administered to the ani- mals were in accordance with institutional guidelines. Tumor volumes were calculated by measurements of the short and long axis of the mass of the prominent metastasis, where V = π/6 × a2 × b (a: short axis of the tumor, b: long axis of the tumor). Immunohistochemistry/Immunofluorescence About half of the complete liver tissue was fixed in 4% buffered formalin for histological examination and