Extended Data Fig. 6: GABA signaling mediates neuron-tumor interactions.

(a) Coculture of DRG/enteric neurons with SW480/CMT93-GFP cells. Tuj1⁺ neurons and GFP⁺ tumor cells imaged. n = 3 biological replicates. (b) TEM images of CMT93 rectal tumors. n = 12 ultra-thin sections from 3 mice. (c) sEPSCs/sIPSCs in CMT93-GFP/GL261 cells implanted in mPFC. n = 6 cells from 3 mice for each recording. (d) DRG-conditioned medium effects on SW480 (n = 9, 12 biological replicates for Control and DRG medium respectively)/CMT93 (n = 9 biological replicates per group) viability. (e) BTXA-treated DRG-conditioned medium effects on SW480 (n = 9 biological replicates per group)/CMT93 (n = 14 biological replicates per group) viability. (f-i) ACh (f, SW480; g, CMT93; n = 5 biological replicates per group) and glutamate (h, SW480, n = 10 biological replicates for Control, 5 μM, 10 μM, 25 μM, 50 μM and 7 biological replicates for 100 μM; i, CMT93, n = 10 biological replicates for Control, 5 μM, 10 μM, 25 μM, 50 μM and 8 biological replicates for 100 μM) effects on cell viability. (j-k) GABA level in DRG medium (j, n = 6 biological replicates per group)/BTXA-treated DRG medium (k, n = 5 biological replicates per group). (l-p) GABA level in rectal tumors after inhibiting (l, n = 5 mice per group)/activating (m, n = 5, 7 mice for Control and hM3Dq respectively) LS^GABA neurons, inhibiting enteric neurons with BTXA (n, C57BL/6 J mice, n = 8, 6 mice for Saline and BTXA respectively; o, Nude mice, n = 6, 5 mice for Saline and BTXA respectively), or chemogenetically modulating enteric cholinergic neurons (p, n = 5, 4, 6 mice for Control, hM4Di and hM3Dq respectively). (q-s) GABA supplement in male C57BL/6 J CDX model. Tumor morphology (q), weight (r), volume (s). n = 5, 6 mice for BTXA+Saline, BTXA + GABA respectively. Green dashed line: tumor margin. (t-v) GABA concentration in ChA-treated DRG medium (t, n = 5 biological replicates per group). ChA (u, n = 6 biological replicates per group) and ChA-treated DRG medium effects on CMT93 viability (v, n = 10, 9 biological replicates for DRG medium, ChA-treated DRG medium respectively). (w-y) ChA treatment in male C57BL/6 J CDX model. Tumor morphology (w), weight (x), volume (y). Vehicle, n = 8 mice; ChA, n = 5 mice. Data are presented as mean ± SEM. Two-tailed unpaired student’s t test (d, SW480 cells in e, j-l, o-p, t, v), two-tailed Mann Whitney test (CMT93 cells in e, m-n, r-s, x-y), and Ordinary one-way ANOVA with Tukey’s multiple comparisons test (f-i, u) are used. n.s., not significant.

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