Figure 1: IFN-γ induces TRC dormancy in vitro.

(a) B16 cells in conventional rigid plate or B16 TRCs seeded in soft 3D fibrin gels were cultured for 3 days in the presence of different concentration of IFN-γ. Cell viability were analysed by flow cytometry. (b,c) B16 cells were seeded in soft 3D fibrin gels for 3 days, and then treated with different doses of IFN-γ for additional 2 days (d2), 4 days (d4) or 6 days (d6). For the bottom wells, IFN-γ was provided for the first 4 days and withdrawn for the last 2 days. Colony size was indicated (b), and cell cycle was analysed after 3 days IFN-γ treatment (100 ng ml⁻¹) (c). The relative colony size was calculated by comparing the colony size in other groups with the colony size in the group of IFN-γ (50 ng ml⁻¹) treated for 2 days, which was set to 1. Bar, 50 μm. (d,e) IFN-γ treatment decreased the expression of PCNA mRNA in B16 TRCs (d), and the glucose consumption (e). (f) B16 TRCs were treated with IFN-γ (100 ng ml⁻¹) or IFN-γ/TNF-α (10 ng ml⁻¹) for 72 h. SA-β gal staining was conducted. Bar, 50 μm. (g) control B16 cells from rigid plate, B16 TRCs, or dormant TRCs (72 h IFN-γ treatment) were treated with methotrexate (MTX, 250 ng ml⁻¹) or paclitaxal (PAX, 2 μg ml⁻¹) for 24 h. Cell viability was measured by flow cytometry. (h) TRC dormancy induced by high dose of IFN-γ was maintained by low dose of IFN-γ. B16 TRCs were treated with 200 ng ml⁻¹ IFN-γ for 2 days, then treated with different low doses of IFN-γ as indicated and cultured for another 2–4 days. The relative colony size was calculated by comparing the colony size in other groups with the colony size in the group of IFN-γ (0 ng ml⁻¹) treated for 2 days, which was set to 1. Data shown are representative of three independent experiments and error bars represent mean±s.e.m., NS, no significant difference (Student’s t-test).