Crucial to designing angiostatic and vascular targeting agents is the identification of target molecules. Because angiogenesis is not limited to pathologic conditions, careful evaluation of putative therapeutic targets is warranted to prevent adverse effects associated with impaired physiologic angiogenesis. To identify tumor-specific angiogenesis markers, we compared transcriptional profiles of angiogenic endothelial cells isolated from malignant and nonmalignant tissues with those of resting endothelial cells. We identified 17 genes that showed specific overexpression in tumor endothelium but not in angiogenic endothelium of normal tissues, creating a therapeutic window for tumor vasculature-specific targeting. Antibody targeting of 4 cell-surface-expressed or secreted products (vimentin, CD59, HMGB1, IGFBP7) inhibited angiogenesis in vitro and in vivo. Finally, targeting endothelial vimentin in a mouse tumor model significantly inhibited tumor growth and reduced microvessel density. Our results demonstrate the usefulness of the identification and subsequent targeting of specific tumor endothelial markers for anticancer therapy.
Here, we present a protocol for the isolation of endothelial cells (ECs) from tissues. ECs make up a minor population of cells in a tissue, but play a major role in tissue homeostasis, as well as in diverse pathologies. To understand the biology of ECs, characterization of this cell population is highly desirable, but requires the availability of purified cells. For this purpose, tissues are mechanically minced and subsequently digested enzymatically with collagenase and dispase. ECs in the resulting single-cell suspension are labeled with Abs against EC surface antigens and separated from the remainder of the cells and debris by capture with magnetic beads or by high-speed cell sorting. Purified ECs are viable and suitable for characterization of diverse cellular properties. This protocol is optimized for human tissues but can also be adapted for use with other species. Depending on the tissue, the procedure can be completed in approximately 6 h.
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