Vasculature within the immune aggregates specifically expresses the proangiogenic molecule CD105. A–C: Omenta were removed and stained simultaneously with anti-CD45 to label immune cells (red) (A), anti-CD31 to identify blood vessels (green) (B), and anti-CD105 (orange) shown as an overlay on blood vessels (C). An outline (white line) of the boundary of immune cells from A is also illustrated in B and C. Vessels outside of the aggregate were low or negative for CD105 expression (arrows). Images are representative of four independent experiments. Scale bar = 25 μm.

Proposed model. As described, omental immune aggregates contain mesothelial cells, some of which are hypoxic (red) and therefore secrete VEGF-A (black asterisks). In a normal physiological setting (top), VEGF-A production can stimulate blood vessels to produce vascular sprouts (green outlined in red), resulting in the induction of angiogenesis and recovery of once-hypoxic mesothelial cells. The increase of vessels delivers more immune cells, along with additional oxygen and nutrients necessary to support the influx of cells. In a metastasis model (bottom), tumor cells invade aggregates and co-opt the existing dense vasculature. Because angiogenesis is already induced, new blood vessel formation is rapid, resulting in aggressive tumor growth.

Macrophages and mesothelial cells stain positively for the hypoxic marker EF5. Omental single-cell suspensions were obtained from mice treated with EF5 and stained with surface markers for macrophages (CD45⁺, F4/80⁺), mesothelial cells (CD45⁻, VCAM⁺), and EF5 (ELK 3.51). Hypoxic fractions (shaded) were defined as the percent positive over no drug controls (dotted lines). A: Representative histograms are shown. B: Arithmetic means and SEM were calculated for each subset based on five (F4/80⁻ and F4/80⁺ populations) or three (mesothelial cells) independent experiments. The percentage of hypoxic cells in the CD45⁺, F4/80⁻ population is significantly different from both the CD45⁺, F4/80⁺ population and the mesothelial cells with P < 0.05 as determined by analysis of variance and Bonferroni’s multiple comparison test.

Tumor cells infiltrate immune aggregates and exhibit rapid growth. B16/GFP tumor cells (1 × 10⁵) were injected intraperitoneally into mice. On the indicated days, omenta were removed and stained simultaneously with anti-CD45 to label immune cells (red) and anti-CD31 to mark blood vasculature (yellow) and visualized in separate fluorescence channels using whole mount microscopy. Images of the same field of view were taken on each day for immune cells alone (top), immune cells and GFP⁺ tumor cells shown as an overlay (middle), or blood vessels alone (bottom). Representative images from three separate experiments from each day are depicted. Arrowheads show morphological changes indicative of angiogenesis. Scale bar = 100 μm.

VCAM⁺ mesothelial cells have a defined patchy architecture over the immune aggregate. Mesothelial cells were identified within immune aggregates using anti-VCAM (red) (A), and their outer boundary was traced. In B, immune cells labeled with anti-CD45 (green) and mesothelial cells (red) are shown as an overlay. C: Within the same field of view, immune cells and blood vessels labeled with anti-CD31 (red) are shown as an overlay with the boundary of the mesothelial cells shown in white. D: A higher magnification of mesothelial cells from a different immune aggregate illustrating large, cobblestone-like cells. E: Confocal analysis of an immune aggregate stained for immune cells (green) and mesothelial cells (orange). Descending confocal slices (0 to 10 μm) of the immune aggregate with mesothelial cells (arrows) visible near the top of the aggregate. Conventional microscopy experiments were repeated three times, whereas confocal experiments were preformed twice. Scale bars: 100 μm (A–C); 25 μm (D).

Definitive vascular sprouting can be observed within omental immune aggregates from naïve mice. A: Omenta were stained with anti-CD45 (red) or anti-CD31 (green) to demonstrate a typical immune aggregate consisting of immune cells and blood vessels, respectively. B: The same field of view stained simultaneously with anti-collagen type IV (yellow) identifying vascular sprouts near the boundaries of the aggregate vasculature (arrows). C: Higher magnification of the inset in B. In a separate immune aggregate, a more detailed examination of the vascular sprouts was performed on the same field of view in images D to F, illustrating anti-collagen type IV (red) staining with the sprout outlined (D), blood vessels lacking CD31 reactivity in the area of the sprout (white line) (E), and an overlay of both images (F). Images are representative from six independent experiments. Scale bars: 50 μm (A, B); 25 μm (C–F).

Tumor cells attach to vascular dense immune aggregates on the omentum. A: The growth of tumor within the peritoneal cavity of a mouse injected intraperitoneally 6 days earlier with 2 × 10⁵ B16 tumor cells. The intestines were removed and are shown in the inset. B: An overview of the entire omentum from a mouse injected 10 hours earlier with 10⁶ B16. The omentum was stained simultaneously with anti-CD45 to label immune cells (red), anti-CD31 to mark blood vessels (green), and anti-LYVE-1 (yellow) to identify lymphatic vessels. The tissue was prepared as a whole mount, viewed by fluorescence microscopy, and is shown as a montage overlay. C: Magnification of the white box, now including tumor cells (blue). D and E: An immune aggregate (anti-CD45-red) with a dense network of blood vessels (anti-CD31-green) (D) and at higher magnification (E). F: Image analysis of vascular density (percent vessel area) of the areas surrounding immune aggregates, such as the boxed area in D compared with surrounding areas. Images are representative of at least three independent experiments. *Significant at P < 0. 001 as determined by the Wilcoxon signed rank test. Scale bars: 1 cm (A); 1 mm (B); 200 μm (C); 100 μm (D, E).

VEGF-A production by cell types found within the omentum or peritoneal cavity. A: Cells (1 × 10⁶) from the spleen, mesenteric LN, peritoneal lavage, or omentum were incubated in normoxic, hypoxic, or hypoxic plus brefeldin A (Golgi Plug, +GP) conditions for 16 hours, and VEGF-A in the supernatant was measured by ELISA. Subsets of cells from either the peritoneal cavity (B) or the omentum (C) were tested for VEGF-A production using intracellular staining and reported as the fold increase over controls as described in Materials and Methods. Cells not producing VEGF-A would have a fold increase of 1 (dotted line). Macrophages from the omentum were divided into those expressing low [Macs (L)] or high [Macs (H)] levels of F4/80. D: Phenotypic and morphological analysis of mesothelial cells isolated from single cell suspensions of the omentum. Gating on a population of VCAM⁺ cells (left dot plot) revealed this to be CD45⁻ (middle dot plot). Further analysis of this VCAM⁺, CD45⁻ population demonstrated the cells were CD31⁻ (top histogram), and CD44⁺ (bottom histogram). The digital image is representative of these cells when grown in vitro after isolation by FACS (see below) showing a cobblestone morphology, which is typical of mesothelial cells. E: Mesothelial cells or CD45⁺ immune cells were sorted using flow cytometry and cultured in normoxic conditions, and VEGF-A production was measured after 16 hours of incubation as above. Each experiment described above was repeated at least three times with the exception of the sorted cells, which was done twice. Statistical significance was determined using a one-way analysis of variance followed by Bonferroni’s multiple comparison test.