Gassmann et al. BMC Cancer 2010, 10:177 http://www.biomedcentral.com/1471-2407/10/177 tip placed centrally to the heart. An open tracheotomy was performed and the animal was mechanically venti- lated through a canula placed and fixed in the trachea by a small animal respirator (Harvard Small Animal Ventila- tor, Harvard Apparatus, Hugstetten, Germany) at a rate of 35-40/minute and a tidal volume of 3,5 - 4 ml. Adequate ventilation was confirmed by arterial blood gas analysis. The anterior chest wall was carefully removed without disturbances of the lung surface avoiding atelectases. The animal was placed under an upright in vivo fluorescence microscope (Zeiss, Oberkochen; Germany) and fluores- cence microscopy was performed through a thin glass cover slip mounted on a specially designed holder, care- fully placed upon the lung's surface with minimal pres- sure. For positive contrast of the blood vessels, the animals were slowly injected with 500 μg fluorescine (FITC) labelled dextran 10,000 kD. Using a similar proto- col, earlier studies demonstrated stable hemodynamic conditions and constant microcirculation in the lungs for at least 60 minutes [19]. Microscopy was performed Figure 1 Set-up for in vivo microscopy of the ventilated rat lung. A: After placement of a catheter central to the heart through the right carotid artery, a tracheostomy and canulation of the trachea was per- formed for establishing mechanical ventilation. The chest wall was re- moved and the right lower lobe was exposed. B: A glass cover slip, mounted on a specially designed holding device, was placed upon the right lower lobe with minimal pressure to serve as artificial pleura, and the animal was placed under an upright in vivo microscope. Time frame (10 sec.) of stably adherent and sticking human colon cancer cell. C: Full scale microscopic pictures representative for in vivo microscopy of the ventilated and physiologically perfused lung in-situ. The present- ed pictures were taken at the indicated time intervals after tumor cell injection. One fluorescence-labeled cell is stably adherent to the vas- cular wall over the 10 second interval of observation. The other cell ("sticking cell") is adherent for a few seconds to the wall without estab- lishing stable adhesion and finally recirculates through the pulmonary capillaries. For quantitative analysis of tumor cell adhesion, the lung surface was screened for stably adherent cells in a standardized fashion in 10 minute intervals for a total of 40 minutes. Page 3 of 11 through a water immersion objective with 20× magnifica- tion (Figure 1A/B). Semiquantitative analysis of tumor cell adhesion in vivo 106 CalceinAM labeled human HT-29LMM colon carci- noma cells were injected as a single cell suspension in 1 ml CMF-PBS over 1 minute. Standardized lung surface microscopy was begun immediately after the injection was completed. The lung surface was continuously screened in a standardized manner recording the number of single adherent tumor cells per microscopic field in inspiration as described before [10]. Tumor cells adhering for ≥10 seconds were defined as stably adherent. Micros- copy was performed in 10 minute intervals for a total of 40 minutes. Emboli of cell clumps were excluded from analysis. The cell numbers given are the number of adherent cells per 20 microscopic fields for each 10 minute time interval and are expressed as means ± standard deviation from n independent experiments (animals injected). Involvement of Cell Adhesion Molecules For inhibition experiments cells were incubated either with function blocking monoclonal antibodies (mAbs), type V-neuraminidase or conditioned medium. For some experiments the animals were treated with 0.8 mg fucoidane per kg bodyweight 15 minutes prior to cell injection as described by Preobrazhenskaya et al. [20] for inhibition of selectin mediated adhesion. For integrin and galectin-3 inhibition cells were incu- bated with the appropriate antibodies (1-3 μg/ml, 60 min) during reconstitution to block specific integrins prior to the assays. The following mAbs were used: anti-β1 integ- rin (clone P4C10, Chemicon, Hofheim, Germany), anti β4-integrin (clone ASC-8; Chemicon), anti-αv (clone 272- 17E6, Calbiochem, Darmstadt, Germany), anti-galectin-3 (clone B2C10; Santa Cruz Biotechnology). This pretreat- ment has been shown to interfere with the regulation of cell adhesion in HT-29 cells in-vitro [21] and in vivo [6,17]. Unspecific mouse IgG (Sigma) was used as con- trol. Enzymatic hydrolisation of cell surface glycoprotein sialic residues to inhibit Selectin ligands was achieved by incubating cells with 0.01 U/ml type V-neuraminidase (from C. perfringens; Sigma, Deisenhofen; Germany). This treatment has been shown to interfere with tumor cell adhesion in vivo [6]. Vehicle (untreated) cells were used as control. The inhibition of the Thomson-Frieden- reich antigen (TF antigen) was realized by, incubating cells with conditioned medium from the supernatant of JAA-F11 hybridoma cells that produce function-blocking mouse IgG against the TF antigen [22,11]. Control cells were incubated with unconditioned medium.