Fig 2: DIO increased expression level of CX3CL1 and CX3CR1 in liver metastasis of PDAC. (a) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106 Panc02 cells (n = 6/group). (b) Representative immunohistochemical images of CX3CL1 staining in liver of ND and DIO. Scale bar, 20 µm. (c) Bar graph showed the relative CX3CL1 staining positive rate. (d) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106 Panc02 cells (n = 6/group). (e) IHC staining images of CX3CR1. Scale bar, 20 µm. (f) Bar graph showed the relative CX3CR1 staining positive rate. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: Down-regulation of CX3CR1 with siRNA attenuated the M1 polarization of macrophages induced by hypoxia/reoxygenation. (A and B) Representative Western blots and summarized data showing the protein levels of CX3CR1, TNF-a, and ARG-1 in different groups of RAW264.7 macrophages. (C) The expression analysis of CD80 and CD206 by flow cytometry in different groups of RAW264.7 macrophages. aP < 0.05, b P < 0.01 vs Control; cP < 0.05, dP < 0.01 vs H/R; eP < 0.05, fP < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein used was 50 ng/mL.

Fig 4: CX3CL1 promoted recruitment of CX3CR1-positive pancreatic tumor cells. (a) mRNA levels of CX3CR1 in PDAC cells. (b) Western blot analysis of CX3CR1 in human PDAC cell lines. (c) Flow cytometry analysis of the surface expression level of CX3CR1 in different human cell lines. (d)The expression level of soluble CX3CL1 was examined by using ELISA. Protein samples were obtained from the mouse liver. (e) Effect of CX3CL1 on the migratory ability of mouse PDAC cell lines Panc02; relative transferred cell numbers were analyzed. Cell migration experiments in blocker group were performed by using the blocking antibody of CX3CL1. The neutralization dose (ND50) is 1 µg/ml in the presence of 30 ng/ml mouse recombinant CX3CL1. Scale bar 10 µm. (f) Effect of CX3CL1 on the migratory ability of human PDAC cell lines AsPC-1 and Capan-1. Different concentration of CX3CL1 recombinant protein was used in cell transwell experiments; relative transferred cell numbers were analyzed. The neutralization dose (ND50) is 3 µg/ml in the presence of 100 ng/ml human recombinant CX3CL1. Scale bar 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 5: Hypoxia/reoxygenation increased the protein expressions of TNF-a, CX3CL1, and CX3CR1, and inhibited the protein expression of ARG-1 in a time-dependent manner in RAW264.7 cells. (A and B) The proteins of RAW264.7 macrophages were tested for TNF-a, ARG-1, CX3CL1, and CX3CR1 at different hypoxia times. (C) The protein expression of TNF-a and ARG-1 in RAW264.7 macrophages under hypoxia/reoxygenation combined with different concentrations of CX3CL1 recombinant protein. (D, E, and F) The quantitative analysis of A, B and C, respectively. aP < 0.05, bP < 0.01 vs control or H/R. H/R: hypoxia/reoxygenation, hypoxia at 2, 4, 6 or 8 h and then reoxygenates for 24 h.