Grimm et al. BMC Cancer 2010, 10:82 http://www.biomedcentral.com/1471-2407/10/82 (DAKO) and incubated with the Cy3 conjugated primary antibody anti-CD8-mAb diluted in TBS plus 0.5% BSA overnight at 4°C in a humidified chamber. The sections were washed and covered with Polyvinyl-alcohol mount- ing medium (DABCO) (Sigma-Aldrich) and analyzed using a Zeiss camera (Jena, Germany). Photographed Images using the Metamorph software (Visitron Systems, Puchheim, Germany) package were imported into Micro- soft Office Picture Manager. For negative controls, sec- tions were incubated with TUNEL reaction mixture without TdT. For positive controls, sections of female mammary gland were used. The apoptotic index (AI) was defined as the ratio of TUNEL-positive infiltrated cells to all counted infiltrated cells × 100. For each group, the number of stained cells was counted in at least ten 400 × high-power fields. Cells were defined as apoptotic if the whole nuclear area of the cell labelled was positive. Statistical analysis Results were expressed as mean ± SD. Each of the above mentioned studies was performed in seven mice per group. Comparisons were performed by analysis of var- iance or paired and unpaired t test when appropriate. Bonferroni’s correction for multiple comparisons was used to determine the level of significance. p < 0.05 was considered significant. Results Induction of liver metastases by intraportal injection of CT26.WT colon carcinoma cells The injection of 1 × 105 CT26.WT cells in 100 μl PBS- solution resulted in development of hepatic metastases in all animals. There was a continuous increase in tumor volume overtime (day 5: 8.4 ± 2 mm3, day 10: 150.7 ± 32 mm3, day 15: 435.4 ± 37 mm3, day 20 680.3 ± 41 mm3). Therefore, the injection of 1 × 105 CT26. WT cells was used for this study. None of the animals showed pulmonary metastases. Analysis of T cell infiltration at early and late tumor stages Real Time PCR analysis following intraportal injection of 1 × 105 CT26.WT cells (n = 7 at each time point) showed significantly higher gene expression level of spe- cific T cell markers during metastatic growth. A signifi- cant increase of the expression of CD4, Foxp3 and CD8 could be shown at all points of time compared to the previous time segment. Additionally, the expression of CD4 and CD8 at day 5 was already significantly increased compared to control tissue. The expression of CD25 showed a significant increase from day 5 to day 10 and from day 15 to day 20. Relative quantification value, fold difference, is expressed as 2-ΔΔCt (Figure 1a). Page 4 of 11 These findings were immunohistologically confirmed showing an expression pattern according to the increased gene expression of CD4, Foxp3, CD8, and CD25 during formation of liver metastases. Immunohis- tologically increasing numbers of Foxp3+ T cells as well as CD4, CD25, and CD8 were detected during carcino- genesis (Figure 1b). Especially the number of CD4+ T cells increased over proportionally on day 20 post injec- tionem (Figure 2a). Using immunofluorescence Foxp3+ expressing cells were identified as CD4+ phenotype which were increased at day 20 compared to day 10 (Figure 2b). Cytokine expression Following injection of 1 × 105 CT26.WT cells Real Time PCR analysis showed significantly increasing gene expression of IL-10 from day 10 to day 20. The expres- sion of TGF-b was significantly increased on day 10 and 15 compared to the previous point of time, whereas TNF-a showed a significant increase in expression only until day 10. From this time on no significant changes occurred with TNF-a. In contrast, IFN-g gene expres- sion increased significantly until day 10. From day 10 on, however, a significant decrease of expression could be observed until day 20 (Figure 3). Analysis of FAS/FAS-L expression on tumor cells during metastatic growth In Real Time PCR analysis, down regulation of FAS gene expression in metastatic liver tissue could be detected from day 10 on (p < 0.05 day 10 to day 15) fol- lowing an initial increase in FAS gene expression at day 5 and 10. FAS-L expression was increased during tumor development (p < 0.05, day 10 compared to day 15 and day 20 respectively, Asterisk) (Figure 4a). Immunohisto- logically FAS-L expression was morphologically attribu- ted to the tumor cells in liver metastases at late tumor stages (p < 0.05, day 10 compared to day 15, day 20 and normal tissue, respectively, Asterisk; Figure 4b, 5a). Additionally the immunohistological detection of FAS receptor in liver metastases showed a significant decrease from day 10 to day 15 following an initial increase in expression (Fig 4b, 5b). The expression of FAS and FAS-L by tumor cells was confirmed by posi- tive control staining of the tumor cell line CT.26 WT. FAS expression was observed predominantly on CD8+ T cells infiltrating the perimetastatic margin: CD8+/FAS day 10: 12 ± 4.9%, day 15: 15 ± 5.4%, day 20: 21 ± 4.7% (day 20 shown in Figure 6a). TUNEL assay confirmed apoptotic events in these perimetastatic areas and showed an increased rate of apoptotic CD8+ cells during tumor growth: CD8+/TUNEL day 10: 5.6 ± 2.9%, day 15: 13 ± 4.7%, day 20: 20 ± 3.4% (day 20 shown in Figure 6b).