Tumor-infiltrating nociceptor neurons promote immunosuppression

Abstract

Small extracellular vesicles (sEVs) released from tumors recruit nociceptor neurons to the tumor bed. Here, we found that ablating these neurons in mouse models of head and neck carcinoma and melanoma reduced the infiltration of myeloid-derived suppressor cells (MDSCs). Moreover, sEV-deficient tumors failed to develop in mice lacking nociceptor neurons. We investigated the interplay between tumor-infiltrating nociceptors and immune cells in head and neck squamous cell carcinoma (HNSCC) and melanoma. Upon exposure to cancer-derived sEVs, mouse dorsal root ganglion (DRG) neurons secreted increased amounts of substance P, IL-6, and injury-associated neuronal markers. Patient-derived sEVs sensitized DRG neurons to capsaicin, implying enhanced nociceptor responsiveness. Furthermore, nociceptors cultured with sEVs induced an immunosuppressed state in CD8⁺ T cells. Incubation with conditioned medium from cocultures of neurons and cancer cells resulted in increased expression of markers of MDSCs and suppressive function in primary bone marrow cells, and the combination of neuron-conditioned medium and cancer sEVs promoted checkpoint receptor expression on T cells. Together, these findings reveal that nociceptor neurons facilitate CD8⁺ T cell exhaustion and bolster MDSC infiltration into HNSCC and melanoma. Consequently, targeting nociceptors may provide a strategy to disrupt detrimental neuroimmune cross-talk in cancer and potentiate antitumor immunity.

Figure 1.. Small extracellular vesicle (sEV)-mediated tumor innervation is essential for tumor growth.

Eight-week-old male wild-type mice or nociceptor neuron-ablated (Trpv1^cre::DTA^fl/wt) mice were injected orthotopically in the oral cavity with 10⁵ mEERL or mEERL Rab27^−/− cells. Data are presented as means ± SEM. Statistical differences were calculated using two-way ANOVA with Tukey post hoc test and are denoted by ** indicating p < 0.01 and *** indicating p < 0.0001. n = 5 mice per group. Representative data of three independent experiments.

Figure 2.. Head and Neck Squamous Cell Carcinomas Alters the Transcriptome of Tumor-Infiltrating Neurons.

Eight-week-old male wildtype mice were orthotopically injected in the oral cavity with 10⁵ mEERL cells. On day 25 post-tumor implantation, the ipsilateral and contralateral trigeminal ganglia were harvested, neurons purified, and their expression profile assessed using qPCR. Gene expression was normalized to the housekeeping gene GusB (A) or α-actin (B). A second cohort of mice underwent the same initial procedure. On day 25 post-tumor implantation, ipsilateral and contralateral trigeminal ganglia were harvested, fixed, and the expression of ATF3 and Substance P (SP) proteins were assessed using immunofluorescence staining (C-D). (E) In a third, similar experiment, tumor-bearing mice were euthanized on day 20 post-tumor implantation. Ipsilateral and contralateral trigeminal ganglia were harvested, lysed, and the lysate analyzed by ELISA for Substance P. (A-E) Data are presented as means ± SEM. Statistical differences were calculated using Student's t-test. Representative data from two independent experiments. n as follows: A: n = 4 per group; B: n = 3 per group; C, D: n = 6 per group; E: n = 3 per group. Scale bar, 100 μm.

Figure 3.. mEERL cancer cells promote nociceptor neuron release of IL-6.

(A) Trigeminal ganglia (TGM) neurons from wildtype male C57BL/6 mice were harvested and cultured in the presence or absence of sEV competent or compromised (Rab27^−/−) mEERL cancer cells. After 48 hours, the conditioned media were harvested and substance P (SP) release measured using a commercial ELISA. (B) mEERL cancer cells were cultured and stimulated with recombinant mouse SP (50 nM) in the presence of the NK-1R antagonist aprepitant (100 μM) or its vehicle. After 48h culture, the conditioned media were harvested, and IL-6 release measured using a commercial ELISA. (C) Wildtype male murine TGM neurons were harvested and cultured in the presence or absence of sEV competent or compromised (Rab27^−/−) mEERL cancer cells. After 48h, the cells were exposed to the NK-1R antagonist, aprepitant (100 uM) or its vehicle and the conditioned media harvested. IL-6 release was measured using a commercial ELISA. (D) DRG from wildtype or IL-6 knockout (IL-6^−/−) male mice were harvested and cultured in the presence or absence of sEV competent or compromised (Rab27^−/−) mEERL cancer cells. After 48h, the conditioned media were harvested, and IL-6 release measured using a commercial ELISA. (E) Wildtype TGM were harvested and cultured alone or co-cultured with increasing numbers of mEERL cells. The conditioned media were harvested 48h later and analyzed using a commercial IL-6 ELISA. Data are presented as means ± SEM. Statistical differences were calculated using one-way ANOVA with a post hoc Tukey test to compare means between groups. n = 3–6 mice per group. Representative data from n=3 independent experiments with n=4 biological replicates for each.

Figure 4.. HNSCC-innervating nociceptor neurons promote MDSCs infiltration.

(A). Eight-week-old male wildtype mice or nociceptor neuron-ablated (Trpv1^cre::DTA^fl/wt) mice were injected orthotopically in the oral cavity with 10⁵ mEERL cells. Twenty-five days post-tumor implantation, tumors were harvested and analyzed by flow cytometry. (B) Additional markers were used to distinguish between subsets of MDSCs. Data are presented as means ± SEM. P values were determined by two-sided unpaired Student’s t-test. Experiments were independently repeated two times with similar results. n as follows: A-B: n = 4 per group.

Figure 5.. Melanoma-innervating nociceptor neurons promote MDSCs infiltration.

Eight-week-old male and female littermate control (Trpv1^wt::DTA^fl/wt) and nociceptor neuron-ablated (Trpv1^cre::DTA^fl/wt) mice were injected intradermally in the left flank with 2x10⁵ B16F10-OVA melanoma cells. Tumor growth was monitored (A). On day fifteen post-tumor implantation, the tumor was harvested, and immune infiltrate profiled using a 12-color flow cytometry antibody panel (B). In a separate experiment, monocytic MDSC (defined as CD11b⁺Ly6G^highLy6C⁻) from nociceptor intact and ablated mice were FACS-purified, transcriptome analyzed by RNA sequencing and differentially expressed genes calculated. Data are presented as means ± SEM (A, B), heatmap showing normalized gene expression (log₂ (0.01 + transcripts per million reads (TPM)) − mean (C) or volcano plot (p < 0.05; D). P values were determined by two-sided unpaired Student’s t-test (A, B). Experiments were independently repeated two (A, B) times with similar results. The sequencing experiment was not repeated (C, D). n as follows: A, B: n = 8 per group; C, D: n = 4 per group.

Figure 6.. TRPV1⁺ neurons promote the differentiation of myeloid-derived suppressor cells (MDSCs).

(A) Schematic of experiments. (B-D) Bone marrow-derived cells were harvested from wildtype mice and were treated with conditioned media from cultured nociceptor intact (Trpv1^wt::DTA^wt/wt) trigeminal (TGM) neurons, cultured nociceptor ablated (Trpv1^cre::DTA^fl/wt) TGM neurons, sEV-competent mEERL cells or sEV-compromised (Rab27^−/−) mEERL cells, either alone or in combination with neurons. Four days after treatment, the cells were harvested and immunophenotyped using flow cytometry (B), had their transcriptome profiled using qPCR (C) or migration assessed (D). Data are presented as schematics (A), or as mean ± SEM (B-D). Statistical differences were determined by one-way ANOVA with a post-hoc Tukey (B-D). Experiments were independently repeated two (B-D) times with similar results. n are as follows: B: n = 4 per group; C, D: n = 3 per group.

Figure 7.. Nociceptor neuron and mEERL-derived small extracellular vesicles impair CD8⁺ T-cells function.

(A-C) Splenocyte-derived CD8⁺ T cells were cultured under T_c1-stimulating conditions (ex vivo activated by anti-CD3 and anti-CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured alone for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8⁺ T cells were challenged (1:2 dilution) with conditioned medium from vehicle, fresh KCl-induced wildtype DRG neurons, sEV-purified from mEERL cells, or a combination of DRG neurons with mEERL sEVs. T cells were analyzed by flow cytometry for the indicated markers. Data are presented as mean ± SEM. Statistical differences were determined by one-way ANOVA with a post-hoc Tukey. Experiments were independently repeated three times with similar results. n = 4–5 per group.

Figure 8.. mEERL-derived small extracellular vesicles tune the transcriptomic landscape of nociceptor neurons.

Splenocyte-derived CD8⁺ T cells were cultured under T_c1-stimulating conditions (ex vivo activated by anti-CD3 and anti-CD28, IL-12, and anti-IL4) for 48h. Subsequently, DRG from 8-week old male TRPV1^cre::tdTomato^fl/wt mice were cultured either alone or co-cultured with: cytotoxic CD8+ T cells (A, B), mEERL-derived sEVs (C, D) or in combination of both (E, F) for a period of 48h. The TRPV1-expressing neurons (tdTomato⁺) were collected, purified via FACS sorting, and their RNA was subjected to sequencing analysis. Hierarchical clustering (A, C, E) of differentially expressed genes are shown as volcano plots (B, D, F) to identify specific populations of transcripts that were distinctly enriched in TRPV1+ neurons after exposure to the different conditions. (G) 8-week-old male and female C57Bl6 mice were euthanized, DRG harvested and cultured for 24h. The neurons were then exposed (24h) to sEV purified from the plasma of male (♂) and female (♀) healthy controls or from HNSCC patients. The neurons were then exposed to a low concentration of capsaicin (300nM) and KCl (50mM) and calcium influx recorded. Data are presented as heatmap showing normalized gene expression (log2 (0.01 + transcripts per million reads (TPM)) – mean (A, C, E) or volcano plot (p<0.05; B, D, F). Experiments were not independently repeated. n = 4 per group.

Figure 9.. Tumor cells, infiltrating neurons and immune cell interactions.

Tumor cells and infiltrating nociceptors engage in complex interactions which include small extracellular vesicles and soluble factors. Together these feed-forward loops promote differentiation and recruitment of MDSCs to the tumor bed, resulting in an immune suppressive environment.