文献标题：Escherichia coli promotes colorectal cancer metastasis by maintaining enhancer-promoter loops through releasing neutrophil extracellular traps
发表期刊：Nat Commun. (IF=15.7)
DOI：https://doi.org/10.1038/s41467-026-69005-y
使用 Absin 产品：人正常肝类器官培养基（货号：abs9529）

Figure 1

a PCA revealed the similarity of bacterial compositions in CRC tissues with and without LM (n=12). b 2bRAD-M-Seq assessed E. coli abundance in CRC tissues (n=12). c FISH assay showed E. coli rRNA levels in CRC tissues with and without LM (n=6). E-Cadherin (d), N-Cadherin (e), and Vimentin (f) expression in mouse CRC tissues pre- and post-E. coli injection (n=6). g NET levels in mouse CRC tissues pre- and post-E. coli injection (n=6), using CitH3, DAPI, and MPO markers. h Correlation between E. coli abundance and NET presence in patient CRC tissues (n=136). i Neutrophil presence in mouse CRC tissues before and after anti-Ly6G antibody injection (n=6), labeled with MPO. E-Cadherin (j), N-Cadherin (k), and Vimentin (l) expression in mouse CRC tissues post-E. coli or anti-Ly6G antibody injection. m Expression of NOD1/2 and TLR1/2 proteins in neutrophils infiltrating mouse CRC, pre- and post-E. coli injection (n=3). n Effects of E. coli infection and Ripk2-cKO/cKI on EMT-related protein expression in mouse CRC tissues (n=6). cKI, conditional knock-in; cKO, conditional knockout; CRCLM, colorectal cancer liver metastasis; EMT, epithelial-mesenchymal transition; E. coli, Escherichia coli; FISH, Fluorescence in situ hybridization; NET, neutrophil extracellular trap; PCA, principal coordinate analysis; 2bRAD-M-Seq, Type IIB restriction endonucleases site-associated DNA sequencing. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). Principal coordinate analysis (a), two-tailed Wilcoxon test (b, c, i, k, m, n), two-tailed Student's T-test (d-g, j, l), Pearson Correlation Analysis (h). Significance: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Source data and exact P values are provided as a Source Data file.

Figure 2

a SAHMI showed microbial abundance in neutrophils from CRC tissues (n=3). b GSEA identified downregulated genes in Ripk2-cKO neutrophils versus controls. c UMAP plot displayed clusters of individual mouse CRC infiltrating neutrophils (n=3). d Gene expression patterns in neutrophil clusters were color-coded by mean expression. e UMAP plot of neutrophils by genotype (n=3). f Proportions of neutrophil clusters in CRC tissues (n=3). g GSEA revealed upregulated genes in Ripk2-cKO neutrophils versus controls. h SCENIC analysis identified regulators in neutrophils. i Ripk2-cKO impacted ATF3/RelB protein expression in neutrophils (n=3). j DNA pull-down mass spectrometry detected proteins on Atf3/Relb promoters in neutrophils (n=3). k Docking model showed interaction of HNRNPK with Atf3/Relb promoters. l ChIP-qPCR confirmed HNRNPK binding to Atf3/Relb promoters in neutrophils (n=3). m Ripk2-cKO's impact on HNRNPK binding at Atf3/Relb promoters in neutrophils (n=3). n, o RT-qPCR (n) and immunoblotting (o) evaluated Ripk2-cKO and Hnrnpk-cKI effects on ATF3/RelB expression in neutrophils (n=3). For (o), P values compared Ripk2-cKO+Ctrl-cKI vs. Ctrl-cKO+Ctrl-cKI, and Ripk2-cKO+Hnrnpk-cKI vs. Ripk2-cKO+Ctrl-cKI. p Immunoblotting analyzed E. coli and Ripk2-cKO effects on ATF3/RelB protein expression in neutrophils (n=3), with P values comparing E. coli vs. Ctrl, and E. coli+Ripk2-cKO vs. E. coli. q Immunofluorescence of NETs in CRC tissues (n=6). ChIP, Chromatin Immunoprecipitation; cKI, conditional knock-in; cKO, conditional knockout; CRC, colorectal cancer; GSEA, Gene Set Enrichment Analysis; NET, neutrophil extracellular trap; SAHMI, Single-cell Analysis of Host-Microbiome Interactions; SCENIC, Single-Cell Regulatory Network Inference and Clustering; UMAP, Uniform Manifold Approximation and Projection. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). GSEA (b, g), two-tailed Wilcoxon test (i, l–n, q), two-tailed Student's T-test (o, p). Significance: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Source data and exact P values are provided as a Source Data file.

Figure 3

a Distribution of RelB-bound DNA peaks around ATF3-bound DNA peaks in mouse CRC infiltrating neutrophils (n=3). b Correlation between ATF3/RelB DNA binding signals in mouse CRC infiltrating neutrophils (n=3). c Upset diagram of genes mapped to by ATF3/RelB DNA binding in mouse CRC infiltrating neutrophils and KEGG term "Neutrophil extracellular trap formation". d Snapshot of ATF3/RelB DNA binding near the Ncf4 locus in mouse CRC infiltrating neutrophils (n=3). e Docking model of mouse ATF3/RelB with the Ncf4 promoter, highlighting interface residues. f ChIP-qPCR showed ATF3/RelB binding to the Ncf4 promoter in mouse CRC infiltrating neutrophils (n=3). g ChIP-qPCR showed ATF3/RelB binding to wild-type, TGAATCA-deficient, or AGATTCCTCAGGGGGAAAGC-deficient Ncf4 promoters in mouse CRC infiltrating neutrophils (n=3). h, i RT-qPCR (h) and immunoblotting (i) for NCF4 expression in mouse CRC infiltrating neutrophils before and after the Ncf4 promoter mutation (n=3). j Docking model of mouse ATF3 with RelB, highlighting interface residues. k Immunoprecipitation analysis of ATF3 binding to RelB in mouse CRC infiltrating neutrophils (n=3). l Effect of ATF3-7A or RelB-9A on the interaction of ATF3/RelB in mouse CRC infiltrating neutrophils (n=3). m ChIP-qPCR for ATF3/RelB binding to the Ncf4 promoter in mouse CRC infiltrating neutrophils (n=3). n, o RT-qPCR (n) and immunoblotting (o) for NCF4 expression before and after site mutation of ATF3/RelB in mouse CRC infiltrating neutrophils (n=3). p Immunofluorescence of NETs in mouse CRC tissues (n=6). ChIP, Chromatin Immunoprecipitation; CRC, colorectal cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes; NET, neutrophil extracellular trap. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). Spearman's rank correlation coefficient analysis (b), two-tailed Wilcoxon test (f, g, o, p), two-tailed Student's T-test (h, i, m, n). Significance: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. Source data and exact P values are provided as a Source Data file.

Figure 4

a Cellchat analyzed cell interaction patterns in CRC tissues. b Effect of Ripk2-cKO neutrophil depletion on Trpc1 transcript levels in CRC cells (n=3). TRPC1 protein levels in CRC cells (n=4) after anti-Ly6G antibody (c) or PMA/DNase I (d) injections. For d, P values compared PMA~NETs vs. DMSO~NETs and Dnase I~NETs vs. DMSO~NETs. e Calcium levels in CRC cells (n=3). f Pathways affected by Ripk2-cKO neutrophil depletion in CRC cells. Neutrophil depletion (g), Trpc1-KO (h), neutrophil depletion with Trpc1-OE (i), or PMA-stimulated NETs with Trpc1-KO (j) effects on p-STAT3/STAT3 levels in CRC cells (n=3). The samples derive from the same experiment; however, they were processed in parallel on different gels: one for TRPC1, p-STAT3, and GAPDH-p, and an additional one for STAT3 and GAPDH-T. Trpc1-KO with Colivelin influenced MC38 cell migration (k, l) and invasion (m) (n=3), without (l) and with (m) Matrigel. n Interaction analysis of S100A8 and S100A9 in MC38 cells (n=3). o Trpc1-KO effects on S100A8-S100A9 interaction in MC38 cells (n=3). p S100A8 and S100A9 docking model. q Impact of S100A8-4A and S100A9-3A on their interaction in MC38 cells (n=3). r Trpc1-OE or S100A8/9-7A effects on MC38 cell migration (n=3). Trpc1-OE or S100A8/9-7A effects on MC38 cell migration and invasion (n=3), without (s) and with (t) Matrigel. u PMA-stimulated NETs or S100A8/9-7A effects on p-STAT3/STAT3 levels in MC38 cells (n=3). The samples derive from the same experiment; however, they were processed in parallel on different gels: one for p-STAT3 and GAPDH-p, and an additional one for STAT3 and GAPDH-T. v Trpc1-OE or S100A8/9-7A effects on p-STAT3/STAT3 levels in MC38 cells (n=3). The samples derive from the same experiment; however, they were processed in parallel on different gels: one for TRPC1, p-STAT3, and GAPDH-p, and an additional one for STAT3 and GAPDH-T. w S100a8/9-KO or Colivelin effects on MC38 cell migration (n=3). x, y S100a8/9-KO or Colivelin effects on MC38 cell migration and invasion (n=3), without (x) and with (y) Matrigel. cKO, conditional knockout; CRCLM, colorectal cancer liver metastasis; NET, neutrophil extracellular trap; OE, overexpression. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). Two-tailed Student's T-test (b–d, g–k, m, r, s, x), two-tailed Wilcoxon test (e, f, l, t–w, y). Significance: *P<0.05; **P<0.01; ***P<0.001. Source data and exact P values are provided as a Source Data file.

Figure 5

a Immunofluorescence revealed the subcellular localization of STAT3, S100A8, and S100A9 in MC38 cell nuclei (n=3). b Docking model between mouse STAT3 and S100A8 or S100A9. Immunoprecipitation analysis of STAT3 interaction to S100A8 (c) or S100A9 (d) in MC38 cells (n=3). Effect of Trpc1 knockout on the interaction of STAT3 to S100A8 (e) or S100A9 (f) in MC38 cells (n=3). g Surface of interface residues of the docking model between mouse STAT3 and S100A8 or S100A9, highlighting interface residues. Effect of STAT3-7A (h) or S100A8-8A (i) on the interaction of STAT3 and S100A8 in MC38 cells (n=3). Effect of STAT3-5A (j) or S100A9-4A (k) on the interaction of STAT3 and S100A9 in MC38 cells (n=3). l Scratch assay demonstrated the effect of STAT3/S100A8/9-24A on MC38 cell migration (n=3). Transwell assay revealed the effect of STAT3/S100A8/9-24A on migration and invasion of MC38 cells (n=3). (m) No Matrigel. (n) With Matrigel. o Structure of the Chromosome 1-wide EPLs in MC38 cells (n=3). p Number of EPLs in MC38 cells (n=3). q Distribution of intensity and length of EPLs in MC38 cells (n=3). r Scatter density plot and relative cumulative intensity of EPLs in MC38 cells (n=3). s Rank plot of EPLs in MC38 cells (n=3). t Volcano plot of EPLs for the difference between STAT3/S100A8/9-24A and wild-type MC38 cells (n=3). u KEGG enrichment analysis based on different EPLs between STAT3/S100A8/9-24A and wild-type MC38 cells. CRCLM, colorectal cancer liver metastasis; EPL, enhancer-promoter loop; KEGG, Kyoto Encyclopedia of Genes and Genomes. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). Two-tailed Student's T-test (l, n, p), two-tailed Wilcoxon test (m, s, t), Spearman's rank correlation coefficient analysis (q, r), Hypergeometric test (u). Significance: *P<0.05; **P<0.01; ***P<0.001. Source data and exact P values are provided as a Source Data file.

Figure 6

a EPL map of Tns1 locus in MC38 cells (n=3). b Intensity of STAT3/S100A8/9-EPLs in Tns1 locus in MC38 cells (n=3). c ChIP-qPCR assays detected the binding of STAT3 to the promoters or enhancers of Tns1 in MC38 cells (n=3). Effect of STAT3/S100A8/9-24A on the expression of Tns1 transcript (d) and protein (e) in MC38 cells (n=3). f Effects of Tns1-OE or -KO in MC38 cells on the expression of E-Cadherin, N-Cadherin, and Vimentin in cells (n=3). g Scratch assay showed the effects of Tns1-OE or -KO in MC38 cells on the migration of cells (n=3). Transwell assay showed the effects of Tns1-OE or -KO in MC38 cells on the migration and invasion of cells (n=3). (h) No Matrigel. (i) With Matrigel. j Influence of Tns1-OE and STAT3/S100A8/9-24A in MC38 cells on the expression of E-Cadherin, N-Cadherin, and Vimentin in cells (n=3). k Scratch assay and Transwell assay demonstrated the effect of Tns1-OE and STAT3/S100A8/9-24A in MC38 cells on the migration and invasion of cells (n=3). ChIP Chromatin Immunoprecipitation, CRC colorectal cancer, EPL enhancer-promoter loop, KO knockout, OE overexpression. n represented the number of independent biological repetitions. Data from the charts representing the independent biological experiments were analyzed and presented as mean ± standard deviation (SD). Two-tailed Student's T-test (b-i, k), two-tailed Wilcoxon test (j). Significance: *P<0.05; **P<0.01; ***P<0.001. Source data and exact P values are provided as a Source Data file.

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