Fig. 3

TAMs of Ly6C⁻CD206^high and Ly6C⁺CD206^−/low synergistically spur tumor fibrosis. (A) Transwell migration assay of CAFs, in which CAFs sorted from tumors were added to upper chambers, and the lower chambers contained serum-free medium (Culture Medium, CM) with or without TAMs. Shown are representative membrane staining (left, scale bars = 20 μm), and numbers of CAFs (right) migrating to the lower compartment (n = 3 biological replicates). Original magnification, ×500. (B) Relative mRNA levels of CCL5 in 4T1^WT cells and CD11b⁺F4/80⁺ macrophages which were sorted from tumors measured by RT-qPCR (n = 3 biological replicates). (C) Relative mRNA levels of CCL5 in four subtypes of macrophages sorted from tumors measured by RT-qPCR (n = 6 biological replicates). (D) Transwell migration assay of CAFs, in which CAFs sorted from tumors were pretreated with or without CCR5 antagonist (Maraviroc, 100 nm/ml) and added to upper chambers, and TAMs were placed in the lower chambers. Shown are representative membrane staining (left, scale bars = 20 μm), and numbers of CAFs (right) migrating to the lower compartment (n = 3 biological replicates). Original magnification, ×500. (E) Relative mRNA levels of TGFβ1 in four subtypes of TAMs measured by RT-qPCR (n = 6 biological replicates). (F) The levels of TGFβ1 in cultured medium derived from Ly6CCD206^−/low or Ly6CCD206^high TAMs detected by ELISA (n = 3 biological replicates). (G) Representative flow cytometric plots (upper), frequency and number (bottom) of Ly6C⁺CD206^−/low and Ly6C⁻CD206^high cells in tumors (n = 5). (H) Relative mRNA levels of αSMA, FSP1, FAP, ITGβ1 and PDPN in CAFs pretreated with TGFβ1 (10 ng/ml) or PBS for 72 h measured by RT-qPCR (n = 3 biological replicates). (I) Representative αSMA, FSP1 and PDPN western blots using lysates of CAFs pretreated with TGFβ1 (10 ng/ml) or PBS for 72 h. (J) Relative mRNA levels of αSMA, FSP1 and PDPN in CAFs co-cultured with TAMs treated with anti-TGFβ1 antibody or isotype control antibody for 72 h (n = 3 biological replicates). (K) Representative αSMA, FSP1 and PDPN western blots using lysates of co-cultured CAFs from (J). Data are presented as means ± SEM, or representative staining profiles or photographs. p values were determined by one-way ANOVA (C, E), unpaired two-tailed Student’s t test (A, B, D, F), or two-way ANOVA (G, H, J). Shown are data from a representative of three independent experiments (A–K). *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significance