Fig. 2: Netrin-1 expression in metaplastic cells induces sympathetic axon remodeling.

a-c, Representative immunohistochemistry (IHC) staining for Netrin-1 and Iba1 (a pan-macrophage marker) in sections of healthy pancreas (a), and cerulein-treated pancreas (b: adjacent asymptomatic tissue; c: metaplasia). Yellow arrows indicate enlarged macrophages in the insets. DAPI for nuclear staining. Scale bar: 20 μm and 5 μm (inset).

d, Quantification of Netrin-1 fluorescence labeling in the exocrine pancreas of healthy (H) mice, metaplastic lesions (M) and adjacent exocrine tissue (A) of cerulein-treated mice. Data are presented as mean ± SEM. n = 15 ROIs from 3 healthy mice, 15 metaplastic, and 20 adjacent ROIs from 3 cerulein-treated mice. p-values are indicated on the graph (Kruskal-Wallis test).

e, Quantification of the proportion of Iba1⁺ macrophages expressing Netrin-1 in the healthy (H) pancreas, metaplastic lesions (M) and adjacent exocrine tissue (A) of cerulein-treated mice. Data are presented as mean ± SEM. n = 18 ROIs from 3 healthy mice, 18 metaplastic, and 13 adjacent ROIs from 3 cerulein-treated mice. p-values are indicated on the graph (Kruskal-Wallis test).

f-i, Representative images of TH⁺ sympathetic neurons from the CSMG cultured in PBS (vehicle) or recombinant Netrin-1 protein, together with an anti-Netrin-1 blocking antibody (αNetrin-1) or an isotype control antibody (Iso-antibody). Scale bar: 50 μm.

j, Quantification of total neurite length in cultures shown in (f–i). Data are presented as mean ± SEM and normalized to 1 for the Netrin-1 condition. n = 94 (vehicle), 132 (Netrin-1), 94 (Netrin-1 + αNetrin-1), and 89 (Netrin-1 + Iso-antibody) neurons from four independent experiments. p values indicated (ns: p > 0.05) (Kruskal–Wallis test).

k, Experimental design for cerulein injections and subsequent treatments with αNetrin-1or Iso-antibody.

l-m, Maximum intensity projection of 3D images of TH⁺ sympathetic axon networks in CK-19+ metaplastic pancreatic lesions treated with Iso-antibody (l) or αNetrin-1 (m). Right panels show 3D reconstruction of sympathetic axons and branching points (purple dots) within the yellow-segmented volume. Scale bar: 30 μm.

n-o, 3D quantification of TH⁺ sympathetic axon density (n) and branching (o) in metaplastic lesions (M) treated with αNetrin-1 (αNet) or Iso-antibody (Iso). Data are presented as mean ± SEM. n = 6 ROIs from 3 mice per condition. p-values indicated (Mann–Whitney test).

p, Experimental design for cerulein treatment of LysM-Cre–Ntn1^lox/lox and control mice.

q-r, Representative IHC staining for TH and SOX9 in metaplastic lesions (q’ and r’) and adjacent tissue (q and r) of cerulein-treated LysM-Cre–Ntn1^lox/lox and control mice. DAPI for nuclear staining. Scale bar: 25 μm.

s, Quantification of TH⁺ sympathetic axon density in metaplastic pancreatic lesions (M) and adjacent tissues (A) of cerulein-treated control (ctr) and LysM-Cre–Ntn1^lox/lox (cKO) mice. Data are presented as mean ± SEM. n = 25 metaplastic and 13 adjacent ROIs from 3 control mice; 26 metaplastic and 16 adjacent ROIs from 3 cKO mice. p values indicated (ns: p > 0.05) (Kruskal–Wallis test).

t, Experimental design for cerulein treatment of Pdx1-Cre–Ntn1^lox/lox and Cre-negative control mice.

u-v, Representative IHC staining for TH and Sox9 in metaplastic pancreatic lesions (u’ and v’) and adjacent exocrine tissue (u and v) of cerulein-treated control and Pdx1-Cre–Ntn1^lox/lox mice. DAPI for nuclear staining. Scale bar: 25 μm.

w, Quantification of TH⁺ sympathetic axon density in metaplastic pancreatic lesions (M) and adjacent tissues (A) of cerulein-treated control (ctr) and Pdx1-Cre–Ntn1^lox/lox (cKO) mice. Data are presented as mean ± SEM. n = 28 metaplastic and 15 adjacent ROIs from 3 control mice; 26 metaplastic and 14 adjacent ROIs from 3 cKO mice. Significant p values are indicated on the graph (ns: p > 0.05) (Kruskal–Wallis test).