Fig. 1.

CSCs induce MFG-E8 expression from TAM. (A) F4/80⁺CD11b⁺ macrophages were isolated from tumors, tumor-draining lymph nodes (TDLs), and splenocytes in mice bearing CD44⁺ALDEFLOUR⁺ MC38-CSCs, or CD133⁺ALDEFLOUR⁺ 3LL-CSCs, or their non-CSC counterparts (n = 3 per group). Murine MFG-E8 mRNA was quantified by RT-PCR. The results are shown as fold induction of target genes relative to a reference gene (GAPDH). (B) MFG-E8 expression in F4/80⁺CD11b⁺ macrophages isolated from MC38-CSCs or non-CSCs was evaluated by intracellular flow cytometry. The splenic macrophages from CSC-bearing mice (spleen) serve as negative control. (C) MC38-CSCs or non-CSCs, bulk MC38 cells were cultured with F4/80⁺ splenic macrophages (Mac) for 24 h, and MFG-E8 in macrophages (F4/80⁺) or MC38 tumor cells (F4/80⁻) were quantified by flow cytometry. Splenic macrophages or MC38 tumor cells without coculture served as a negative control. The expression was shown as mean fluorescence intensity (MFI). (D) MFG-E8 in CD68⁺ macrophages (Mac) isolated from pleural effusion (PE) or peripheral blood (PBMC) of two nonsmall cell lung cancer patients (Pt-1 and Pt-2). The MFG-E8 mRNA (Left) or protein (Right) levels were analyzed by RT-PCR or flow cytometry, respectively. Data are representative of three independent experiments.