Extended Data Fig. 10: Nociceptive neurons activate Rb/E2F signalling depending on CaMK and PI3K.

(a) Neuron-connected spheroids and unconnected spheroids were split. Expression levels of chemical synaptic genes, electrical synaptic genes and neurodevelopmental genes were analysed (n = 3 experiments). (b) Concentrations of CGRP peptide in orthotopic ACKP-ChR2 tumours from Trpv1-Cre; GCamp6s; tdTomato mice (n = 3 mice/group). (c) PCA plot of bulk RNA sequencing data from cancer spheroids alone, cancer spheroids cocultured with DRG, and cancer spheroids cocultured with CNO-activated DRG (n = 3 experiments/group). (d) GSEA plot of top 20 enhanced pathways in each comparison. (e) Heat map of proliferation genes, Rb-E2F signal enhancer genes and Rb-E2F signal repressor genes. (f) Relative E2F activity in ACKP cells treated with CGRP or kinase inhibitors. Wortmannin, PI3K inhibitor (n = 3 experiments/group). KN-93, CaMK inhibitor. ASN007, ERK inhibitor. ST034307, PKA inhibitor. (g) Representative images and (h) quantification of p-Rb staining in orthotopic tumours from CNO-treated mice (n = 5 mice/group). Scale bar, 100 μm. (i) Representative images and correlation test of sensory nerve and p-Rb staining in human GC tissue microarray (n = 50 cases). Scale bar, 100 μm. (j) Graphical abstract: Gastric cancer is innervated by nociceptive neurons from ipsilateral jugular nucleus complex and dorsal root ganglia (T7-T13). Gastric cancer cells increase the expression of NGF which attracts the expansion of nociceptive nerves. NGF binds to TrkA receptor causing CGRP synthesis and release from nociceptive nerves. In turn, CGRP activates Calcrl/Ramp1 receptor and promotes E2F activity through PI3K signalling and CaMK signalling in gastric cancer cells. Data represent mean ± SEM, and P values were calculated by ANOVA in f, by t test in b and h, and by spearman’s rank correlation test in i. The statistical tests were two-sided. The diagram in j was created using BioRender.

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