Fig 2: Increase in M2 macrophages proximal to nerves in human PDA–expressing SEMA3D.(A) Percentages of M1 or M2-like TAMs distal or proximal to nerves in human PDA. (B and C) Percentages of M1 (B) or M2-like (C) TAMs distal or proximal to nerves in SEMA3D-positive and SEMA3D-negative human PDA samples. (D) Representative images of multiplex immunohistochemical staining of SEMA3D-negative and SEMA3D-positive human PDA samples in areas proximal or distal to nerves, respectively. Nerves (N) identified with anti-TUJ1 staining and immune cells identified with anti-CD45, CD163, CSF1R, and CD68 staining. An amplified region in each panel was shown (white box). The data were presented as means ± SEM and compared by unpaired t test; *P < 0.05 and ****P < 0.0001; all others, not significant.

Fig 3: SEMA3D reprograms macrophages indirectly though ARF6 signaling and lactate production in PDA tumor cells.(A) PLXND1 protein expression in KPC tumor cells (KPC), KPCS tumor cells (KPCS), M0, M1, and M2 macrophages, respectively. (B) Western blot analysis of the expression of ARF6 and active ARF6-GTP protein in CTRL-AP–and SEMA3D-AP–treated KPC tumor cells. (C) qRT-PCR analysis of the expression of LDHB, LDHC, and LDHD genes in CTRL-AP–or SEMA3D-AP–treated KPC cells first pretreated with anti-PLXND1 neutralizing antibody (Ab) or isotype immunoglobulin G (IgG) control Ab. (D) Lactate concentration in the supernatant from KPC and KPCS cells treated with CTRL-AP or SEMA3D-AP, respectively. (E and F) qRT-PCR analysis of gene expression markers for M1 (E) and M2 (F) polarization, respectively, in BMDM cells polarized with M1- or M2-polarizing cytokines and subsequently cocultured with CTRL-AP–or SEMA3D-AP–treated KPC cells in the double chamber. (G) qRT-PCR analysis of gene expression markers for M1 and M2 polarization in polarized M1 or M2 macrophages after coculture with KPC or KPCS cells that have been treated with CTRL-AP or SEMA3D-AP recombinant protein, respectively. The data were presented as means ± SEM and compared by unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; all others, not significant. GAPDH, glyceraldehyde phosphate dehydrogenase.

Fig 4: KPCS mouse model demonstrates a delayed tumor initiation and growth compared to KPC mouse model.(A) Representative SEMA3D and H&E immunohistochemical staining of PanIN and invasive PDA samples from KPC (n = 17) and KPCS (n = 11) mice with percentage of mice expressing SEMA3D-positive tumor cells. (B) Percentage of KPC and KPCS mice with normal, PanIN1, PanIN2, PanIN3, or PDA at various ages. KPC mouse sample sizes were n = 4, 6, 7, 9, 12, and 6 for weeks 4, 8, 12, 16, 24, and 32, respectively. KPCS mouse sample sizes were n = 2, 2, 5, 6, 8, and 6 for weeks 4, 8, 12, 16, 24, and 32, respectively. (C) Time of first tumor detection in KPC (n = 27) and KPCS (n = 29) mice. (D) Spaghetti plots of tumor volumes measured by the small animal ultrasound for KPC (n = 19) and KPCS (n = 29) mice. (E) Tumor size of KPC and KPCS mice at age of 32 weeks. (F) Predicted tumor growth curves for KPC and KPCS mice using log-transformed tumor volumes measured by ultrasound. The data were presented as percentage of mice or means ± SEM and compared by a chi-square or unpaired t test; *P < 0.05 and ***P < 0.001.

Fig 5: Nerve-derived SEMA3D affects macrophage polarization in vitro.(A) ELISA of secreted SEMA3D from media cultured with and without DRG cells. (B) Western blot and densitometry analysis of ARF6 and active ARF6-GTP proteins in KPC cells treated with control and DRG-conditioning media. ARF6-GTP/ARF6 densitometry analysis based on corresponding Western blot, and values were normalized to GAPDH expression. (C) Western blot and densitometry analysis of ARF6 and active ARF6-GTP proteins in KPC cells treated with CTRL-AP, SEMA3D-AP, or the DRG-conditioning media supplemented with anti-PLXND1 neutralizing Ab or isotype IgG control. ARF6-GTP/ARF6 densitometry analysis based on corresponding Western blot, and values were normalized to GAPDH expression. (D) qRT-PCR analysis of gene expression of GLUT-1, HK-2, and LDHA in KPC cells treated with CTRL-AP, SEMA3D-AP, or the DRG-conditioning media supplemented with anti-PLXND1 neutralizing Ab or isotype IgG control. (E) qRT-PCR analysis of gene expression markers for M1 and M2 polarization in M1- and M2-polarized macrophages, as indicated, after coculture with KPC or KPCS cells treated with control or DRG-conditioning media. The data were presented as means ± SEM and compared by unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; all others, not significant.