Characterization of tumor-infiltrating CD11b⁺Gr-1⁺ MDSCs. CD11b⁺ cells were enriched from subcutaneous 3LL tumors when tumor grew to 6-10 mm in diameter, and the subpopulations were subsequently sorted by a cell sorter. (A) The sorted cells were subjected to Giemsa or macrophage-specific esterase staining. Dotted line in the bottom right image indicates that different parts of the same slide were grouped to show the cells with representative morphology. Original magnification, ×1200. (B) The percentage of MDSC subpopulations in tumor-infiltrating CD45⁺ leukocytes in various tumor models. B16 melanoma, Meth A fibrosarcoma, and 3LL were used. (C) mRNA expression of iNOS, ARG1, VEGF-A, and MMP9 in tumor-infiltrating CD11b⁺ cells, CD11b⁺Gr-1^int/dullLy-6C^hi macrophages, and CD11b⁺Gr-1^hiLy-6C^dull neutrophils were analyzed by real-time RT-PCR. (D) Expression of intracellular MMP9 was analyzed by a flow cytometry. Solid lines indicate staining with anti-MMP9 mAb, shaded histograms indicates isotype control. In panels A, C, and D, cells were collected from tumor of 5-8 mice. In B, graphs represent the mean (± SD) of 3 mice. Data are representative of 2 independent experiments.