Fig 2: Detection of MAC-1 in lung homogenates. Western blot analysis of MAC-1 was performed in the lung of each group mice. *P < 0.05, **P < 0.01 between the groups indicated

Fig 3: CD11b deficiency reduced myeloid-derived suppressor cells (MDSC) recruitment in the tumor environment.The accumulation of Gr-1+CD11b+ MDSC cells in the tumor microenvironment of the ApcMin/+ mice compared with the normal intestine tissues from the C57 mice; the Gr-1+ cells were decreased in the tumor microenvironment of the ApcMin/+;CD11b-/- mice (a). The number of the Ly6G+Ly6Clow subset of MDSCs (gated on CD45+ cells) in the bone marrow of C57, CD11b-/-, ApcMin/+ and ApcMin/+;CD11b-/- mice (b) and in the peripheral blood, spleen and tumor tissues of the ApcMin/+ and ApcMin/+;CD11b-/- mice (c) was measured by FACS. The results of the IF staining and FACS represent of 11 independent mice (all mice were 16–weeks-old). The proteins expression of IFN-? and CXCL9 in the tumor tissues from the ApcMin/+ and ApcMin/+;CD11b-/- mice was examined by immunoblotting (d). The protein bands intensities were quantitated and normalized to those of GAPDH. The statistical data are expressed as the means ± S.D. *P<0.05, **P < 0.01 and ***P < 0.001. Scale bars: 50 µm (a).

Fig 4: CD11b deficiency inactivated the Wnt/ß-catenin pathway.The expression of E-cadherin, ß-catenin and cyclin D1 was examined in the tumor tissues of the ApcMin/+ and ApcMin/+;CD11b-/- mice by IHC (a) and immunoblotting (b), and the IHC results were quantitatively determined using IPP the software and expressed as the means ± S.D. The subcellular location of ß-catenin was examined in the tumor tissues of the ApcMin/+ and ApcMin/+;CD11b-/- mice by IF (c). All IHC and IF staining in the mouse tissues represent 11 independent mice (all mice were 16-week-old). The IHC results were quantified using the IPP software and are expressed as the means ± S.D. **P < 0.01, ***P < 0.001. Scale bars, 20 µm (a–c) and 25 µm (e).

Fig 5: Clinical validation of tumor-infiltrating immune cells and target genes in clinical osteosarcoma tissues with and without metastasis. (A) Representative images and (B) statistical analyses of immunohistochemical staining for CD56, CD22, CD86, CD163, CD11B, GATA3, LPAR5, EVI2B, RIAM and CFH. Magnification, ×200. *P<0.05.