fgf19  (R&D Systems)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 1. High FGF19 expression is correlated with liver metastasis in colorectal cancer. A) Flowchart of CRLM-related gene screening using the bioinfor- matics method. Left panel: Venn diagram showing the 33 overlapping DEGs (log2 |fold change|≥2 and p-value < 0.05) in three CRC- and CRLM-related datasets from the Human Genome Array. Right panel: The prognostic value of the 33 overlapping genes was further validated in TCGA CRC cohort. Ten candidates were screened out. B) Among the 10 candidates, FGF19 was confirmed to have the strongest prognostic value. C) Kaplan‒Meier survival curves of progression-free survival for CRC patients with low (n = 247) and high (n = 161) FGF19 expression. Patients were stratified into FGF19-high or FGF19-low groups according to the cutoffvalue. D) Representative IHC staining of FGF19 expression in normal colon epithelium, CRC, and paired primary CRC and CRLM tissues. Scale bar: 50 μm. E) FGF19 expression was higher in CRC tissues (n = 122) than that in normal colon epithelium tissues (n = 122), and higher in LM(+) CRC tissues (n = 12) than that in LM(−) CRC tissues (n = 110). F) FGF19 expression was higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). G) Kaplan‒Meier survival curves of overall and liver metastasis-free survival of CRC patients with low (n = 59) and high (n = 63) FGF19 expression. H) The serological level of FGF19 was higher in patients with LM(+) CRC (n = 12) than that in those with LM(−) CRC (n = 110). I) Kaplan‒Meier survival curves of overall and liver metastasis-free survival of CRC patients with low (n = 71) and high (n = 51) serological levels of FGF19. *p < 0.05, **p < 0.01, ***p < 0.001. In (E) and (H), data were subjected to Mann–Whitney test (data not normally distributed). In (F), data were subjected to paired Student’s t-test. In (B), (C), (G), and (I), data were subjected to Cox proportional hazards regression. See also related Figure S1, Supporting Information.

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 2. FGF19 promoted CRLM in a mouse model. A–C) Splenic injection of HCT-15 cells with FGF19 overexpression for liver metastasis experiments (n = 6 mice). A) IVIS images and quantification, B) liver weight and the proportion of tumor metastases in livers, and C) animal overall survival are shown. Scale bar: 1 mm. D–F) Splenic injection of KM12SM cells with FGF19 knockdown for liver metastasis experiments (n = 6 mice). D) IVIS images and quantification, E) liver weight and the proportion of tumor metastases in livers, and F) animal overall survival are shown. Scale bar: 1 mm. G) Mice were administrated intravenously with AAV-shCtrl or AAV-Tbg-shFgf15 (1.0 × 1012 genome copies per mouse) for 4 weeks and then injected FGF19- overexpressing HCT-15 cells for liver metastasis experiments (n = 5 mice). The proportion of tumor metastases in livers is shown. H) Experimental design of rhFGF19 (50 μg kg−1) treatment and sequential splenic injection of HCT-15 cells in a liver metastasis mouse model. Liver specimens (n = 5 mice) were harvested 14 days after splenic injection of HCT-15 cells. Scale bar: 1 mm. I,J) Liver metastasis of mice with splenic injection of FGF19- overexpressing HCT-15 cells treated with or without FGF19 neutralizing antibody (25 μg per mouse) (n = 6 mice). I) Liver weight and the proportion of tumor metastases in livers and J) animal overall survival are shown. *p < 0.05, **p < 0.01, ***p < 0.001; and n.s., nonsignificant. In (A), (B), (D), (E), (G), and (I), data were subjected to Student’s t-test (data normally distributed). In (C), (F), and (J), data were subjected to log-rank test. See also related Figure S2, Supporting Information.

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Over Expression, Knockdown

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 3. FGF19 mediated the formation of inflammatory CAFs in liver metastatic niches. A,B) mRNA sequencing of LX-2 cells stimulated with 0, 50, or 100 ng mL−1 rhFGF19 for 24 h. A) Bubble plot showing the GO signatures enriched in rhFGF19-stimulated LX-2 cells. B) Heatmap showing the mRNA expression of iCAF and myCAF markers in rhFGF19-stimulated LX-2 cells. C) mRNA expression of iCAF markers in LX-2 cells treated with FGF19- containing CM in the presence or absence of FGF19 neutralizing antibody (10 μg mL−1) for 24 h (n = 6). D) mRNA expression of iCAF markers in LX-2 cells treated with rhFGF19 (50 ng mL−1) for 24 h (n = 6). E) IF staining of iCAFs (PDPN+ and IL-6+ cells) formed by LX-2 cells treated with FGF19- containing CM or rhFGF19 for 48 h. Scale bar: 20 μm. F) IF staining of iCAFs (PDPN+ and IL-6+ cells) in primary CRC and CRLM tissues. Scale bar: 50 μm. G) Correlation between FGF19 expression and the frequency of iCAFs in human CRLM tissues (n = 30). H) Phosphorylation of FGFR4, JAK2, and STAT3 in LX-2 cells treated with rhFGF19 or FGF19-containing CM in the presence or absence of FGF19 neutralizing antibody for 24 h. I) Phosphorylation of FGFR4, JAK2, and STAT3 in LX-2 cells treated with FGF19-containing CM in the presence or absence of fisogatinib (100 nм) or BLU9931 (10 μм). J) Extracellular expression of IL-1𝛼in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM in the presence or absence of fisogatinib or BLU9931 (n = 4). K) mRNA expression of iCAF markers in LX-2 cells treated with FGF19-containing CM in the presence or absence of anakinra (20 mg mL−1) for 24 h (n = 6). L) Phosphorylation of JAK2 and STAT3 in LX-2 cells treated with CM obtained from LX-2 cells pretreated with FGF19-containing tumor cell CM, in the presence or absence of anakinra. M) mRNA expression of iCAF markers in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sequencing, Expressing, Staining, Phospho-proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 4. FGF19 regulated NET formation in liver metastatic niches to promote the liver metastasis of colorectal cancer. A) IF staining of NETs (H3Cit+ and MPO+ structures) formed by neutrophils treated with CM derived from FGF19-overexpressing HCT-15 and FGF19-knockdown KM12SM cells for 12 h (n = 18 RMFs from 6 experimental replicates per group). Scale bar: 50 μm. B) IF staining and quantification of NETs in livers of mice injected with FGF19-overexpressing HCT-15 and FGF19-knockdown KM12SM cells (n = 18 RMFs from 6 mice per group). Scale bar: 50 μm. C) IF staining of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with CM derived from FGF19-overexpressing HCT-15 cells in the presence or absence of anakinra for 12 h (n = 18 RMFs from 6 experimental replicates per group). Scale bar: 50 μm. D) IF staining and quantification of NETs in the livers of mice injected with FGF19-overexpressing HCT-15 cells with or without anakinra treatment (n = 18 RMFs from 6 mice per group). Scale bar: 50 μm. E–G) Liver metastasis of mice with splenic injection of FGF19-overexpressing HCT-15 cells, with or without DNAse I treatment (5 mg kg−1) (n = 6 mice). IF staining and quantification of NETs in the livers of mice (n = 18 RMFs from 6 mice per group), F) liver weight and the proportion of tumor metastases in livers, and G) animal overall survival are shown. Scale bar: 50 μm. H–J) Liver and serum specimens obtained on the indicated days after

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Derivative Assay, Knockdown, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 6. NET formation was correlated with FGF19 in liver metastases, and targeting FGF19 signaling with fisogatinib prevented CRLM. A) IF staining and quantification of NETs in primary CRC and CRLM tissues (n = 30). Scale bar: 50 μm. B) Correlation between serological FGF19 and MPO-DNA (n = 122). C) Kaplan‒Meier survival curves of liver metastasis-free survival of patients with CRC with low (n = 72) and high (n = 50) serological levels of MPO-DNA. D–I) Splenic injection of KM12SM and FGF19-overexpressing HCT-15 cells for liver metastasis experiments (n = 6 mice). D) IVIS images and quantification, E) ultrasound images, F) liver weight and the proportion of tumor metastases in livers, G,H) IF staining and quantification of NETs in livers, and I) animal overall survival are shown. Scale bar for H&E: 1 mm. Scale bar for IF: 50 μm. **p < 0.01; ***p < 0.001. In (D), (F), and (H), data were subjected to Student’s t-test. In (A), data were subjected to paired Student’s t-test. In (C), data were subjected to Cox proportional hazards regression. In (I), data were subjected to log-rank test. See also related Figure S7, Supporting Information.

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Injection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 7. Schematic depiction of the mechanism of FGF19-mediated colorectal cancer liver metastasis via induction of iCAF and NET formation. Created with BioRender.com.

Article Snippet: Recombinant human FGF19, IL-1α, CXCL11, MIF and PAI-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: