Ein neues Modell zur Quantifizierung der Tumorzellmigration in vivo

Abstract

AbstractIntroduction: Tumor cell migration and extravasation were recognized as fundamental cell properties involved in the formation cancer metastasis. A variety of different in vitro models has been established to monitor tumor cell motility and chemotaxis in 2- and 3-dimensional systems. Nevertheless in vitro systems are insufficient to analyze the complexity of the in vivo microenvironment. Using in vivo fluorescence video microscopy we therefore established an intravital model to semi-quantitatively analyze tumor cell migration within the rat liver. Materials and methods: Rat colon (CC531) and human colon carcinoma cell lines (HT-29 P, KM-12 C; their corresponding highly metastatic subclones: HT-29 LMM, KM-12 L4) were used. In male CD rats (250 g) a median laparotomy was performed and the left liver lobe placed under an in vivo fluorescence microscope without disturbance of the microcirculation. 106 fluorescence labeled (CalceinAM®) tumor cells were injected intraarterially. 30 standardized microscopic fields were analyzed in 5 min intervals for an observation period of 30 min. The migration rate was calculated as rate of migrated/extravasated cells in relation to all arrested cells. In some animals arrested cells were also detected by immunhistochemistry using a human specific anti-EPCAM antibody. Results: Using in vivo microscopy passing and arrested tumor cells within liver sinusoids were identified. Furthermore, arrested cells could be localized within the lumen of the sinusoids (»adherent cells«) or were seen extravasated (migrated cells) within the parenchyma. Immunhistochemistry confirmed the early extravasation of tumor cells. The rates of migrated cells increased over the 30 min observation period. Human and rat colon carcinoma cells demonstrated similar behavior in terms of cell adhesion and migration within the liver. Furthermore the high metastatic subclones demonstrated significantly higher migration rates than the parent cells: KM 12C (n = 7) 38% ± 7% vs. KM-12L4 (n = 4) 45% ± 5%, (p < 0.05) and HT-29 P (n = 9) 16% ± 11% vs. HT-29 LMM (n = 7) 29% ± 10%, (p < 0.05). Conclusions: Using this method metastatic tumor cell migration can be quantified in vivo. Cell lines with different metastatic potential demonstrated different migration rates suggesting tumor cell extravasation/migration as a rate limiting step of metastasis formation. This model makes it possible to study different factors that may be involved in metastatic tumor cell migration in vivo.