Abstract
We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro.
Highlights
Summary We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen
This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F1O murine melanoma or MDA-MB-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16FO cell line
The anti-angiogenic compound suramin inhibits in a dose-dependent manner the migration and differentiation of BAE cells on type I collagen in response to B16F1O CM
Summary
Bovine aortic endothelial (BAE) cells and the B16F1O murine melanoma cell line were obtained from Farmitalia Carlo Erba (Nerviano, Italy). B16FO murine melanoma and MDAMB-231 human breast carcinoma cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Cell lines were cultured at 37°C, in a 5% carbon dioxide atmosphere, in Dulbecco's modified Eagle medium (DMEM) (Gibco, UK) (BAE cells, B16FO and MDA-MB231) or RPMI-1640 (Flow, UK) (Bl6F1O), supplemented with 10% fetal calf serum (FCS) (Seralab, UK), 2 mM glutamine (Seralab, UK), 100 IU mli penicillin and 0.1 mg ml-' streptomycin. Basement membrane Matrigel and type I collagen were purchased from Collaborative Biomedical Products (Bedford, MA, USA). Human recombinant basic fibroblast growth factor (bFGF) was obtained from Farmitalia Carlo Erba. Anti-mouse laminin and anti-human bFGF antibodies were obtained from Collaborative Biomedical Products
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