Vascular-specific quantification in an in vivo Matrigel chamber angiogenesis assay.
The study of angiogenesis as a therapeutic target requires reliable in vivo assays that can provide physiologically relevant data. A murine in vivo Matrigel-based angiogenesis assay is presented here which includes the quantitative assessment of vascular-specific indicators of neovascularization. Matrigel containing 175 ng/ml bFGF is encapsulated in synthetic chambers which are implanted subcutaneously in C57/B16J mice. Ex vivo implants can be imaged to qualitatively view perfused vasculature within the chambers, or histologically processed to confirm the presence of vascular-specific tissue within the Matrigel. Viable cells are recovered from the excised chambers and quantified cytometrically using endothelial cell-specific markers CD34 and CD144, and for a marker of nucleated cells, Hoechst 33342. Thalidomide, 200 mg/kg/day, was tested using the assay and was found to inhibit angiogenesis by 46%. Angiogenesis inhibitors secreted by LL/M27 tumors were also characterized, where tumor-bearing mice showed a 73% inhibition of angiogenesis compared to tumor-free controls. Analysis of the number of nucleated cells in these samples failed to show a strong correlation with the number of endothelial cells, indicating that quantification of nonvascular-specific tissue in in vivo angiogenesis assays may not be sufficient. This new assay provides an objective, comprehensive determination of the vasculature-specific response of both endogenous and exogenous angiogenesis inhibitors in vivo, and also creates new opportunities for obtaining primary murine endothelial cells.
Department of Medical Biophysics, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3.
Jennifer H E
JH
Baker
Lynsey A
LA
Huxham
Alastair H
AH
Kyle
Kevin K
KK
Lam
Andrew I
AI
Minchinton
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D004338:Drug Combinations; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012884:Skin Window Technique; D013792:Thalidomide / Q000494:pharmacology; D014184:Transplantation, Homologous
D020533:Angiogenesis Inhibitors; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D016222:Fibroblast Growth Factor 2; C059485:matrigel; D013792:Thalidomide; D003094:Collagen