Cancer Biology

Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of primary cultured endothelial cells. A major advantage of this assay is the possibility to study angiogenesis in a 3D environment. Endothelial cells are cultured as hanging drops to form spheroids. Those spheroids are embedded into a collagen matrix and tube formation is analyzed 24 h later. By analyzing sprout number and sprout length the effects of genetic manipulation or drug treatment on angiogenesis can be investigated.

Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to their receptors, release of proteases to dissolve the basement membrane, migration towards an angiogenic signal, proliferation, and an increase in cell number for new blood vessel formation. Finally, reorganization of ECs forms the three-dimensional vasculature. HUVEC tube-formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of ECs to form three-dimensional capillary-like tubular structures, when cultured on a gel of growth factor-reduced basement membrane extracts. During the assay, ECs differentiate, directionally migrate to align, branch, and form the tubular polygonal networks of blood vessels.