Prostate Cancer Lecture 3.doc

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Anatomy and Cell Biology
Anatomy and Cell Biology 4461B
Geordie Shepherd

JL Lecture 3: Angiogenesis and Androgen Ablation Vasculogenesis and Angiogenesis 1. Vasculogenesis 2. Angiogenic Remodeling 3. Stabilization and Maturation 4. Destabilization 5. Regression 6. Sprouting Angiogenesis is regulated through multiple signaling pathways. Vascular Endothelial Growth Factor • Glycoproteins consisting of A-E forms and placental growth factor (PIGF) • Loss of even a single VEGF allele results in embryonic lethality due to cardiac compli- cations • Researchers showed that anti-human VEFG monoclonal antibody inhibited growth of tumor cell lines Angiogenesis Assays in Research Endothelial Cell Tube Formation Assay • Endothelial cells are cultivated on a gel matrix (Matrigel) and form tubes and nodes af- ter a few hours. • Images are taken using an inverted microscope • Assay in quantified by counting the branch points or average tube lengths Ex Vivo Rat Aortic Ring Angiogenesis Assay • Isolated rat aorta is cut into segments that are embedded in gel matrix (Matrigel) • Over the next 7-14 days, the explants are monitored for the outgrowth of endothelial cells. • Assay is quantified by measurement of the length and abundance of vessel-like exten- sions. Chicken Embryo CAM onplant assay • Collagen onplant • Collagenous matrix sandwiched between 2 squares of laser cut nylon mesh • Test articles are incorporated directly into the collagen • Each onplant is of identical size and volume Collagen is allowed to polymerize before use • Study Design • 4 onplants are placed on the CAM of each embryo • Groups of 8-12 embryos are generally used, giving 32-48 data points per group • New vessels sprout from the CAM up into the onplant • Functional neovasculature becomes visible from the top • Grid squares containing identifiable vasculature are scored positive • Scores are expressed as % positive grid squares • With tumor cells - 50,000 tumor cells are incorporated into the collagen onplant as the angiogenic stimulus Mouse Corneal Pocket Angiogenesis Assay • A pocket is made in the cornea where the test articles are introduced, which induce the ingrowth of new vessels from the peripheral limbal vasculature • Visualization of the new vasculature is achieved by intravenously injecting a fluores- cent dye. • Assay is quantified by measuring the area of vessel penetration or the progress of ves- sels toward the angiogenic stimulus over time Matrigel Plug Angiogenesis Assay • Matrigel containing test cells or substances is injected subcutaneously where it solidi- fies to form a plug • Plugs are recovered after 7-21 days and examined histologically to determine the ex- tent to which blood vessels have entered it • Bulk quantification can be made by injecting a fluorescent dye before removing the plugs Prostate Cancer Clinical Progression Avastin failed clinical trials for prostate cancer. Androgen Receptor Signaling Androgen Action Testosterone ci
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