Gassmann et al. BMC Cancer 2010, 10:177 http://www.biomedcentral.com/1471-2407/10/177 Prior to injection cells were washed and resuspended as single cell suspension in CMF-PBS at a final concentra- tion of 1 × 106 cells/ml. Laser scanning microscopy for ex situ evaluation of specific adhesion Cells were labeled with CellTracker Green CMFDA fluo- rescence staining (Molecular Probes) (0.01 mg/ml) for 3D-reconstruction of adherent tumor cells within the pulmonary microcirculation and the animal was injected with Vybrad-Dil-LDL (0.04 mg/animal) for labeling of the endothelium. Both dyes are stable during formalin fixa- tion and paraffin-embedding. 40 minutes after the tumor cell injection in situ fixation was carried out by infusing CMF free PBS at a physiologic pressure of 10-15 mmHg through the inferior vena cava, followed by a 3.75% form- alin infusion. The inflated lungs were removed carefully avoiding the creation of atelectases and kept in formalin for three days. 25 μm sections were mounted on glass slides after paraffin embedding. Three dimensional reconstructions of fluorescence-labeled sections were processed using a laser-scanning confocal microscope (Nikon, Düsseldorf; Germany) and the lucia5-software package (Nikon). Results HT 29 LMM adhere to the pulmonary microvasculature The first fluorescence labeled cells were seen in the pul- monary microcirculation as soon as 1 minute after the initiation of the tumor cell injection. While some cells passed the pulmonary capillaries without any sign of mechanical size restriction, first adhesive interaction of the injected cells with the pulmonary capillary system were observed within minutes. All adherent cells were localized within the pulmonary capillaries and no adhe- sive cells were observed within larger pre- or post-capil- lary vessels. The mean diameter of pulmonary capillaries determined by in vivo microscopy was 14.3 μm ± 2.5 μm. Perfusion of the capillaries was usually maintained despite the presence of adhering cells. Once tumor cells were adherent for at least 10 seconds, significant recircu- lation was not observed within the observation period. In contrast to earlier observations under comparable condi- tions regarding the liver [23], some cells were found stick- ing to the lung vessel walls and eventually recirculating before establishing a firm adhesion as it has been described for leucocytes (Figure 1C). Rolling of cells prior to adhesion was not observed. Three dimensional reconstructions of the lungs after in situ fixation and confocal laserscanning microscopy revealed specific adhesive interactions between tumor cells and the microvasculature with persistent free capil- lary lumen (Figure 2). In vivo microscopy of these areas demonstrated remaining perfusion within the capillaries Page 4 of 11 Figure 2 A/B: Laser scanning microscopy of adherent tumor cell (formalin fixed). After in situ perfusion with PBS with physiologic pres- sure through the inferior V. Cava, lungs were fixated in situ by formalin infused with physiologic pressure. After paraffin embedding, 25 μm sections were analysed by 3D laser scanning microscopy. Three di- mensional reconstruction revealed tumor cells (A, yellow cell with white arrow head) adherent to the microvascular endothelium (B, white arrow heads) and leaving a perfused vessel lumen without signs of mechanical size-restricted cell arrest. next to the adhering tumor cells (Figure 1C). It is worth- while to mention that extravasated tumor cells, e.g. cells in the alveolar lumen, were not detected by confocal laser scanning microscopy 40 min after cell injection. Adhesive properties of HT-29LMM and T84 colon cancer cells The human HT-29LMM colon cancer cell line was derived from repeated in vivo passaging in mice [24]. To validate the cell line's adhesive properties within the rodent model used here, we compared the pulmonary tumor cell adhesion of HT-29LMM with the adhesive properties of human T84 colon cancer cells derived from a lung metastasis. After injection of T84 cells treated with unspecific mouse IgG (n = 6) 16.7 ± 9.0; 15.8 ± 5.1; 14.1 ± 5.6; and 13.0 ± 1.9 cells/20 microscopic fields were found adherent in the lung at 10; 20; 30; and 40 minutes after injection, respectively. Similar processing of HT-29LMM cells resulted in comparable numbers of adhesive cells within the pulmonary microcirculation at all time inter- vals (Table 1). HT-29LMM cells were used for further experiments due to better comparability with results from earlier studies using a similar microscopic tech- nique within the liver. Expression of adhesion molecules on human HT 29 LMM colon cancer cells Identification of potentially involved sets of adhesion molecules was performed by flow cytometry analysis defining adhesion molecule expression at the tumor cell surface. Human HT-29LMM colon cancer cells showed significant expression of β1-, β4-, α1-, α2-, α3-, α6-, and αv-integrin subunits. While only weak α5-integrin