Einleitung: Tumorzellen weisen unterschiedliche Potentiale im Hinblick auf ihr organspezifisches Metastasierungsverhalten auf. Entscheidend scheinen vor allem ihre adhäsiven und invasiven Eigenschaften zu sein.

Einleitung: Adhäsion und Migration von Tumorzellen stellen entscheidende Schritte bei der organspezifischen Entstehung von Metastasen dar. Die adhäsiven Interaktionen werden hierbei insbesondere von den Integrinen entscheidend beeinflusst. Mithilfe eines neu entwickelten Modells wurden die Regulation der frühen Metastasierungsschritte in vivo analysiert.

Tumour cell adhesion within the microvasculature of host organs, its stabilisation and cell invasion into the host organs, appear to be important steps in the formation of distant metastases. Intravital fluorescence-video microscopy was used to investigate the early steps in metastasis formation of colon carcinoma cells within the liver, which is the main target organ of colorectal carcinomas. The involvement of alphav-integrins was analysed in vivo using HT-29 cells after treatment with different function-blocking antibodies [pan-alphav (n=9 animals), specific alphavbeta3 (n=8 animals) and alphavbeta5 (n=8 animals)] or linear Arg-Gly-Asp (RGD)-containing peptides (RGD-peptides) (n=6 animals). Treatment with anti-alphav and anti-alphavbeta5 antibodies resulted in significantly (P<0.001) decreased tumour cell adhesion in vivo within the hepatic microvasculature. Cells treated with anti-alphavbeta3 antibodies or unspecific immunoglobulin-G (IgG) did not show significant changes in their adhesive properties. Furthermore, inhibition of cell adhesion was achieved by linear RGD-peptides in a dose-dependent manner. Relative numbers of migrated cells were not affected by any of the treatments. These results suggest that alphav-integrins, especially alphavbeta5, can influence the ability of circulating tumour cells to adhere within the hepatic microvessels. In contrast, migration of adherent cells into the liver parenchyma was not affected by alphav-integrin inhibition. Our findings support the hypothesis that specific interactions between circulating tumour cells and host organs are required for organ-specific tumour cell arrest.

Tumor cell adhesion within host organ microvasculature, its stabilization and invasion into host organ parenchyma appear to be important steps during formation of distant metastasis. These interactions of circulating tumor cells with the host organs occur in the presence of fluid shear forces and soluble and cellular environmental conditions of the blood that can modulate their cellular responses and possibly their metastatic efficiency. Cytoskeletal components, such as actin filaments and microtubules, can regulate biophysical characteristics and cellular signaling of the circulating cells. Therefore, we investigated the role of these cytoskeletal structures for early steps during metastasis formation in vivo and in vitro. Using an intravital observation technique, tumor cell adhesion of colon carcinoma cells within the hepatic microcirculation of rats and their invasion into liver parenchyma was observed. Disruption of actin filaments increased cell adhesion, whereas tubulin disruption inhibited adhesive interactions in vivo. The impairment of the cytoskeleton modulated adhesion-mediated cell signaling via focal adhesion kinase (FAK) and paxillin under flow conditions in vitro. In the presence of fluid flow, focal adhesions were enlarged and hyperphosphorylated, whereas stress fibers were reduced compared to static cell adhesion. Disruption of microtubules, however, partially inhibited these effects. Combining the in vivo and in vitro results, our study suggested that changes in cell rigidity and avidity of cell adhesion molecules after disruption of cytoskeletal components appear to be more important for initial adhesive interactions in vivo than their interference with adhesion-mediated cellular signal transduction.

Tumor cells can show different malignant properties regarding their ability for organ-specific metastasis formation. Their adhesive and invasive characteristics mediated by various cell adhesion molecules appear to be crucial for this process. Using intravital fluorescence microscopy, we analyzed the adhesive and invasive interactions of circulating human colon carcinoma cells within the microvasculature of the liver in rats. The involvement of different cell adhesion molecules in specific tumor cell-host organ interactions was investigated. Single-cell suspensions of human colon carcinoma with low (HT-29P) and high (HT-29LMM) metastatic potential were fluorescence labeled with calcein-AM and intra-arterially injected into Sprague-Dawley rats. Initial interactions between different cell lines and the microvasculature of the liver were observed over 30 minutes and semiquantitatively analyzed. Different integrin subunits, carbohydrate ligands, and vascular cell adhesion molecule-1 were inhibited using function-blocking antibodies or by enzymatic removal. Inhibition of sialyl-Lewisa (sLea) or enzymatic removal of selectin carbohydrate ligands significantly reduced metastatic cell adhesion. In addition, α6-, β1-, and β4-integrins can directly mediate cell adhesion within the hepatic microcirculation. Furthermore,α2-,α6-,β1-, and β4-integrins are involved in early tumor cell extravasation into the liver parenchyma. Organ-specific formation of colorectal metastases appears to be mainly mediated by specific interactions between circulating carcinoma cells and the vessel wall of target organs but not mechanical entrapment. Selectins Lea interactions with sinusoidal endothelial cells can play a key role in organ-specific targeting, but direct integrin-mediated cell adhesion to extracellular matrix components in the space of Disse appears to be required for the successful formation of liver metastases.

Tumor cell adhesion within the microcirculation of host organs is a fundamental event in the formation of distant tumor metastases. Circulating tumor cells have to adhere within the microcirculatory vessels, quickly stabilize their adhesion and probably leave the circulation to avoid toxic effects of hydrodynamic shear forces of circulating blood. However, in vivo controversial results have been reported for the complex interactions between circulating tumor cells and host organs in different experimental models.

Organ-specific tumor cell adhesion within the microcirculation of host organs is an important step in the metastatic cascade. Circulating tumor cells have to adhere within the microcirculatory vessels, quickly stabilize their adhesion and probably leave the circulation to avoid toxic effects of hydrodynamic shear forces of circulating blood. Using intravital fluorescence microscopy we established a new model for the intravital observation of colon carcinoma cell adhesion within the hepatic microcirculation. HT-29 (human) and CC531 (rat) colon carcinoma cells were fluorescence labeled using CalceinAM. Single cell suspensions were injected intraarterially in Sprague-Dawley rats. Using intravital fluorescence microscopy adhesive interactions of circulating tumor cells within the hepatic microcirculation were observed at the liver surface. These interactions were analyzed regarding their time course and the localization within the vascular tree. Autofluorescence of liver parenchyma was sufficient for distinction of hepatic sinusoids. Intravital microscopy enabled the differentiation of early events in adhesion formation within hepatic sinosoids, adhesion stabilization, and extravasation of the tumor cells into the liver parenchyma. Tumor cell adhesion occurred almost exclusively within sinusoidal capillaries; however, the diameter of these vessels was usually larger than that of the tumor cells leaving remaining perfused lumen of the capillaries. Colon carcinoma cells rapidly migrated into the liver parenchyma after successful adhesion within the sinusoids. In contrast to common endpoint assays of the metastatic cascade, this in vivo model allows investigations of metastatic colon carcinoma cell adhesion within the liver microcirculation as specific steps during the formation of hematogenous metastasis and their underlying mechanisms.