Fig 2: Genetic depletion of pericytes ablates CXCL14 and TAM infiltration in the TME.(A) Growth rates of 4T1-vector and 4T1–FGF-2–overexpressing tumor cells in vitro. (B and C) Cell migration (n = 8 samples per group) and chemotactic ability (n = 6 samples per group) of 4T1-vector and 4T1–FGF-2–overexpressing tumor cells. (D) Tumor-bearing WT and NG2-TK mice were administrated with ganciclovir when the tumor reached 0.5 cm3. H&E staining and immunofluorescence localization of CD31 (red), NG2 (green), and DAPI (blue) signals in 4T1-vector and 4T1-FGF-2–overexpressing tumor–bearing WT and NG2-TK mice (n = 6 mice per group). Scale bar in upper panel: 50 µm. Scale bar in lower panel: 100 µm. Quantification of CD31+ signals, NG2+ signals, pericyte coverage, and average vessel diameters (n = 8 random fields per group). (E) qPCR quantification of Cxcl14 mRNA levels of 4T1-vector and 4T1–FGF-2–overexpressing tumor tissues from WT and NG2-TK mice (n = 6 mice per group). (F) F4/80 (brown) IHC in vector and FGF-2 tumor with or without NG2+ pericyte depletion and in CXCL14-administrated, NG2+ pericyte–depleted FGF-2 tumor (n = 6 mice per group). Scale bar: 50 µm. Quantification of F4/80+ signals (n = 8 random fields per group) (G) CD206 (brown) IHC in FGF-2 tumor with or without NG2+ pericyte depletion or CXCL14 administration (n = 6 mice per group). Scale bar: 50 µm. Quantification of CD206+ signals (n = 8 random fields per group) (H) qPCR quantification of Cd206 mRNA levels in F4/80+ TAMs from various tumor groups (n = 3 samples per group). *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired 2-tailed Student’s t test (A–D) or 1-way ANOVA with Tukey’s multiple-comparison analysis (E–H). Data are presented as mean ± SD.

Fig 3: FGF-2 induces CXCL14 expression in pericytes via FGFR1/ERK/AHR signaling.(A) Heatmap of selected genes by inflammatory cytokine/chemokine profiling of vehicle- and FGF-2–treated primary mouse pericytes (n = 3 samples per group). Arrow points to upregulated Cxcl14 gene. (B) Volcano plot of inflammatory gene profiling of vehicle- and FGF-2–stimulated pericytes (n = 3 samples per group). (C and D) Expression levels of Ccl11 and Cxcl14 in vehicle- and FGF-2–stimulated isolated primary pericytes and MS5 fibroblasts (n = 3 samples per group). (E) qPCR quantification of Cxcl14 mRNA levels in F4/80+ TAMs, NG2+ pericytes, CD31+ endothelial cells, and NG2– population isolated from T241-vector and T241–FGF-2 tumors (n = 3 samples per group). (F) qPCR quantification of Cxcl14 mRNA levels in vehicle- and FGF-2–stimulated pericytes in the presence or absence of FGFR1, FGFR2, and FGFR3 specific inhibitors, and pan-FGFR inhibitor (n = 3 samples per group). (G) After 0, 15, 30 minutes of stimulation, FGF-2 induced phosphorylation of AKT and ERK in pericytes. ß-Tubulin marks the loading level in each lane. These experiments were repeated twice. (H) qPCR quantification of Cxcl14 mRNA levels in vehicle- and FGF-2–stimulated pericytes in the presence or absence of MEK1/2, ERK1/2, and AKT specific inhibitors (n = 3 samples per group). (I) Volcano plot of predicted transcription factors which bind to Cxcl14 promoter in genome-wide expression profiling of vehicle- and FGF-2–stimulated pericytes (n = 3 samples per group). (J) qPCR quantification of Cxcl14 mRNA levels in vehicle- and FGF-2–stimulated pericytes in the presence or absence of Control or Ahr-specific siRNA (n = 3 samples per group). (K) ChIP assay of AHR binding to the Cxcl14 gene promoter. Nonimmune IgG and Cxcl14 exon 2 regions served as controls (n = 3 samples per group). (L) Mechanistic diagram of the FGF-2/FGFR1/ERK/AHR/CXCL14 signaling pathway. **P < 0.01, ***P < 0.001 by unpaired 2-tailed Student’s t test (C–E and K) or 1-way ANOVA with Tukey’s multiple-comparison analysis (F, H, and J). Data are presented as mean ± SD.

Fig 4: FGF-2 is distinctively expressed and correlates with TAM infiltration in human NPC.(A) Cross–data set quantitative heatmap of selected genes of various types of cancer and their adjacent control healthy tissues. Arrow points to distinctively upregulated genes in NPC. Log2 fold changes were used for quantification. (B) Transcriptomic expression levels of FGF2 in human LUAD tissues, BRCA tissues and their adjacent healthy tissues. Sample number: control-LUAD/LUAD/control-BRCA/BRCA=347/483/291/1085. (C) Transcriptomic expression levels of FGF2 in various stages of human NPC tissues and their adjacent healthy tissues. Sample number: control/StageT1/StageT2/StageT3=10/16/11/4. (D) Human normal nasopharyngeal tissues (NNT), rhinitis tissues, and NPC tissues were stained with H&E and an anti–FGF-2 antibody (brown). Sample number: NNT/Rhinitis/NPC=3/10/6. Scale bar in upper panel: 500 µm. Scale bar in middle and lower panels: 50 µm. Quantification of FGF-2+ signals and FGF-2+ signals in stromal and epithelial components (n = 8 random fields per group). (E) NPC cancer cells were sorted by MACS from freshly tissues. qPCR quantification of FGF2 mRNA (n = 3 samples per group). (F) NNT rhinitis tissues and NPC tissues were stained. Sample number: NNT/Rhinitis/NPC=3/10/6. Scale bar in upper and middle panels: 50 µm. Scale bar in lower panel: 100 µm. Quantification of FSP1+ (brown), CD163+ (brown), CD31+ (red), and NG2+ (green) and coverage rate of NG2+ pericytes (n = 8 random fields per group). (G) qPCR quantification of FGF2, CD163, CD31, NG2, and FSP1 mRNA in freshly collected tissues. Sample number: Rhinitis/NPC=5/6. (H) Correlation of FGF2 and CD163 expression of human NPCs and their control healthy tissues. Sample number: Control/NPC=10/31. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired 2-tailed Student’s t test (B, D, E, G, and H) or 1-way ANOVA with Tukey’s multiple-comparison analysis (C, D, and F). Data are presented as mean ± SD.

Fig 5: CYN blocks the activation and development of liver fibrosis in vitro. (A) Evaluation of the inhibitory effect of CYN on the fibrosis-promoting effect of FGFR2. The fibrotic transformation of wild-type and FGFR2-OE LX-2 cells and Huh-7 cells was induced through TGF-ß activation, followed by an intervention with CYN for the relevant groups. The expression changes of the fibrosis markers ACTA2 and COL1A1 were analyzed by qPCR. (B) Wild-type cell lines were employed in the aforementioned experiments, and the activation of FGFR2 was triggered by supplementation with the exogenous basic fibroblast growth factor (bFGF) factor. (C) Analysis of the extent of antagonism of CYN towards TGF-ß Signaling. Activation induction models were established by adding or not adding TGF-ß to the cell culture environment with or without the CYN intervention, and the expression of liver fibrosis markers was determined by qPCR, (D) Western blot, and (E,F) ELISA analyses. (G) A co-culture model was used to evaluate the blocking effect of CYN on liver fibrosis activation of signaling transmission. The activation intensity of lower-layer wild-type cells was measured and compared using a-SMA expression and collagen secretion. The results are marked as significant “*” when p < 0.05, “**” when p < 0.01, and not significant (ns) if p = 0.05.