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. 2022 Nov 2;611(7935):405–412. doi: 10.1038/s41586-022-05374-w

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — (a–c) When co-cultured with B16F10-eGFP cells (green), TRPV1⁺ nociceptor (Trpv1^cre::tdTomato^fl/WT; orange) neurons form neuro-neoplasic contacts (a), show longer neurites (b), and exhibit reduced arborization (c) than when cultured alone or with non-tumorigenic keratinocytes (b–c). (d) L3–L5 DRG neurons were collected from mice 2-weeks after they were inoculated (left hindpaw; i.d.) with B16F10- or non-tumorigenic keratinocytes, cultured and calcium flux to ligands tested (ATP (10 μM), and capsaicin (1 μM)). Compared to neurons from keratinocytes-injected mice, the one from tumour-bearing mice showed increased sensitivity to capsaicin. (e) Naive DRG neurons (Trpv1^cre::CheRiff-eGFP^fl/WT) were cultured alone or in combination with B16F10-mCherry-OVA. After 48h, the cells were collected, FACS purified, and RNA sequenced. Hierarchical clustering of sorted neuron DEG show distinct groups of transcripts enriched in TRPV1⁺ neuron vs cancer-exposed TRPV1⁺ neuron populations. Pairwise comparison of naive TRPV1⁺ neuron vs cancer-exposed TRPV1⁺ neuron populations showing differentially expressed transcripts as a volcano plot (p<0.05). Among others, Calca (gene encoding for CGRP) was overexpressed in TRPV1⁺ (FACS-purified eGFP-expressing cells) neurons when co-cultured with B16F10-mCherry-OVA. Data are shown as representative image (a), mean ± S.E.M (b–d), or volcano plot (e). N are as follows: a: n = 4, b: neuron (n=8), neuron + keratinocytes (n = 7), neuron + B16F0 (n = 7), neuron + B16F10 (n = 7), c: n = 15/groups, d: keratinocytes inj. + ATP (n=5), B16F10 inj. + ATP (n=36), Keratinocytes inj. + caps (n = 6), B16F10 inj. + caps (n = 44), e: n = 4/groups. Experiments were independently repeated two (d) or three (a–c) times with similar results. Sequencing experiment was not repeated (e). P-values are shown in the figure and determined by one-way ANOVA post-hoc Bonferroni (b–c) or two-sided unpaired Student’s t-test (d). Scale bar = 100 μm (a).

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