Fig 2: (A) Weight of WT and CSf1op/op mice (P = NS; Mann–Whitney test; N = 7 mice for each group). (B and C) Major histocompatibility complex class II (MHCII) macrophages (green) and Protein gene product 9.5 (PGP 9.5) fibers in smooth muscular layers (upper panels) and myenteric plexus (lower panels). The small panels show orthogonal views generated by projecting the z-series in the x (right) and on the y plane (above). Arrows point to macrophage/fiber interactions and squares show macrophage/fiber interactions in orthogonal views. PGP 9.5 immunoreactivity was unusually bright in the cell bodies of myenteric neurons in CSf1op/op mice when compared with WT tissues. Scale bars: (B) 20 µm, (C) 10 µm.

Fig 3: IHC assessment of hind paw and forelimb samples after MC38 injection. a Changes in IENF density with time in the hind paws of control vs MC38-injected mice. Colon cancer initiates a significant decrease in IENF density after the 3rd week, indicating significant neuronal damage. (**: p < 0.01; n = 9–13/group, two-way ANOVA with Šídák's multiple comparisons test). b Representative images of hind paw samples, collected 3 weeks after inoculation. White arrows mark the counted intraepidermal nerve fibers, stained with PGP9.5 antibody (green). Nuclei are stained with DAPI (blue). Scale bar: 100 µm c Changes in IENF density, 3 weeks after inoculation, in the forelimb samples of control vs MC38-injected mice. Similarly to the hind paw samples, our data reveal a significant decrease in IENF density, 3 weeks after local MC38 cell injection (two-tailed unpaired t test). d Representative images of forelimb samples, collected 3 weeks after tumor inoculation. White arrows mark the counted intraepidermal nerve fibers marked by PGP9.5 antibody. Nuclei are stained with DAPI. e Signal intensity of the CD68 macrophage marker in the skin of control vs MC38-injected mice, depicted as binary area/measured area. Based on the CD68 marker, there is no significant change in the number of macrophages in the epidermis, around the intraepidermal nerve fibers (p = 0.75, two-tailed unpaired t test). f Representative images of hind paw samples show no change in the CD68 signal intensity of the epidermis. Nuclei are stained with DAPI. Scale bar: 100 µm