Fig 2: CXCL14 recruits, activates, and polarizes TAMs.(A and B) Mouse macrophage migration (n = 8 samples per group) and chemotactic ability (n = 6 samples per group) of macrophage treated with or without CXCL14. (C) Quantification of CD45+ cells in xenograft shScrambled- and shFGF2-transfected NPC tumors (n = 5 samples per group). (D) Pie charts of percentage of various inflammatory cells in xenograft shScrambled- and shFGF2-transfected NPC tumors (n = 5 samples per group). CD45+CD11b+F4/80+ macrophage population, CD45+MHCII+CD11b+CD11c+ DC population, CD45+CD11b+Ly6GhiLy6Cint granulocytic subsets of myeloid-derived suppressor cell population, CD45+CD11b+Ly6G–Ly6C+ monocytic subsets of myeloid-derived suppressor cell population, CD45+B220+ B cell population, and CD45+CD11b–CD49b+ NK cell population were analyzed. (E and F) Quantification of CD45+CD11b+F4/80+ TAM population, CD45+CD11b+ F4/80+CD206+ M2-like TAM population, and CD45+CD11b+F4/80+CD86+ M1-like TAM population (n = 5 sample per group). (G) qPCR quantification of CD206 and CD86 mRNA levels in F4/80+ TAMs isolated from xenograft shScrambled- and shFGF2-transfected NPC tumors (n = 3 samples per group). (H) Tumor tissues were stained with an anti-CD206 antibody (brown). Scale bar: 50 µm. Quantification of CD206+ signals (n = 8 random fields per group). (I and J) qPCR quantification of CD206 and CD86 mRNA levels in macrophages that were activated with FGF-2–treated pericyte conditioned medium or CXCL14. Vehicle- and FGF-2–stimulated macrophages serve as controls (n = 3 samples per group). (K) CXCL14- or FGF-2–treated pericyte conditioned medium–induced CD206 upregulation and CD86 downregulation in macrophages. ß-Actin marks the loading level in each lane. These experiments were repeated twice. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired 2-tailed Student’s t test (A–C and E–J). Data are presented as mean ± SD.

Fig 3: 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 4: 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.