Fig 1: Inhibition of the SAA1–TAM–CXCL1 axis suppresses ovarian cancer metastasis. (A) Experimental scheme for the C57BL/6 mouse model of ovarian cancer peritoneal dissemination. (B) Western blot of SAA1 in luciferase‐labelled ID8 cells. Protein levels were compared between shSAA1‐ID8‐LUC and shCtrl‐ID8‐LUC. (C) Bioluminescence imaging at Week 7 after intraperitoneal injection of ID8‐LUC or shSAA1‐ID8‐LUC cells, treated with control liposomes (Ctrl Lip), clodronate liposomes (CLO Lip), vehicle (DMSO), or the CXCL1 receptor inhibitor SB225002, n = 4. (D) Quantification of average luminescence intensity. (E) Immunohistochemistry of F4/80 in spleen sections from mice bearing shCtrl‐ID8‐LUC or shSAA1‐ID8‐LUC tumours, with or without Ctrl Lip, CLO Lip, DMSO, or SB225002. Left: representative fields from red pulp; right: quantification of F4/80+ area ratio (%). Scale bar, 100 µm. (F) Multiplex immunofluorescence of peritoneal tumour sections under the indicated interventions. Staining includes Pan‐CK (red), SAA1 (green), F4/80 (cyan), CD206 (magenta), CXCL1 (grey), and nuclei (blue). Merged images are shown in the bottom row. Scale bar, 20 µm. Quantitative data in D–F are shown as mean ± SD. Statistical significance was determined by one‐way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 2: SAA1–FPR2 axis drives CXCL1 production in macrophages via JAK2–STAT3 signalling. (A) Proteomic profiling of secreted factors in TeMCM generated with TCM from SKOV3Cas9WT versus SKOV3SAA1–/– cells. (B) KEGG pathway enrichment (bubble plot) of DEGs from macrophages exposed to SKOV3Cas9WT versus SKOV3SAA1–/– TCM. (C) Heatmap of cytokine/chemokine‐related DEGs from RNA‐seq of macrophages educated with SKOV3Cas9WT TCM (A1–A3) or SKOV3SAA1–/– TCM (B4–B6). (D) Venn diagram showing overlap of upregulated genes from RNA‐seq and proteomics. The 38 shared genes are listed below. (E) Differential expression of the 38 overlapping genes. (F) CXCL1 expression in macrophages under different treatments. Upper: densitometric quantification; lower: representative immunoblots. (G) ELISA quantification of CXCL1 in macrophage supernatants under indicated treatments, n = 3. (H) Predicted STAT3‐binding sites within the CXCL1 promoter (−2000 bp to TSS) are shown, along with the consensus STAT3 motif. (I) ChIP–qPCR showing STAT3 enrichment at nine predicted binding sites on the CXCL1 promoter in macrophages treated with SKOV3Cas9WT TCM, SKOV3SAA1–/– TCM, or SKOV3SAA1–/– TCM + rSAA1. (J) Dual‐luciferase assay of CXCL1 promoter activity upon STAT3 overexpression, n = 3. Reporter groups: empty vector, CXCL1 WT promoter, STAT3 + WT, and STAT3 + MUT. (K) Immunoblot of p‐JAK2, JAK2, p‐STAT3, STAT3, and CXCL1 in macrophages treated with SKOV3Cas9WT or SKOV3SAA1–/– TCM with or without WRW4, rSAA1, Fedratinib, or Stattic. Quantitative data in F, G, I and K are shown as mean ± SD (n = 3 independent experiments). Statistical significance was determined by one‐way ANOVA. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: CXCL1 enhances the invasive and metastatic potential of ovarian cancer cells. (A–B) Wound‐healing assays of SKOV3Cas9WT and SKOV3SAA1–/– cells treated with rCXCL1 or PBS, n = 3. Representative images (left) and quantification of migration rate (right). Scale bar, 100 µm. (C) Transwell migration of SKOV3Cas9WT and SKOV3SAA1–/– cells treated with rCXCL1 or PBS, n = 3. Representative images (left) and quantification (right). Scale bar, 50 µm. (D) Matrigel invasion assay of SKOV3Cas9WT and SKOV3SAA1–/– cells with rCXCL1 or PBS, n = 3. Representative images (left) and quantification (right). Scale bar, 50 µm. (E) Left: schematic illustration of the inverse invasion model; middle: laser‐induced fluorescence imaging; right: quantitative analysis of the effect of CXCL1 on SKOV3‐GFP invasion. (F) Representative three‐dimensional reconstruction based on Z‐stack scanning, presented as a depth‐coded heatmap. (G) Immunoblot of E‐cadherin and vimentin in SKOV3Cas9WT and SKOV3SAA1–/– cells treated with PBS or rCXCL1. (H) Immunoblot of E‐cadherin, β‐catenin, and vimentin in SKOV3 cells treated with increasing concentrations of rCXCL1 (0–100 ng/mL, 24 h). (I) Representative tissue microarray (TMA) cores stained for CXCL1 by immunohistochemistry. Scale bar, 2 mm. Kaplan–Meier overall survival analysis of the same cohort stratified by CXCL1 IHC score (High vs Low). Log‐rank p = 0.029. (J) Kaplan–Meier analysis of TCGA‐OV cohort stratified by CXCL1 mRNA expression (High vs Low). Log‐rank p = 0.0083; HR = 1.60, 95% CI 1.13–2.27. Quantitative data in A–E are shown as mean ± SD (n = 3 independent experiments). Statistical significance was determined by Student's t‐test in A–D and by one‐way ANOVA in E. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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