Intravital fluorescence videomicroscopy to study tumor angiogenesis and microcirculation

Abstract

Angiogenesis and microcirculation play a central role in growth and metastasis of human neoplasms, and, thus, represent a major target for novel treatment strategies. Mechanistic analysis of processes involved in tumor vascularization, however, requires sophisticated in vivo experimental models and techniques. Intravital microscopy allows direct assessment of tumor angiogenesis, microcirculation and overall perfusion. Its application to the study of tumor-induced neovascularization further provides information on molecular transport and delivery, intra- and extravascular cell-to-cell and cell-to-matrix interaction, as well as tumor oxygenation and metabolism. With the recent advances in the field of bioluminescence and fluorescent reporter genes, appropriate for in vivo imaging, the intravital fluorescent microscopic approach has to be considered a powerful tool to study microvascular, cellular and molecular mechanisms of tumor growth.

Figure 1

Microvasculature of normal liver (A) and of hepatic metastasis of a CC531-WAG colon carcinoma (B) as visualized by intravital epi-illumination fluorescence videomicroscopy. Normal liver shows the hexagonal arrangement of acini with dense sinusoidal network, draining blood flow into postsinusoidal venules (A). The vasculature of the hepatic tumor is supplied by and drained into host hepatic vessels. Note the high vascular density in the periphery, and the low vascular density in the centre of the tumor (B). Contrast enhancement by sodium fluorescein i.v. Original magnification, x40.

Figure 2

Microvasculature of striated muscle (A) and C6 glioma on day 18 after implantation into the dorsal skin fold chamber of a nude mouse (B) as visualized by intravital epi-illumination fluorescence videomicroscopy. Note the major feeding arteriolar (right) and draining venular vessels (left) as well as the parallelly arranged capillaries of the muscle tissue (A). In contrast, the glioma microvasculature presents with chaotic angioarchitecture, tortuous and dilated vessel loops, and extravasation of the high-molecular weight fluorescent marker (B). Contrast enhancement by fluorescein isothiocyanate (FITC)-Dextran 150,000 i.v. Original magnification, x 100.

Figure 3

Microvasculature of cerebral surface (A) and C6 glioma on day 14 after implantation into the chronic cranial window model of a nude mouse (B) as visualized by intravital epi-illumination fluorescence videomicroscopy. Note the feeding arteriole, the cortical capillaries, and the draining venule, representing the cerebral microcirculation (A). The glioma microvasculature presents with chaotic and heterogeneous angioarchitecture, tortuous and dilated vessel loops, and extravasation of the high-molecular weight fluorescent marker (B). Contrast enhancement by FITC-Dextran 150,000 i.v. Original magnification, x200.

Figure 4

Detachment and spreading (migration) of individual glioma cells 24 hours after C6 spheroid implantation into the dorsal skin fold chamber preparation of a nude mouse. Intravital fluorescence microscopy using epi-illumination techniques. Fluorescent labeling of C6 glioma cells with Oil before implantation. Original magnification, x400.