Fig. 6

Macrophages mediate acGM-1.8’s anti-tumor activity. a–f Profiling of immune cell populations in the S180 tumor of in mice. After the 14-day treatment by acGM-1.8 or PBS, the tumor tissue was harvested, processed, and analyzed with flow cytometry for the proportion of (a) leukocytes (CD45⁺) in tumor cells, (b) M1-type macrophages (F4/80⁺ CD11c⁺) in CD45⁺ cells, (c) M2-type macrophages (F4/80⁺ CD206⁺) in CD45⁺ cells, (d) CD4⁺ T lymphocytes in CD45⁺ cells, (e) CD8⁺ T lymphocyte in CD45⁺ cells, and (f) regulatory T cells (CD4⁺ Foxp3⁺) in CD45⁺ cells. g Determination of pro-tumor cytokines (IL-10, VEGF-A, and TGF-β1) and anti-tumor cytokines (TNF-α, IL-12 p70, and IFN-γ) by ELISA. h, i Evaluation of the anti-tumor effect of acGM-1.8 in S180-bearing mice with macrophages depleted by clodronate liposomes: measurement of (h) the tumor size and (i) weight after the 14-day treatment; circles and squares denote the PBS and acGM-1.8 treatment, respectively; solid and hollow signs represent mice without and with macrophage depletion, respectively. j–l Evaluation of the anti-tumor effect of acGM-1.8 in S180-bearing nude mice: measurement of (j) the tumor size and (k) weight after the 14-day treatment; *P < 0.05 versus the PBS treatment; n = 5; and (l) the proportion of M1/M2-type macrophages within CD45⁺ cells in the tumor tissue in the nude mice. m, n Representative images for co-staining of Ly6c⁺ (green) and (m) CD11c⁺ (red) or (n) CD206⁺ (red) cells in the tumor tissue of the S180-bearing mice; the cell nuclei were counter-stained with DAPI (blue); scale bar: 100 μm. o The effect of acGM-1.8 on the phenotype change of tumor-associated macrophages (TAM) ex vivo: macrophages were isolated from the tumor, cultured in vitro, treated with acGM-1.8 for 48 h, and analyzed for (o) their M1/M2 phenotype change by the flow cytometry; *P < 0.05 vs. the PBS treatment; ns: no significance; n = 5