Fig 2: Acetoacetate suppresses TAM infiltration and metastatic lung cancer by downregulating IL-6 and CSF1 levels. (A-B). Raw264.7 cells were treated with the indicated concentrations of IL-6 and CSF1 for 24 hours, and cell viability was assessed using ATP Lite. (C-D). A Raw264.7 cell migration assay was performed at different concentrations of IL-6 and CSF1 to evaluate chemotactic efficacy. Representative images are shown on the left. Statistical analysis of randomly selected biological replicates is shown on the right. (E-F). The correlations of IL-6 and CSF1 with EMT, ECM, and angiogenesis were analyzed in pTNM stage IIB lung cancer patients using data from Home for Researchers (https://www.home-for-researchers.org). (G). C57BL/6 mice were intravenously injected with 2 × 10^5 LLC lung cancer cells via the tail vein. On the second day after injection, they were randomly divided into four groups (n = 6 per group): negative control (NC), Acetoacetate (AC), Acetoacetate combined with IL-6 (AC+IL-6), and Acetoacetate combined with CSF1 (AC+CSF1). Acetoacetate (100 mg/kg, daily), IL-6 (8 µg/kg, every 2 days), and CSF1 (50 µg/kg, every 3 days) were administered by intraperitoneal injection. On day 21, mice were euthanized, and lung tissues were harvested. (H-I). IL-6 and CSF1 protein levels in lung tissues were measured by ELISA. (J-K). Statistical analysis of metastatic lung tumor counts and lung weight in mice. (L). Lung tissues were photographed and then fixed for H&E staining. (M). Statistical analysis of F4/80+ cells across the four groups. (N-O). The count of Ki-67-positive macrophages (F4/80+ Ki-67+) and their changes among the four groups. Values are presented as mean ± SD or mean ± SEM, with * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. indicating no significance, determined by one-way ANOVA and Welch’s one-way ANOVA (I-K)

Fig 3: A GPR43-dependent metabolic signaling axis connects acetoacetate to the repression of IL-6 and CSF1. (A). The results of GPR43 expression in immune cells from the Cell-omics Data Coordinate Platform. (B). Monocytes were identified by CD163 staining, and the colocalization of GPR43 with monocytes was visualized by confocal fluorescence microscopy (DAPI, blue; GPR43, green; CD163, red; merge, orange). (C). A schematic drawing of the RNA-seq analysis of acetoacetate-administered LLC metastatic lung tissues and RAW264.7 cells (acetoacetate 20 mM treated cells for 24 hours, with LPS 5 µg/mL added 6 hours before the cells were harvested). (D-E). Volcano plot analysis was performed on lung tissues and RAW264.7 cells. (F). A Venn diagram showing co-down-regulated genes in mouse lung tissues and RAW264.7 cells after acetoacetate treatment. (G-H). Gene Ontology Biological Process (GO-BP) analysis was conducted using the DAVID website, focusing on the frequency of representative genes associated with the enriched pathways. (I-J). The mRNA levels of IL-6 and CSF1 were quantified by qPCR in lung tissues from the LLC and 4T1 metastatic lung tumor models. (K). RAW264.7 cells were treated with acetoacetate (AC, 10 mM and 20 mM) for 24 hours, with LPS added 6 hours before harvesting. The mRNA levels of IL-6 and CSF1 were measured by qPCR. (L). LLC cells were treated with acetoacetate (AC, 5 mM, 10 mM, 20 mM) for 24 hours, then harvested and analyzed through qPCR. (M). RAW264.7 cells were treated with acetoacetate (AC, 20 mM) and combined with GPR43 inhibitors (AC+GLP0974 80 µM or AC+CATPB 40 µM) for 24 hours, then harvested and assessed by qPCR. (N). The mRNA levels of IL-6 and CSF1 were quantified by qPCR in lung tissues from the LLC model across four groups. Values are presented as mean ± SD or mean ± SEM, with * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. indicating no significance, determined by one-way ANOVA and Student’s t-test (I, J)

Fig 4: The acetoacetate working model inhibits monocyte-derived IL-6 and CSF1 via GPR43-mediated P-ERK1/2-MAT2A-SAM elevation and DNA hypermethylation, suppressing TAM infiltration and metastatic lung cancer