Figure 3

KLF4 ablation in CCR2⁺ MDSCs reduced pulmonary metastasis that was associated with decreased numbers of fibrocytes. (a) Rosa26CreER/KLF4 (flox^+/+)/β-actin-EGFP mice were inoculated under the dorsal skin with 5 × 10⁵ B16F10-Luc2 cells. Three to four weeks later, CCR2⁺ MDSCs were sorted from the lung of the tumor-bearing mice, mixed with B16F10 melanoma cells and injected into WT mice intravenously, followed by induction of KLF4 knockout as described in Figure 1. The mice in the control group were injected with B16F10 melanoma cells only, but were still treated with tamoxifen or sunflower seed oil. Mice were sacrificed 1 week later for various analyses. (b) Left, representative lung macrometastases (top) and hematoxylin and eosin stained sections with red arrows pointing metastatic lesions (bottom) from the mice as described in a were shown (scale bars=100 μm). Quantification of percentages of metastatic areas in each group was showed on the right. (c) and (d) Representative IF staining of lung tissues with COL1A1 (1:200) and α-SMA (1:200). COL1A1⁺EGFP⁺ and α-SMA⁺EGFP⁺ double positive cells (yellow arrows) within dotted lines circling metastatic lesions were counted for every five HPF and compared (scale bars=50 μm). Mean±s.e.m., **P< 0.01, ***P< 0.001. HPF, high power fields; IF, immunofluorescent; NS, not significant; WT, wild type.