Extended Data Fig. 4: Chemogenetic inactivation of LS^GABA neurons inhibits colorectal tumorigenesis.

(a-b) Viral targeting validation. (a) Co-localization of mCherry⁺ and GFP⁺ neurons in LS after AAV-DIO-mCherry injection in GAD67-GFP mice. n = 2 mice. (b) CNO-induced c-Fos’s expression in hM3Dq⁺ LS neurons (arrowheads; n = 3 mice per group). (c-k) CDX model studies. (c-e) Chemogenetic activation of LS^GABA neurons via hM3Dq/CNO. Tumor morphology (c), weight (d), and volume (e). Saline, n = 3 mice; CNO, n = 5 mice. (f-h) Control virus injection in LS. Tumor morphology (f), weight (g), and volume (h). n = 3 mice per group. (i-k) Chemogenetic activation of LS^GABA neurons via hM3Dq/DCZ. Tumor morphology (i), weight (j), and volume (k). Control, n = 5 mice; hM3Dq, n = 4 mice. (l-o) Chemogenetic inactivation of LS^GABA neurons in male AOM/DSS model. NF-L⁺ nerve fibers in tumors (l), tumor morphology (m), number (n), and area (o). Red boxes: tumor regions. n = 8 mice per group. (p-t) Apc^Min/+ model. (p-q) HSV-eGFP tracing from LS in male Apc^Min/+ mice at 24 weeks old. eGFP⁺ LS neurons (p) and tumor nerve fibers (q) at 48 hours post-injection. n = 3 mice. (r-t) Chemogenetic inactivation of LS^GABA neurons in Apc^Min/+ model with CNO intraperitoneal injection once every week for 12 times. Tumor morphology (r), number (s), and area (t) in male Apc^Min/+ mice at 24 weeks old. n = 7 mice per group. Data are presented as mean ± SEM. Two-tailed unpaired student’s t test (b, g-h, k, n, t), two-tailed unpaired student’s t test with Welch’s correction (d, s), and two-tailed Mann Whitney test (e, j, o) are used. n.s., not significant.

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