Gassmann et al. BMC Cancer 2010, 10:177 http://www.biomedcentral.com/1471-2407/10/177 Page 7 of 11 LMM control IgG T84 control IgG LMM control IgG in RNU vehicle control IgG Neuraminidase Fucoidan A B 30 s d ilfe25 c i p cso20 o r c im15 0 2 / lles10 c t n ree 5 h d a 0 s25 d l e i f ic20 p o c s rco15 i m 0 /210 s l l e c tn 5 e r e h d a 0 0-10 * 11-20 21-30 time after cell injection [min] 31-40 control IgG anti-beta 1 anti-beta 4 anti-alpha v 0-10 11-20 21-30 time after cell injection [min] 31-40 0-10 11-20 21-30 time after cell injection [min] control IgG anti-Galectin-3 anti-TF anti-Galectin 3 + anti-TF 0-10 11-20 21-30 time after cell injection [min] ** ** 31-40 ** ** 31-40 C D s25 d l e i f ic20 p o c s rco15 i m 0 /210 s l l e c tn 5 e r e h d a 0 s30 d l e if 25 c i p o sc20 o r c im15 0 2 / lls10 e c t ren 5 e h d a 0 Figure 4 Pulmonary tumor cell adhesion in vivo. A: Adhesion of HT-29LMM and T84 colon cancer cells. Determined by quantitative in vivo mi- croscopy, human highly metastastic HT-29LMM and lung metastasis derived T84 colon cancer cells show similar adhesive properties in the rat lung. B: Integrin - inhibition Using quantitative in vivo microscopy of the lung, inhibition of colon cancer cells' integrins did not impair in vivo tumor cell adhesion in the pulmonary microvasculature. C: Selectin - inhibition Enzymatic hydrolisation of sialylated glycoprotein structures by Neumaminidase- V significantly impaired tumor cell adhesion within the pulmonary microvasculature. Furthermore, tumor cell arrest was impaired after treatment of the animals with fucoidan 15 minutes prior to cell injection for inhibition of P- and L-selectins. D: Thomson-Friedenreich antigen inhibition Tumor cell - endothelial cell adhesion in vivo was significantly reduced, but not completely lost, after inhibition of TF-antigen and galectin-3 at tumor cells. fields were counted in the respective time intervals. Enzy- matic hydrolization of sialylated cell surface glycopro- teins by incubation with neuraminidase-V significantly impaired early cell adhesion in vivo and the number of adherent cells was reduced: 6.5 ± 2.6 to 7.9 ± 3.0 cells/20 microscopic fields (p < 0.05 - 0.001). To confirm the involvement of selectins, animals were treated with fucoidan (0.8 mg/kg) 15 minutes prior to cell injection [19]. This treatment also significantly impaired adhesion of vehicle treated cells in the pulmonary microvascula- ture (Figure 4C; Table 1). Tumor cell adhesion is partly mediated by combined activity of Thomson-Friedenreich (TF) antigen and galectin-3 The TF antigen binding cell surface molecule Galectin-3 is likely involved in the adhesion of metastasizing tumor cells to endothelial cells [11,21]. We therefore treated control cells (n = 5) with unconditioned control medium prior to injection. At 10; 20; 30; and 40 minutes after cell injection 10.9 ± 5.1; 18.3 ± 10.1, 16.4 ± 3,9; and 14.8 ± 6.2 cells/20 microscopic fields were adherent within the pul- monary microcirculation. After incubation of the cells with unconditioned medium and addition of function blocking anti-galectin-3 antibodies (n = 5) prior to injec- tion no significant differences in the numbers of adherent cells were observed. After incubation with JAA-hybri- doma medium containing function blocking anti-TF mouse IgG, significantly reduced adhesion of HT- 29LMM cells was observed. Simultaneous anti-galectin-3 and anti-TF treatment of HT-29LMM cells (n = 5) also resulted in a significant reduction of adherent cells within the lung (p < 0.05 - p < 0.001) (Figure 4D; Table 1), similar to anti-TF treatment alone. Discussion The organ specificity of metastatatic TC arrest is not only determined by the repertoire of available adhesion mole- cules expressed at the cells' surfaces but in addition is influenced by the histological architecture, the availabil- ity, and organ specific exposure of possible adhesion sites within the targeted organ. In the case of colon cancer, the lung, next to liver and lymph nodes, is one of the most important metastasic targets. In this study, we demon- * * ** ** * * * **