Gassmann et al. BMC Cancer 2010, 10:177 http://www.biomedcentral.com/1471-2407/10/177 strate that metastatic cell arrest in the lung is exclusively initiated by adhesive interactions between circulating TC and the pulmonary microvascular endothelium. In con- trast to the relevance of initial TC-ECM interactions for cell arrest within the hepatic sinusoids [6,17] direct matrix binding is not responsible for initial pulmonary tumor cell arrest. This points out the organ specific nature of metastatic tumor cell adhesion within potential target organs. The lung's microvasculature is lined by a continuous endothelium, underlined by a basal membrane mainly composed of fibronectin, collagen IV and laminin. Ana- tomically defined fenestrae as documented for the kid- neys' glomerula or the hepatic sinusoidal endothelium cannot be found in the pulmonary endothelium [25]. Although TC-EC adhesive interactions are of solely func- tional nature and earlier reports failed to observe mor- phologic correlations [26], the data presented herein support the role of TC - EC interactions in the initial phase of metastatic lung colonization. Adherent TCs leave a remaining perfused vessel lumen suggesting spe- cific interactions rather than size restricted arrest. Fur- thermore, TCs passing the pulmonary capillaries were repeatedly observed in our study. Size restricted arrest, cannot be completely excluded by two dimensional in vivo microscopy, but 3D examination of in situ fixed and physiologically perfused lungs supports specific adhesion of colon cancer cells to the pulmonary capillary endothe- lium. Despite the controversies about the potential role of size-restricted mechanical arrest for the initiation of organ colonization [4] a large body of data supports the role of specific adhesive interactions of circulating TCs within the capillary microenvironment of potential meta- static target organs [27]. This initial adhesion can be mediated by heterotypic TC - EC interactions and/or TC - ECM interactions [17,26,28]. Furthermore, the coagula- tory system [29,30], including platelets [31] and possibly leucocytes [32] seems to interfere with metastatic tumor cell arrest. Under physiological conditions, terminally fucosylated glycans such as sLe-x function as selectin ligands, e.g. on activated leucocytes [33], and mediate rolling on and adhesion to ECs [34]. But altered glycosylation of cell sur- face molecules is also a prominent feature of malignant cells [9,35]. These molecules appear to be critically involved in the organ tropism of metastasis formation. For example, sLe-a and sLe-x can mediate adhesion of differentiated epithelial cancer cells to endothelial E- selectin in vitro [36] and in vivo [28]. SLe-x was shown to initiate liver metastasis of human colon cancer cells [37] and high levels of sLe-x expression were correlated with poor survival of colo-rectal cancer patients [38]. E-selec- tin mediated adhesion triggers numerous functional Page 8 of 11 alterations in adhering TCs [39] as well as in ECs. For example, adhering TCs induce further E-selectin expres- sion in ECs [40], while adhering TCs are subjected to shear forces, inducing intracellular signalling like focal adhesion kinase phosphorylation and further adhesion stabilisation [41]. Confined disturbances in microvascu- lar physiology may further be caused by local induction of nitric oxide after TC -EC interaction [42], as well as altered mechanical properties of TC by cytoskeletal rear- rangement after cell adhesion [43]. Nevertheless, details of these biophysical alterations remain unclear. In this study pre-treatment of the animals with fucoidan and pre-treatment of the injected cells with neuraminidase significantly impaired pulmonary TC arrest in vivo. Fucoidan mainly affects P-selectin and L- selectin with minor effects on E-selectin [44]. In contrast, neuraminidase inactivates sialylated adhesion molecules, such as sLe-a that is known to bind E- and P- selectin, and to a lesser extent also L-selectin. Nevertheless, adhesion of epithelial TCs to the microvascular endothelium may also be mediated by indirect cell adhesion, using addi- tional cell types or soluble molecules within the blood [31,32]. Therefore, our results suggest the involvement of platelets and possibly leucocytes in in vivo adhesion of TCs in the lung. This is also supported by previous reports from other groups [31,32]. Direct heterotypic TC - EC adhesion seems to be also mediated by the TF and galectin-3 system [45]. The inhi- bition of tumor cell adhesion to EC by anti-TF treatment resulted in increased survival in a mouse model for spon- taneous breast cancer metastases by impairing TC adhe- sion without affecting tumor cell proliferation [46]. Similar to earlier reports [9,11,17,32], we were able to detect a significant contribution of sLe-a - selectin medi- ated adhesion and TF - galectin-3 mediated TC adhesion within the lungs. Beside TC - EC interactions, TC - ECM interactions, mediated by integrins were shown to be critically involved in the initiation of distant metastasis formation [12,47,48]. But in contrast to other typical metastatic tar- get organs of colon cancer [6,10,17] we could not detect any significant contribution of ECM binding integrins to the initiation of pulmonary tumor cell adhesion. Although α3β1-integrin mediated TC adhesion to the basal membrane's laminin through single spot-like gaps of the endothelium was reported [49], we could not detect TC-ECM interaction in the early time frame ana- lyzed. The ECM components underlining the endothelium as the basal membrane are composed in an organ specific manner. Furthermore, the endothelial lining has specific characteristics in different organs that affect tumor cell adhesion. We recently reported that HT-29LMM passes the renal microcirculation without any signs of cell arrest