Fig 2: PD-L1 blockade increases production of sVEGFR1 from GBM-educated macrophages. (a) sVEGFR1 levels on the supernatant of healthy control or GBM monocyte-derived macrophages (both n = 6), treated with isotype control or anti-PD-L1. * P < 0.05, ** P < 0.01, isotype control vs. anti-PD-L1. (b) Typical flow-cytometry results of VEGFR1 surface expression on GBM-educated macrophages, treated with isotype control or anti-PD-L1. (c) Relative sVEGFR1 mRNA levels of in healthy control or GBM monocyte-derived macrophages (both n = 6), treated with isotype control or anti-PD-L1. * P < 0.05, ** P < 0.01, isotype control vs. anti-PD-L1. P values are achieved by t-test

Fig 3: Schematic diagram summarizing the regulatory role of the Id1/IGF2/VEGF/VEGFR1 cascade in oesophageal cancer progression.IGF2 secreted by Id1-expressing cancer cells activates fibroblasts to secrete VEGF which exerts paracrine effects in the tumour microenvironment, as well as instigates VEGFR1+ bone marrow cells in the tumour macroenvironment to facilitate distant metastasis.

Fig 4: VEGFR1+ bone marrow cells interact with ESCC cells to promote tumour growth and metastasis.(a) Experimental scheme of bone marrow admixture-tumour xenograft model. Bioluminescence imaging (inset) showed absence of spontaneous metastasis in the lungs 2 weeks after the mice were subcutaneously injected with Luc-expressing KYSE150-Id1 cancer cells. (b) Representative photos and growth curves of tumours among the experimental groups (female 6–8-week-old nude mice, n=6 per group; scale bar, 1 cm). (c) Experimental scheme of the bone marrow admixture-experimental metastasis model (left panel); bioluminescence imaging and quantification of lung metastasis (right panel) (female 6–8-week-old nude mice, n=5 per group; scale bars, 200 µm and 100 µm for top and bottom rows of photomicrographs, respectively). (d) Experimental scheme of the bone marrow admixture-spontaneous metastasis model (left panel); bioluminescence imaging, macroscopic (arrows indicate visible metastases; scale bar, 1 cm) and microscopic pictures of the dissected lungs and quantification of lung weight (right panel) (female 6–8-week-old nude mice, n=5 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.

Fig 5: Blockage VEGFR1 counteracts IFN-?-mediated PD-L1 upregulation in monocyte-derived macrophages. (a) Typical flow-cytometry results of VEGFR1 surface expression on circulating monocytes from a healthy volunteer or their derived macrophages (n = 5). (b) ?MFI values of PD-L1 on GBM-educated macrophages (n = 5), treated with VEGFR1 NAb and/or rhIFN-?. * P < 0.05, ** P < 0.01. (c) ?MFI values of PD-L1 on GBM-educated macrophages (n = 5), co-cultured with syngeneic T cells, and treated with VEGFR1 NAb and/or IFN-? NAb. * P < 0.05, ** P < 0.01. (d) IFN-? concentration in the co-culture supernatant containing CD3+ T cells and allogeneic GBM-educated macrophages, pre-treated by VEGFR1 NAb and/or IFN-? NAb (n = 5). * P < 0.05. P values are achieved by t-test. GEMs, GBM-educated macrophages