Tumor-derived lipids induce an IL-6/STAT3 autocrine pathway to promote glutamine synthesis in NFs. (A) qPCR analysis of relative mRNA expression levels of iCAF signature genes in NFs treated with TCM or MEF CM for 24 h. (B and C) Western blot of STAT3 in NFs treated with TCM with or without anti–IL-6 neutralization antibody for 24 h (B). Quantification calculated by ImageJ (C). (D) qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway associated genes in NFs treated with TCM derived from ER⁺ breast cancer EO771 cells with or without IL-6–blocking antibody for 24 h. (E) Multiplex cytokine array was used to detect IL-6 levels in TCM as compared with the control medium. Positive control was the supernatant collected from NFs after TCM treatment for 24 h. (F and G) NFs were treated with TCM or TCM w/o lipid or TCM w/o lipid supplemented with palmitate conjugated with BSA (200 μM) for 24 h. BSA alone was used as a control. qPCR analysis of expression of glutamine synthesis pathway genes (Got1, Got2, and Bcat2) (F) and ELISA measurements of IL-6 (G). (H and I) NFs were treated with TCM in the presence of anti-CD36 antibody for 24 h at indicated concentrations. qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway associated genes was determined in H, and Il6 mRNA level was checked in I. (J and K) NFs were treated with TCM in the presence of TLR2 inhibitor for 24 h at indicated concentrations. qPCR analysis of relative mRNA expression levels of glutamine synthesis pathway-associated genes was determined in J, and Il6 mRNA level was checked in K. (L) Proportion of Detox-iCAF signature (from Kieffer et al. [2020]) deconvoluted on bulk RNA-seq data on NFs treated with TCM or TCM w/o lipid for 24 h. (M) STAT3 transcription factor activity inference analysis performed on scRNA-seq data from CAFs of breast cancer patients. Data are representative of at least two independent experiments with similar result. All data are shown as mean ± SEM and were analyzed by two-tailed, unpaired Student’s t test (A–C), two-way ANOVA with Sidak’s multiple comparison test (D and H–K), one-way ANOVA with Tukey’s multiple comparison test (F–G), or two-tailed unpaired Student’s t test (L). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). MEF CM, MEF-conditioned medium. Source data are available for this figure: SourceData FS5.