Longitudinal in vivo imaging of tumor angiogenesis

Optical window chambers were implanted onto CD-1 nude mice as described above. Prior to sealing the window with a coverslip, a tagRFP-expressing tumor fragment (~1 mm in diameter) was placed into the window. To obtain tumor fragments, a donor animal was injected with 2.5 × 10⁵ tagRFP-expressing SCC cells 10 days prior to optical window implantation. Tumors were removed, cut into small pieces using a scalpel, and fluorescence checked using an Olympus OV110 whole-animal imaging system. For longitudinal imaging of tumor angiogenesis, mice bearing optical windows were anaesthetized using an isoflurane–oxygen mix. Images were acquired 2, 4, 7, and 9 days post-implantation using an Olympus OV110 whole-animal imaging system set to acquire both GFP and RFP signals using the zoom lens set to 1.6× magnification. For image analysis and identification of blood vessels, autofluorescence images acquired using the GFP channel were inverted in ImageJ. The tubness plugin was used to detect blood vessel structures and apply an image mask over these structures. The accuracy of this was checked manually. The vascular density was determined by calculating the percentage of the field of view covered by the vascular image mask. Data were graphed and statistics calculated using Prism.