PA-TLR4 signal drives IL-6 autocrine and iCAFs formation. (A) Bubble plots indicating lipid-related metabolism in myCAF and iCAF. (B) Bulk RNA-seq of NFs treated with TCM or TCM w/o. lipid. (n = 3 for each group) shown as principal component analysis (PCA) plot. (C and D) GSEA for iCAF signature (C) or IL-6/JAK/STAT3 signaling pathway (D) in NFs treated with TCM versus control. Significant NES > 1; q value <0.05. (E) qPCR analysis of expression of genes associated with glutamine synthesis pathway in NFs treated with TCM or TCM without lipid (TCM w/o lipid) for 24 h. (F) ELISA of IL-6 protein expression level. (G) Palmitate level quantified in medium conditioned by MEFs or Yumm1.7 tumor cells for 24 h. (H and I) NFs were treated with palmitate conjugated with BSA (200 μM) for 24 h. BSA alone was used as a control. qPCR analysis of expression of genes associated with glutamine synthesis pathway (H) and ELISA of IL-6 protein (I). (J) Transcription factor activity prediction from bulk RNA-seq data. (K) IL-6 protein level in supernatants from NFs treated with palmitate conjugated with BSA (200 μM) in the presence of NF-κB inhibitor PDTC for 24 h. BSA alone was used as a control. (K and L) 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 synthase (GS). Data are representative of at least two independent experiments with similar results (E, F, H, I, K, and L). All data are shown as mean ± SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparison test (E, F, K, and L) or two-tailed, unpaired Student’s t test (G–I). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).