Abstract
The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31+ endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans.
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Acknowledgments
We would like to thank Dr. Daniel Gustafson and AstraZeneca for providing the ZD6474 used for these experiments. These studies were supported in part by the Harbers Foundation and by a grant from the Colorado State University Cancer Supercluster.
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10456_2010_9182_MOESM1_ESM.jpg
Supplementary Figure 1: Computer aided analysis of serially sacrificed mice reveals angiogenic collapse of the tumor. Syngeneic mice challenged with MCA-205 tumors (n = 3–4 mice per time point) were serially sacrificed and tumors collected for immunohistochemical determination of microvessel density. Analysis of MVD revealed similar dynamics of CD31+ cells to those observed by FNA (A). Representative immunohistochemical sections immunostained for CD31 are shown (B) (JPEG 11412 kb)
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Sottnik, J.L., Guth, A.M., Mitchell, L.A. et al. Minimally invasive assessment of tumor angiogenesis by fine needle aspiration and flow cytometry. Angiogenesis 13, 251–258 (2010). https://doi.org/10.1007/s10456-010-9182-0
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DOI: https://doi.org/10.1007/s10456-010-9182-0