Abnormalities of vascular morphology resulting from angiogenesis, the formation of new blood vessels, are characteristic of cancerous tumors and are associated with a switch from quiescent to aggressively invasive, metastatic behavior. Thus angiogenic activity, as reflected by morphological microvascular change, is a critical point of assessment in cancer research. Visualization and quantification of microvascular attributes permit monitoring of disease progression and response to therapy, yet there are no currently available sample preparation methods to produce data that are of capillary-resolution and suitable for automated 3D quantitative analysis. Specimen preparation techniques for vascular visualization thus far have primarily been developed to meet the requirements of individual studies and have not addressed the need for standardized quantitative analysis across labs. Routine vascular metrics, such as density, often still rely upon time-consuming manual counting from 2D paraffin sections by multiple observers, in part because of the lack of 3D image data of sufficient quality to allow automated quantification. Moreover, one of the principal techniques allowing the collection of 3D quantitative vascular data, corrosion casting, destroys surrounding tissue, eliminating the possibility of simultaneous probing for molecules of interest. Our goal is to create and refine specimen preparation techniques that allow collection of high-resolution 3D vascular image data within tumors and the supporting peri-tumoral tissue, while simultaneously allowing labeling of related molecules of interest. The methods will be optimized to produce vascular image data that will allow automated quantitative analysis, thus introducing a more rapid, standardized approach for studies of microvascular changes associated with tumor growth and treatment. Methods appropriate to animal models and to fixed archived tissue will be developed toward the ultimate goal of extending these techniques to human biopsy tissue. Our methods will combine and optimize protocols used in vascular biology and adapt procedures developed in other fields, such as non-mammalian developmental biology, that though relevant, have not previously been employed in this realm. We propose to modify these existing specimen preparation techniques, and in concert with optical clearing methods to minimize problems associated with tissue opacity, produce 3D renderings of the microvascular architecture of whole-mount specimens, while permitting labeling of other molecules of interest. Our preparation techniques will employ combinations of: 1) casting of the vasculature by filling with a contrast agent; 2) marking of functional blood vessels by fluorescently labeled lectin administered i.v.; 3) marking of other structures of interest by whole-mount labeling with antibodies or nuclear dyes; and 4) tissue clearing to minimize light scattering for imaging by confocal microscopy so that data can be collected from deep within peri-tumoral and viable tumoral tissue. ? ? ?
