Gassmann et al. BMC Cancer 2010, 10:177 http://www.biomedcentral.com/1471-2407/10/177 tumor cell arrest of human breast cancer cells in a mouse model, following the typical clinical pattern of metastatic breast cancer diseases, and α2- and α6- integrin expres- sion of colon carcinomas was found to correlate with their metastatic potential and patient prognosis [12] Circulating tumor cells adhere to microvascular endothelial cells (EC) and subendothelial extracellular matrix (ECM) proteins by different sets of adhesion mol- ecules. Subsequent and regulated transendothelial cell migration taking place at secondary sites in response to several microenvironmental factors as has been numer- ously reported [6-10,13]. Despite the morphologic simi- larities of leukocyte and tumor cell adhesion, there are distinct differences between inflammatory cells and can- cer cells due to the available surface adhesion molecules and intracellular signalling cascades. Obviously, malig- nant cells originating from a variety of tissues differ with regard to available adhesion molecules for cell - cell or cell - ECM interactions. For example, E-Selectin medi- ated adhesion to endothelial cells has been established for inflammatory leucocytes as well as several human cancer cells [7-9] α4β1-integrin binding of endothelial Vascular Cellular Adhesion Molecule-1 (VCAM) was associated with leu- kocyte adhesion [14] to endothelial cells, as well as adhe- sion of human melanoma cells [15] to the endothelium, but appears not to be involved in prostate cancer cell adhesion [16]. These findings suggest different adhesion mechanisms of hematogenous cells, non-epithelial and epithelial cancer cells during their arrest within capillar- ies. Recently, we reported comprehensive data on cell surface molecules involved in initial metastatic tumor cell adhesion of human colon carcinoma cells within the hepatic microvasculature in vivo [17,6]. These results showed preferential arrest of circulating colon carcinoma cells within the liver via integrins [9,10] The aim of the present study was to evaluate the adhesion molecules and mechanisms involved in the organ-specific adhesion of human colon cancer cells in the lung as their second met- astatic target organ. To this end we established a new in- situ model for quantitative in vivo fluorescence micros- copy of the ventilated and perfused lung. Methods Materials and Cells Media (RPMI1640; DME/F12) and fetal bovine serum (FBS) were purchased from GIBCO-BRL (Karlsruhe, Germany). All other chemicals were purchased from Sigma (Deisenhofen, Germany). Human highly-metastatic HT-29LMM colon carci- noma cells (I. Fidler; Houston, TX) were cultured in RPMI1640 medium and human T84 colon cancer cells (A. Nussrath, Atlanta), derived from a lung metastasis, were cultured in DMEM-F12 medium containing 10% Page 2 of 11 FBS without antibiotics in humidified 5% CO2/95% air at 37°C. Confluent cell monolayers were used during the log-phase of growth. Prior to the experiments cells were rinsed with Calcium-Magnesium-free phosphate buff- ered solution (CMF-PBS), trypsinized and kept in serum- free adhesion medium (RPMI1640 containing bovine serum albumin [BSA] 1%) for 60 min for reconstitution of cell surface proteins. Trypsinized cells were resuspended as single cell suspension in CMF-PBS at a final concentra- tion of 1 × 106 cells/ml. This preparation did not interfere with adhesive and migrative properties in vitro [18]. For inhibition experiments, various antibodies or neuramini- dase-V (C. perfringens) (Sigma) were added during reconstitution of the cells as indicated below. Flow cytometry analyses of adhesion molecules For flowcytometric analysis of adhesion molecules, cells were trypsinized and washed in serum free medium. After reconstitution for 60 min, cells were fixed with fresh 1% paraformaldehyde and processed following a standardized protocol. The following primary antibodies were used for detection of certain cell adhesion mole- cules: anti-human integrin β1 mAb (clone P4C10, Chemi- con, Hofheim, Germany), anti-human integrin β4 mAb (clone ASC-8, Chemicon), anti-human integrin β3 (clone N-20, Santa Cruz,), anti-human αv mAb (clone 272-17E6, Calbiochem, Darmstadt, Germany), anti-human α1 inte- grin mAb (clone SP2/0, Upstate, Lake Placid NY), anti- human α2 integrin mAb (clone 16B4, Serotec, Oxford, UK), anti-human α3 integrin mAb (clone ASC-1, Chemi- con), anti-human α4 integrin mAb (kindly provided by J. Eble; Münster, Germany), anti-human α5 mAb (clone JBS5, Serotec), anti-human α6 mAb (clone 4F10, Serotec), anti-sLea (clone KM93; Chemicon), anti sLex (clone KM231; Chemicon), anti galectin-3 (clone B2C10; Santa Cruz Biotechnology). Negative and isotype controls were similarly processed. Corresponding Alexa-fluor568- labeled secondary antibodies (Molecular Probes, Leiden, Netherlands) were used for flow cytometry analysis. For detection of Thomson-Friedenreich antigen, cell were incubated with fluorescence labeled Lectin from Arachis hypogaea (L0881; Sigma) specifically binding TF-antigen in increasing concentrations. Flow cytometry was done using EPIC XL (Beckman Coulter, Krefeld, Germany). Intravital Fluorescence Video Microscopy Male Sprague-Dawley rats (CD rats; 250-300 g; Charles River) were cared for in accordance with the standards of the German Council on Animal Care, under an approved protocol of the local Animal Welfare Committee (LANUV NRW 9.93.2.10.36.07.122). Rats were anesthe- tized using inhalation of isofluorane (Curamed, Karlsruhe, Germany) and N2O. A permanent catheter was introduced through the right carotid artery and the