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Tumor vasculature was visualized by using transillumination and filters for green light, and tumor vascular networks were mapped by recording 1 4 single frames with Nilotinib a 4 objective lens resulting in a field of view of 3. 80 3. 80 mm2 and a pixel size of 3. 7 3. 7 um2. To study the function of tumor vasculature, first pass imaging movies were recorded after a 0. 2 mL bolus of 50 mg mL tetramethylrhodamine isothiocyanate labeled dextran with a molecular weight of 155 kDa was injected into the lateral tail vein. First pass imaging movies were recorded at a frame rate of 22. 3 frames per second by using a 2 objective lens, resulting in a time resolution of 44. 8 ms, a field of view of 5. 97 5. 97 mm2, and a pixel size of 7. 5 7. 5 um2. All recordings were stored and analyzed offline.
Tumor size was calculated from the number of pixels showing GFP fluorescence. Analysis of vascular morphology Two dimensional projected vascular masks were pro duced manually from transillumination images recorded with the 4 objective lens. Interstitial distance and vessel diameter were computed from the vascular masks. Vessel density was calculated from skeletons of vascular masks. Vessel density of small or large vessels was calculated from skeletons of vascular masks only showing vessels with a diameter smaller or larger than 15 um. The ana lysis of vascular morphology is illustrated for a represen tative tumor in Figure 1. This figure shows high resolution transillumination images, the vascular mask, the skeleton of the vascular mask, and color coded vessel diameter superimposed on the vascular mask.
Vessel segment length and vessel tortuosity were calculated from 50 randomly selected vessel segments. Vessel tor tuosity was defined as T 100% SL, where SL represents the segment length and S represents the shortest distance between the branching points, as illustrated in Figure 1F. Change in vessel diameter was assessed by manually measuring the diameter of the same vessel segments on subsequent days. Analysis of vascular function Two dimensional projected vascular masks were pro duced from the movies recorded with the 2 objective lens as described previously. Blood supply time images were produced by assigning a BST value to each pixel of the vascular masks. The BST of a pixel was defined as the time difference between the frame showing maximum fluorescence intensity in the pixel and the frame showing maximum fluorescence intensity in the main tumor supplying artery, as described in de tail previously. Immunohistochemical detection of tumor hypoxia, microvessels, vascular basement membrane, and pericytes The tumors were resected immediately after the last intra vital microscopy examinations and fixed in phosphate buffered 4% paraformaldehyde.