Figure 5: Systemic instigation of VEGFR1⁺ bone marrow cells by Id1-induced IGF2.

(a) Illustration within the box shows the experimental scheme for bone marrow transplantation; lower panel shows representative fluorescence images of recipient control mice without (labelled ‘No’) and with bone marrow transplantation (labelled ‘Yes’); the red intensity indicated strong GFP signals. (b,c) Quantification of VEGFR1⁺ cells in bone marrow (b) and lung (c) of mice (female 6–8-week-old nude mice, n=3 per group) from each experimental group by flow cytometry analysis. (d) Representative three-dimensional tomography image of mice showing accumulation of GFP-positive cells in the thoracic region and in the subcutaneous tumour indicated by red frames (left panel) and quantification of VEGFR1⁺ cells in the subcutaneous tumours (right panel). (e) Migrating ability of sorted VEGFR1⁺ and VEGFR1⁻ bone marrow cells compared by FBS-gradient induced cell migration assay (scale bar, 100 μm). (f) The migration ability of VEGFR1⁺ bone marrow cells in response to the attraction of conditioned medium from IGF2-induced fibroblasts in the presence or absence of VEGF antibody was compared (scale bar, 100 μm). (g) Outline of experimental scheme and the effect of VEGFR1 blockade on tumour growth (female 6–8-week-old nude mice, n=6 per group; scale bar, 1 cm). (h) Experimental scheme and bioluminescence imaging showing effect of VEGFR1 blockade on lung metastasis; sections of lung tissue (H & E stained) are shown in the bottom panel (female 6–8-week old nude mice, n=8 per group; scale bars, 200 μm and 100 μm for top and bottom rows of photomicrographs, respectively). Bars, s.d.; *P<0.05; **P<0.01, ***P<0.001 by Student’s t-test.