Imaging of Angiogenesis In Vivo with Fluorescent Proteins

Abstract

We have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) in nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. The fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold. The observable depth of tissue is thereby greatly increased. With dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFPexpressing transgenic mice that express GFP in all cells, great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis are visualized. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFPexpressing vessels within the RFP-labeled tumor by whole-body imaging. This new differential dual-colored fluorescence imaging tumor-host model, along with spectral unmixing, can non-invasively visualize in realtime the onset and progression of angiogenesis in a tumor. Thus, fluorescent proteins expressed in vivo offer the highest resolution and sensitivity for real-time whole-body imaging of angiogenesis.