Fig. 2. Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons.

a–c, Naive DRG neurons (Trpv1^cre::-CheRiff-eGFP^fl/WT), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8⁺ T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a, Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b, DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8⁺ T cells, DRG neurons or both populations. c, SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8⁺ T cells, with a maximal effect after 48 h. d–f, Using calcium microscopy, we found that SLPI (10 pg ml⁻¹–10 ng ml⁻¹) activated around 20% of cultured naive DRG neurons (d,e). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP (f). Data are shown as a heat map showing normalized gene expression (log₂(1 + TPM) − mean (a), as box-and-whisters plots (as defined in Fig. 1b,c) (b) or as mean ± s.e.m. (c–f). n as follows: a,b: n = 2–4 per groups; c: n = 3 for all groups except CD8⁺ T cells (n = 8); d: n = 17; e: n = 8 per group; f: 0 ng ml⁻¹ (n = 4), 0.1 ng ml⁻¹ (n = 5), 1 ng ml⁻¹ (n = 5), 5 ng ml⁻¹ (n = 4). Experiments in c–f were independently repeated three times with similar results. The sequencing experiment was not repeated (a,b). P values were determined by one-way ANOVA with post-hoc Bonferroni (b,e,f) or two-sided unpaired Student’s t-test (c). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

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