TUMOR-INFILTRATING NOCICEPTOR NEURONS PROMOTE IMMUNOSUPPRESSION

Nociceptor neurons impact tumor immunity. Removing nociceptor neurons reduced myeloid-derived suppressor cell (MDSCs) tumor infiltration in mouse models of head and neck carcinoma and melanoma. Carcinoma-released small extracellular vesicles (sEVs) attract nociceptive nerves to tumors. sEV-deficient tumors fail to develop in mice lacking nociceptor neurons. Exposure of dorsal root ganglia (DRG) neurons to cancer sEVs elevated expression of Substance P, IL-6 and injury-related neuronal markers while treatment with cancer sEVs and cytotoxic CD8 T-cells induced an immunosuppressive state (increased exhaustion ligands and cytokines). Cancer patient sEVs enhanced DRG responses to capsaicin, indicating increased nociceptor sensitivity. Conditioned media from DRG and cancer cell co-cultures promoted expression of MDSC markers in primary bone marrow cells while DRG conditioned media together with cancer sEVs induced checkpoint expression on T-cells. Our findings indicate that nociceptor neurons facilitate CD8+ T cell exhaustion and enhance MDSC infiltration. Targeting nociceptor-released IL-6 emerges as a novel strategy to disrupt harmful neuro-immune interactions in cancer and enhance anti-tumor immunity. Isolation of CD8+ T cells Six-to-eight-week-old male and female naïve mice were euthanized, and their spleens were collected in ice-cold PBS (5% FBS) and mechanically dissociated. The cells were strained (70 μm), RBC lysed (Life Technologies, A1049201; 2 min), and counted using a haemocytometer. Total CD8+ T cells were magnet sorted (Stem Cell, 19853A) and cultured (DMEM + FBS 10%, penicillin–streptomycin 1% + nonessential amino acids (Corning, 25-025-Cl) + vitamin + β-mercaptoethanol (Gibco, 21985-023) + l-glutamine (VWR, 02-0131) + sodium pyruvate (Corning, 25-000-Cl)). Cell purity was systematically confirmed after magnet sorting and the numbers of CD8+CD62Lhi were immunophenotyped by flow cytometry. To generate cytotoxic T lymphocytes, 2 × 105 CD8+ T cells were seeded and stimulated for 48 h under Tc1 inflammatory conditions (2 μg ml−1 plate bounded anti-CD3 and anti-CD28 (Bio X Cell, BE00011, BE00151) + 10 ng ml−1 rIL-12 (BioLegend, 577008) + 10 μg ml−1 of anti-IL-4 (Bio X Cell, BE0045). Neuro culture. Naïve TRPV1cre::tdTomatofl/WT mice were euthanized, and their dorsal root ganglia (DRG) were immediately transferred into DMEM (Corning, 10 013 CV) supplemented with 50 U ml−1 penicillin, 50 μg ml−1 streptomycin (Corning, MT 3001 Cl) and 10 % fetal bovine serum (Seradigm, 3100). The tissue was dissociated for 80 min at 37 °C in HEPES buffered saline (Sigma, 51558) containing 1 mg ml−1 collagenase IV (Sigma, C0130) and 2.4 U ml−1 dispase II (Sigma, 04942078001). After digestion, ganglia were gently triturated through glass Pasteur pipettes of decreasing diameter, centrifuged through a 10 % BSA cushion and plated onto laminin coated (Sigma, L2020) culture dishes. Approximately 4 × 104 neurons per dish were maintained for 48 h in Neurobasal A medium (Gibco, 21103 049) supplemented with 0.05 ng μl−1 nerve growth factor (Life Technologies, 13257 019), 0.002 ng μl−1 glial cell line derived neurotrophic factor (PeproTech, 450 51 10), 0.01 mM cytosine β D arabinoside (Sigma, C6645), 200 mM L glutamine (VWR, 02 0131) and B 27 (Gibco, 17504044).' Neurons were then co-cultured at a 1:10 ratio with mEERL derived small extracellular vesicles, cytotoxic CD8+ T-cells or both, each condition prepared in T cell medium supplemented with the same concentrations of NGF and GDNF to support neuronal viability. After 48 h, cells were detached with a scraper, washed in flow cytometry buffer (PBS containing 2 % FBS and 1 mM EDTA), passed through a 70 μm filter, and TRPV1+ neurons (FSChigh SSChigh tdTomatohigh) were isolated by fluorescence activated cell sorting directly into TRIzol. RNA extraction, library construction and sequencing were performed at the IRIC Genomics Platform (Université de Montréal). RNA integrity was confirmed with an Agilent Bioanalyzer (RIN > 7.5). Poly adenylated transcripts were captured and libraries prepared with the KAPA mRNA HyperPrep Kit (Kapa Biosystems, KR1352). Bar coded libraries were sequenced on an Illumina NextSeq 500 using 75 cycle single end reads. Adapter trimming and low quality base removal were carried out with Trimmomatic v0.35, and reads were aligned to the mouse reference genome GRCm38 (Gencode M25, Ensembl 100) with STAR v2.7.1. Gene level counts were generated with STAR and RSEM, normalized to fragments per kilobase per million mapped reads, and differential expression was assessed with DESeq2. Genes with a false discovery rate–adjusted P value below 0.20 were considered differentially expressed. Downstream analyses and visualization were performed in RStudio or Microsoft Excel.