Fig 1: CXCL2 in omental adipocytes is critical for GC cell growth/migration and in vitro angiogenesis.Each graph represents the mean ± SE from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. a Gene expression of GRO family in omental adipocyte. Bar graph represents the relative ratio of each RNA expression to CXCL2 expression level, using 2−ΔΔCt, where ΔCt indicates the difference in Ct values between each gene and β-actin [ΔCt = Ct (target gene) − Ct (β-actin)]. Mean, 0.41 (CXCL1), 1.0 (CXCL2), 0.18 (CXCL3). b Western blotting. c Efficiency of siRNA. Omental preadipocytes were transfected with non-targeting siRNA and CXCL2. The left graph represents RNA gene expression of CXCL2 in the OmAd cells. Bar graph represents the relative ratio of siNT and siCXCL2 to control, using 2−ΔΔCt, where ΔCt indicates the difference in Ct values between each gene and β-actin [ΔCt = Ct (target gene) − Ct (β-actin)]. The right graph represents protein concentration of CXCL2 in the OmAd-CM. Mean, RNA level: 1.0 (control), 1.0 (siNT), 0.27 (siCXCL2); protein level: 11.0 (control), 11.0 (siNT), 1.3 (siCXCL2). d Cell growth. Shown are the relative ratios of absorbance under each condition of OmAd-CM to those under control media (n = 5). Mean, AGS: 1.0 (control), 1.2 (control OmAd-CM), 1.3 (siNT OmAd-CM), 1.1 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 1.3 (control OmAd-CM), 1.2 (siNT OmAd-CM), 1.0 (siCXCL2 OmAd-CM). e Representative images of migration assay (×100). f Quantification of migration assay. Migrated GC cells were counted from averages at four microscopic fields, and each result was presented as the mean of at least three independent experiments. Each value represents the mean relative ratio of migrated GC cells under each type of OmAd-CM to those under control media. Mean, AGS: 1.0 (control), 3.8 (control OmAd-CM), 4.3 (siNT OmAd-CM), 1.7 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 2.5 (control OmAd-CM), 2.9 (siNT OmAd-CM), 0.7 (siCXCL2 OmAd-CM). g Representative images of EC recruitment assay (×100). h Quantification of EC recruitment assay. Migrated HMVECs were counted from averages at four microscopic fields, and each result was presented as the mean of at least three independent experiments. Each value represents the mean relative ratio of migrated HMVECs co-cultured with GC cells treated with each type of OmAd-CM to those co-cultured with GC cells treated with control media. Mean, AGS: 1.0 (control), 1.4 (control OmAd-CM), 1.6 (siNT OmAd-CM), 0.7 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 1.8 (control OmAd-CM), 2.2 (siNT OmAd-CM), 1.0 (siCXCL2 OmAd-CM). i Representative images of tube-formation assay (×100). j Quantification of tube-formation assay. Each value represents the mean number of branched tubes under each condition. Mean, AGS: 4.0 (control), 9.0 (siNT OmAd-CM), 3.3 (siCXCL2 OmAd-CM); IM95: 2.0 (control), 7.0 (siNT OmAd-CM), 2.8 (siCXCL2 OmAd-CM). k Semi-comprehensive analysis for angiogenic factors. RNA was extracted from both AGS and IM95 cells before and after OmAd-stimulation. Each bar represents the relative ratios of each gene expression in OmAd-treated GC cells to those in non-treated GC cells. l VEGFA expression in GC cells. Relative ratios are expressed with 2−ΔΔCt, where ΔCt indicates the difference in Ct values between each gene and β-actin. Mean, AGS: 1.0 (control), 1.8 (control OmAd-CM), 2.1 (siNT OmAd-CM), 1.4 (siCXCL2 OmAd-CM); IM95: 1.0 (control), 2.1 (control OmAd-CM), 2.4 (siNT OmAd-CM), 1.9 (siCXCL2 OmAd-CM). m AKT phosphorylation and HIF1α expression in GC cells. Each protein extracted from GC cells incubated with control media, OmAd-CM, siNT OmAd-CM or siCXCL2 OmAd-CM for 24 h was immunoblotted with anti-phospho-AKT, anti-AKT and anti-HIFα antibodies. Each band density was quantified with Image J. β-actin is shown as a loading control.
Fig 2: SLC25A22 knockout abrogates secretion of CXCL1 and CXCL3 in vitro and in vivo.a RNA-seq of SLC25A22 knockout DLD1 cells and gene set enrichment analysis (GSEA) for the identification of common differentially regulated pathways in SLC-KO1 and SLC-KO2 cells (n = 4). b, c GSEA enrichment scores for differentially regulated gene sets unveiled the cytokine-cytokine receptor interaction signaling pathway as the top pathway depleted in SLC25A22 knockout cells. d Inflammatory Response and Autoimmunity PCR array showed that CXCL1, CXCL3 and IL1B were induced in ApcMin/+KrasG12D/+Villin-Cre mice tumors, but were down-regulated by SLC25A22 knockout (FC > 2). e qPCR validated that SLC25A22 knockout inhibited CXCL1/3 mRNA in DLD1, CT26 and Colo26 cells (n = 3). Each dot represents an independent sample. f Antibody array showed SLC25A22 knockout down-regulated cytokine secretion in DLD1 cells. g Densitometry showed CXCL1 and CXCL1/2/3 were top-down-regulated cytokines. h ELISA confirmed SLC25A22 knockout impaired CXCL1/3 secretion in DLD1 (72 h), CT26 (24 h) and Colo26 (24 h) (n = 3). Each dot represents an independent sample. i Detection of CXCL1/3 in serum and tumors of mice implanted with CT26 allografts (left, n = 5) and ApcMin/+KrasG12D/+ organoid allografts (right, n = 10) with or without SLC25A22. Each dot represents an independent mouse. j SLC25A22 mRNA correlates with CXCL1/2/3 mRNA in TCGA CRC (COADREAD) cohort (n = 677). Each dot represents an independent patient. Data are shown as mean ± SD (e, h, i). Two-tailed one-way ANOVA (e, h, i). Two-tailed Student’s t test analysis for two-group comparison i. Pearson correlation test j. Source data are provided as a Source Data file.
Fig 3: IL-26 drives massive CXC chemokine production.(a–d, h, n) mRNA expression levels of CXC chemokines in the human CRC cell line COLO205 following stimulation for 12 h. a Stimulation with IL-26 alone or in combination with TNF, IL-1β, or TNF plus IL-1β. b Stimulation with IL-26, IL-17A, IL-21 or IL-22 alone, or in combination with TNF and IL-1β. c Stimulation with IL-26, TNF and IL-1β in the presence of isotype IgG, anti-human IL-10Rb antibody or anti-human IL-20Ra antibody. d Stimulation with IL-26, TNF and IL-1β in the presence of vehicle, the STAT1 inhibitor fludarabine (50 μM) or the STAT3 inhibitor Stattic (50 μM). e Immunofluorescence and confocal microscopy of COLO205 cells stimulated with vehicle or IL-26 in the presence of TNF and IL-1β for 12 h. IL-26 is shown in green and nuclei are counterstained with DAPI (blue). Representative z-stack views are shown. Scale bars, 5 μm. f Western blot analysis of IL-26 in cytoplasmic and nuclear fractions of cells co-stimulated with TNF, IL-1β and IL-26 for 12 h. g Nuclear extracts from stimulated COLO205 cells were subjected to immunoprecipitation with anti-STAT1 antibody, followed by western blotting for STAT1 and IL-26. h Stimulation with IL-26, TNF and IL-1β in the presence of vehicle, the NF-κB inhibitor JSH-23 (50 μM) or the AP-1 inhibitor T5224 (100 μM). i Nuclear extracts from cells stimulated with TNF and IL-1β (control), or with TNF, IL-1β and IL-26 ( + IL-26), were subjected to immunoprecipitation with an anti-BRD4 antibody, followed by western blotting for p65, c-Fos, c-Jun and IL-26. j CXCL1 protein levels in culture supernatants measured by ELISA after transfection with control siRNA, STAT1 siRNA or BRD4 siRNA, followed by stimulation with the indicated cytokines for 12 h. k ChIP-sequencing analysis of H3K27ac and BRD4 peaks near CXC chemokine-coding regions (CXCL1, CXCL2, CXCL3 and CXCL7) in COLO205 cells stimulated with the indicated cytokines. The vertical axis represents reads per million mapped reads (RPM). l Motif enrichment analysis of peak regions at CXC chemokine loci altered by stimulation with IL-26, TNF and IL-1β, showing enrichment of NF-κB, AP-1 and STAT1 motifs in accessible chromatin regions. m ChIP-sequencing analysis of H3K27ac and BRD4 peaks near CXC chemokine-coding regions in COLO205 cells stimulated with IL-26, TNF and IL-1β in the presence of vehicle or JQ1 for 12 h. n mRNA expression levels of CXCL1 in COLO205 cells stimulated with the indicated cytokines in the presence of JQ1 for 12 h. For bar graphs in (a–d), (h), (j) and (n), bars indicate mean ± s.e.m.; dots represent biologically independent samples; n = 3 biologically independent samples per group. P values in (a), (b), (h), (j) and (n) were calculated using ordinary two-way ANOVA followed by Šídák’s multiple-comparisons test. P values in (c) and (d) were calculated using ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test. Representative results from three independent experiments are shown in (e–g) and (i). Representative results from two biologically independent samples are shown in (k) and (m). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided in the Source Data file.
Fig 4: BRD4 inhibition suppresses CRC in hIL-26Tg mice.a–c MC38 tumor-bearing model. a Schematic protocol of MC38 tumor-bearing hIL-26Tg mice treated with anti–PD-1 antibody with or without JQ1. b Mean tumor volumes of subcutaneous MC38 tumors from hIL-26Tg mice treated with anti–PD-1 antibody plus vehicle or JQ1. Vehicle, n = 6 mice; JQ1, n = 9 mice. c Representative flow cytometry plots (left) and quantification (right) of neutrophils among 104 total single-cell suspensions prepared from MC38 tumors. Vehicle, n = 5 mice; JQ1, n = 7 mice. d mRNA expression levels of Cxcl1, Cxcl2, Cxcl3 and Cxcl7 in MC38 tumors. Vehicle, n = 5 mice; JQ1, n = 6 mice. e–h AOM/DSS CRC model. e, Schematic protocol of AOM/DSS-treated hIL-26Tg mice treated with or without JQ1. f Polyp counts in AOM/DSS-treated hIL-26Tg mice treated with vehicle or JQ1; n = 5 mice per group. g Representative flow cytometry plots (left) and quantification (right) of neutrophils among 104 total single-cell suspensions prepared from colon tumors. Vehicle, n = 5 mice; JQ1, n = 5 mice. h mRNA expression levels of Cxcl1, Cxcl2, Cxcl3 and Cxcl7 in colon tumors. Vehicle, n = 6 mice; JQ1, n = 6 mice. i Schematic illustration showing that nuclear-localized IL-26 forms complexes with BRD4 and transcription factors, inducing epigenetic alterations that drive tumor-promoting inflammation and immune evasion. Created with BioRender.com (https://BioRender.com/tw2bksf). Tumor growth curves in (b) are shown as mean tumor volume ± s.e.m. For box plots in (c), (f) and (g), box plots show the median, 25th and 75th percentiles; whiskers indicate the minimum and maximum values; each dot represents one mouse. For bar graphs in (d) and (h), bars indicate mean ± s.e.m.; dots represent individual mice. P values in (b) were calculated using two-way repeated-measures ANOVA followed by Šídák’s multiple-comparisons test. P values in (c), (d), (f), (g) and (h) were calculated using a two-sided Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided in the Source Data file.
Supplier Page from Abcam for Human CXCL3 ELISA Kit