Figure 2. Imaging blood vessel network and interactions with tumor cells.

(A) Mean projection image of a tumor and associated vascularization acquired through a 20× Water Immersion objective (N.A. = 1.0). One field of view corresponds to the dotted line box; 49 such fields were acquired to cover the full tumor extent. (B) For quantitative pharmacological experiments, a 10× dry objective (N.A. = 0.45) was used to increase data throughput by a factor 4. An overlap of 50 µm between fields of view ensured qualitative image stitching performed with ImageJ 3D Stitching macro, “pcm3D”. (C) Upper part, 3D projection of the vascular network over a depth of 150 µm of the tumor (right of the plain line) and of surrounding healthy brain (left of the plain line). Lower part: zooms of the outlined ROIs. Thin and straight normal brain vessels (left); tortuous and swollen tumor vessels (right). (D) Example of bicolor images showing at a 10 day interval the average vascular density (red) and tumor cell density (green) in a 25 µm thick horizontal section of the tumor located 200 µm below the brain surface. Note that the lower vascular density observed after 10 days is not associated with a reduction of tumor cell density. (E,F) Horizontal mean projection images over 100 µm subdural layers 15–19 days post implantation showing perivascular invasion (arrow heads). (G) Sagittal 3D rendering projection showing superficial tumor cells (arrow heads) migrating toward deeper brain regions along a large vertical vessel. (H) Mean projection of the volume of interest scanned for vascular changes occurring at a 1 day interval. All the changes (white dots) were found inside or within 150 µm off the tumor margin but none in the healthy brain region. Insets show examples of microvascular changes (arrows) at several locations. Scale bars, A&B: 1 mm; C&D: 300 µm, F&G: 50 µm,; H:300 µm, inset 25 µm.