Morphological aspects of angiogenesis in experimental liver metastases

In this work we describe the process of angiogenesis in liver metastases of high- and low-metastatic 3LL mouse carcinoma lines. Fourteen days after intrasplenic inoculation of the tumor lines, two types of metastases were observed; a sinusoidal type, containing large convoluted vessels and devoid of immunohistochemically detectable basement membrane, and a portal type, located in the vicinity of portal tracts, characterized by numerous small vessels, and staining positively for basement membrane components. After intrasplenic inoculation of the high-metastatic tumor cells (portal route) only 18.2% of the metastases were portal type, whereas when the tumor cells were injected into the left ventricle (arterial route), a significantly higher percentage of the metastases (33.2%) proved to be portal type. Detailed analysis of the process of angiogenesis were performed only concerning the main, sinusoidal type metastases. After intrasplenic inoculation of tumor cells, vascularization of tumor colonies started on day 6 by the appearance of intratumoral sinusoids lined by endothelial cells. These sinusoids were directly connected with liver sinusoids. Afterward (11 to 14 days), large convoluted vessels developed within the metastases, in which tumor globules protruded. These globules were covered by factor VIII-related antigen-positive endothelial cells. The functioning vascular nature of these vessels were proven by supravital staining with Hoechst 33342 dye and by bromodeoxyuridine labeling. The first event of the angiogenesis in sinusoids and veins seemed to be the separation of the endothelial cells from their basement membrane, demonstrated by electron microscopic immunohistochemistry (laminin, fibronectin). This process elicited vigorous proliferation of the matrix-deprived endothelial cells, shown by the increased bromodeoxyuridine labeling index and by the increased number of endothelial cell nuclei per mm vessel length. Morphometric analysis of the sinusoids in the perimetastatic zone (up to 100 mu) and in the normal liver parenchyma showed neither dilatation of the vessels nor sprouting of new vessels in the former region. There was no difference in the neovascularization of the liver metastases of the high- and low-metastatic carcinoma lines. The dominant type of angiogenesis in liver metastases can be determined by the unique basement membrane architecture of the liver and by the high affinity of 3LL tumor cells to the endothelial side of basement membrane during invasion.