Figure 1

KLF4 deficiency in mouse bone marrow drastically reduced lung metastasis that was accompanied by decreased recruitment of CCR2⁺ MDSCs in the lung. (a) Six-week old C57BL/6 (B6) wild-type mice (The Jackson Laboratory, Bar Harbor, ME, USA) were lethally irradiated (900 rad) and then transplanted with bone marrow cells (1.0 × 10⁶ cells per recipient mouse) from B6 Rosa26CreER/KLF4 (flox^+/+)/β-actin-EGFP mice, which were generated by crossing tamoxifen-inducible RosaCreER mice (01XAB, NCI) with KLF4 (flox) mice (029877-Mu, MMRRC) and β-actin-EGFP mice (The Jackson Laboratory). Four weeks later, KLF4 knockout of chimeric mice was induced by daily intraperitoneal injection of tamoxifen (TAM, Sigma-Aldrich, St Louis, MO, USA; 50 mg/kg) for five consecutive days, followed by intravenous injection of B16F10-Luc2 tumor cells (Caliper Life Sciences, Middlesex, MA, USA). Sunflower seed oil (Sigma-Aldrich) was used as control. These mice were then monitored by IVIS Spectrum Fluorescence and Bioluminescence Imaging System (Caliper Life sciences) to detect the signals of pulmonary metastasis. Results of representative control (KLF4^+/+) and TAM-induced (KLF4^−/−) mice at day 10 after tumor cell inoculation were shown (n=8, P<0.05). (b) Representatives of lung macrometastases from the chimeric mice as described in a were shown. (c) Left, representative HE stained lung sections with red arrows pointing the metastatic lesions were displayed (scale bars=100 μm). Right, percentages of metastatic areas in control and TAM-induced chimeric mice were quantified. (d) Lungs were minced into small fragments (<1 mm³) and digested in 10 ml of dissociation solution (RPMI 1640 medium supplemented with Collagenase type I (200 U/ml) and DNase I (100 μg/ml)) for 60 min at 37 °C. Lung-infiltrating immune cells were further purified using Percoll (Sigma-Aldrich) according to the manufacturer’s instructions. The cells with intermediate Ly6G (clone RB6-8C5, eBiosciences, San Diego, CA, USA) signals (Ly6G^Int) were gated (left) followed by further flow cytometry analysis (right) using CD11b (clone M1/70, eBiosciences) and CCR2 (R&D, Minneapolis, MN, USA) antibodies. The flow cytometry were performed and data were analyzed using FACSAria II (BD Biosciences, San Jose, CA, USA). Representative dot plots in each group were shown (n=8). Mean±s.e.m., ***P< 0.001.