Fig. 6: Thiostrepton exhibits antitumor activities through reprogramming tumor-associated macrophages in vivo.

a Tumor growth curves in B6 mice bearing subcutaneous B16F10 tumors treated I.P. with DMSO, TA99, thiostrepton (150 or 300 mg/kg), and thiostrepton plus TA99 (n = 6–13 mice per group). b Tumor growth curves in B6 mice bearing subcutaneous B16F10 tumors treated I.P. with TA99, and S.C. with PBS or DMSO or thiostrepton (20 mg/kg) or thiostrepton plus TA99 (n = 9–11 mice per group). In a and b, arrows indicate dosing time points and data are presented as mean ± sem. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-sided T-test are indicated. c, d Flow cytometry analysis of TAM (F4/80⁺CD11b⁺Ly6C⁻Ly6G⁻), inflammatory monocytes (F4/80^intCD11b⁺Ly6C⁺Ly6G⁻) and monocytes (F4/80⁻CD11b⁺Ly6C⁺Ly6G⁺) in the tumors from control, TA99-treated, thiostrepton-treated and thiostrepton plus TA99-treated tumor-bearing mice 18 days after tumor engraftment. Shown are representative F4/80 versus CD11b staining profiles gating on CD45⁺ cells (c) and summarized data (mean ± sd) (d) from three independent experiments (n = 7–10 per group). e Representative immunohistochemistry staining with anti-F4/80 in tumor sections. The bottom panels are the enlargement of marked areas from the top panels. Brown: anti-F4/80 stain; blue: nuclear stain. Scale bar: 100 μm. Shown are representative staining from one mouse per group in a. f Comparison of gene expression changes induced by thiostrepton in tumor-infiltrating macrophages from individual mice following I.P. (n = 4) or S.C. administration (n = 2) of thiostrepton or DMSO (n = 2). Tumor infiltrated macrophages were sorted separately from tumor tissues of each mouse based on CD45⁺F4/80⁺CD11b⁺Gr-1⁻ 18 days after tumor engraftment, followed by separate RNA-seq. I.P. intraperitoneal injection, S.C. paratumor subcutaneous injection.