Figure 1: PDGF-BB induces PDGFRβ-dependent macrophage recruitment in tumour cell line grafts tumours of human and mouse origin.

(a) Expression levels of PDGF-BB in conditioned medium of various human tumour cell lines (n=3 samples per group). (b) Iba1⁺ macrophages (red) in human scrambled shRNA-A431 and Pdgfb shRNA-A431 squamous carcinomas cell line grafts. Arrowheads indicate tumour-infiltrating macrophages. Scale bar, 50 μm. Iba1⁺ TAMs were quantified as areas (n=8 random fields per group). (c) Upper panels: FACS analysis of F4/80⁺ TAMs in vector-T241 and PDGF-BB-T241 fibrosarcoma cell line grafts and quantification of percentages of F4/80⁺ macrophages (n=6 samples per group). Lower panels: quantification of F4/80⁺ TAMs of immunohistochemical micrographs (n=8 random fields per group). (d) Iba1⁺ macrophages (red) in vector-T241 and PDGF-BB-T241, and vector- and PDGF-BB-LLC tumours. The tumour cells express GFP (green). Arrowheads indicate tumour-infiltrated macrophages. Scale bar, 50 μm. Iba1⁺ TAMs were quantified as areas (n=8 random fields per group). (e) Iba1⁺ macrophages (green) in vehicle- or imatinib-treated A431 and PDGF-BB-LLC tumours, and in vehicle-, imatinib-, anti-PDGFRα- or anti-PDGFRβ-treated PDGF-BB-T241 tumours. Tissue sections were counter-stained with DAPI (blue). Arrowheads indicate tumour-infiltrated macrophages. Scale bar, 50 μm. Iba1⁺ TAMs were quantified from eight random fields per group. (f) PDGFRβ (green) and F4/80 (red), or PDGFRα (green) and F4/80 (red) double immunostaining of A431, PDGF-BB-T241 and PDGF-BB-LLC tumours. White arrowheads indicate F4/80⁺ macrophages and yellow arrowheads point to PDGFRα⁺ or PDGFRβ⁺ cells. Scale bar, 25 μm. (g) αSMA (green) and PDGFRβ (red), or NG2 (green) and PDGFRβ (red) double immunostaining of vector- and PDGF-BB-T241 tumours. Arrowheads indicate double-positive signals (yellow). Scale bar, 25 μm. (h) RT–PCR and qPCR analyses of Pdgfrb in various cell types. Beta-actin was used as a standard loading (mean±s.e.m., NS, not significant, Student’s t-test).