Clinical samples and cohorts<\/p>\n
Supplementary Table 10<\/a> summarizes the different clinical cohorts used in this Article, and presents the results of their relevant analysis. The full description of each cohort is depicted in the text and in Supplementary Table 10<\/a>.<\/p>\n Neoadjuvant anti-PD-1 cSCC clinical trial cohorts<\/p>\n The clinical trials protocol, statistical analysis plan, and institutional review board approvals were previously reported19<\/a>,20<\/a>. In brief, the trials were conducted according to the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. All patients provided written informed consent. The authors had unrestricted access to the data and were responsible for all content. Patients older than 18 were eligible if they had resectable stage II\u2013IV (M0) CSCC, for which primary surgery would be recommended in routine clinical practice. Patients with stage II CSCC should have a primary tumour \u22653\u2009cm in longest diameter to be eligible. Patients were additionally required to have adequate organ function (determined by assessment of complete blood cell count and comprehensive metabolic function), at least one measurable lesion on the basis of the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1)57<\/a> and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. After a screening period of up to 28\u2009days, the patients received the neoadjuvant cemiplimab (Regeneron Pharmaceuticals) 350\u2009mg intravenously every 3\u2009weeks until unacceptable toxicity, disease progression or withdrawal of consent. Imaging assessments were performed at the baseline and at weeks 6 and 12. After completion of neoadjuvant treatment, the protocol window for surgery was study days 75\u2013100. If the patient met the criteria for early discontinuation of cemiplimab during the neoadjuvant period, the treating physician could divert the patient to surgery earlier. Pretreatment biopsy specimens were subjected to histopathological assessment for confirmation of diagnosis and to permit morphologic comparison between tumour tissue before treatment and any residual tumour following therapy. Pathologic response was assessed in the post-treatment surgical specimens according to standard pathologic evaluation recommendations58<\/a> and re-reviewed by a dedicated dermatopathologist (P.N.) to standardize reporting. The primary end point was pathologic complete response, defined as the absence of viable tumour in post-treatment surgical specimens, determined on the basis of independent central pathology review. Major pathologic response was a secondary end point defined by the presence of >0% but \u226410% viable tumour cells in post-treatment surgical specimens. The definitions specified for major pathologic response and pathologic complete response were in accordance with the immune-related pathologic response criteria59<\/a>. Further clinical information regarding the trial patients is provided in Fig. 1b<\/a>. The allocation of the trial patient tumour samples into different analyses is described in Extended Data Fig. 1<\/a>.<\/p>\n Immunohistochemistry analysis of cSCC anti-PD-1 clinical trial samples<\/p>\n Immunohistochemical analysis was performed using 4-\u03bcm-thick formalin-fixed and paraffin-embedded (FFPE) cSCC tissue samples obtained before and after anti-PD-1 therapy. Staining was performed using antibodies against CD8 (Thermo Fisher Scientific, MS-457s), PD-1 (Abcam, ab137132) and PD-L1 (Cell Signaling Technology, 13684S) using a BOND-RX instrument with a Bond Polymer Refine Detection Kit (Leica Biosystems, DS9800). Stained slides were scanned and digitalized using a Scan Scope XT system (Aperio\/Leica Biosystems). Single-stain annotations were performed by a pathologist, and staining quantification was done using HALO software (v.2.3.2089.70; Indica Labs). The number of marker-positive cells was calculated and expressed as positive-cell density (number of positive cells per mm2). Statistical analysis was performed using two-tailed Mann\u2013Whitney U-tests using Prism (v.9). P\u2009<\u20090.05 was considered to be significant.<\/p>\n High-plex, high-dimensional IF staining and analysis<\/p>\n We used the Lunaphore Comet to stain and image the FFPE tissue samples. In brief, FFPE slides were placed in tanks containing BioGenex EZ Elegans AR 2 buffer, enclosed inside a BioGenex EZ Retriever microwave system. The samples were heated in the microwave to 107\u2009\u00b0C for 15\u2009min to dewax, rehydrate and retrieve the antigens. After heating, the slides were cooled to room temperature, loaded into the Comet system, and covered and sealed by a fast-fluidic exchange microfluidics chip for staining and imaging in the instrument. The Comet system performs a sequential IF-based staining and imaging followed by an elution of the primary and secondary antibodies. Optimal staining conditions for each antibody panel were determined using the \u2018Characterization Part 2\u2019 instrument protocol, in which antibodies are titrated for optimal signal and the elution efficiency is verified by an iterative procedure comprised of staining, imaging, elution and re-imaging for elution confirmation for all antibodies. This study used four mIF panels, described in Supplementary Table 10<\/a> (neuroimmune panel 1; myelin degradation panel; neuroimmune panel 2; and neuroimmune panel 3) for patient-derived tissues. Two panels (m-neuroimmune panel; and m-myelin degradation panel) were used for mouse-derived tissues (Supplementary Table 10<\/a>). The Comet collected images in OME-Tiff formats, which were scanned and then visualized in Comet Viewer (Lunaphore). Image analysis was performed using Oncotopix Discovery (Visiopharm, v.2023.01)60<\/a>,61<\/a>. A deep learning classifier was trained using specific tissue features from annotated reference images to guide the identification of ROIs. In brief, a tissue ROI was created after training two variables (tissue and non-tissue regions). Tissue and ROI boundaries were then delineated using image analysis smoothing. Aberrant signals, such as those caused by dust particles, tissue folds or air bubbles, were excluded manually from the ROIs. Another deep learner classifier was developed and trained for nerve identification following two training variables (nerve and non-nerve structure) within the delineated tissue ROI regions. Staining for the nerve markers NFH and B3T delineated the nerve within the tissue ROIs. The following training parameters were used to develop the nerve identification app: input: NFH and B3T; learning parameters: learning rate-1.0e-05; Mini-batch size-2; loss function: cross-entropy; iteration: 300,000. Cells in non-nerve areas (NFH\u2212B3T\u2212) were identified by watershed desegmentation of the nuclear DAPI signal and removal of incomplete nuclei by size exclusion. The resulting nuclear mask was enlarged by 1.25\u2009\u03bcm (5 pixels) to capture nuclear and adjacent cytoplasmic fluorescence signals. A further estimation of the cytoplasm was generated using a dilation of the nuclear mask to a maximum of 3.75\u2009\u03bcm (15 pixels). Cell types were identified using a hierarchical decision tree with empirically determined thresholds visually verified across the tissues. NFH+B3T+ nerves were segmented using the NFH\/B3T signal intensity. Nerve nuclei were excluded a priori from the initial cell classification strategy, and only those that exceeded the threshold for NFH+B3T+ levels were incorporated into the nerve mask. Neural niches were determined to have a 150-\u03bcm ROI diameter around each nerve (NFH+B3T+). All object-based phenotyping resulting tables were exported as CSV files for downstream analyses. Staining was performed 4\u20138\u2009min per cycle for primary antibodies, diluted 1:50\u20131:2000 in a Multistaining Buffer (Lunaphore). Secondary antibodies, AlexaFluor 555 and AlexaFluor 647 (Thermo Fisher Scientific) were diluted 1:200 and 1:400, respectively, in a multistaining buffer. Tissues were counterstained with DAPI solution (Thermo Fisher Scientific) at every cycle. Imaging was performed on the Comet at 80\u2009ms for DAPI, 400 ms for AlexaFluor 555, and 200 ms for AlexaFluor 647 at each cycle. Elution was performed for 4\u2009min using an elution buffer (Lunaphore).<\/p>\n GeoMx DSP experimental design<\/p>\n cSCC anti-PD-1 clinical trial FFPE tumour samples were used for DSP of the neural and immune protein expression (see below the description of assays and markers used). First, we mapped the neural niches using haematoxylin and eosin (H&E)-stained tissue sections, including the tumour and surrounding tissue. These slides were scanned using a Scan Scope digital pathology system (Aperio) in SVS format at a 40\u00d7 magnification and visualized using Image Scope software (Aperio). All nerve profiles in the specimen were annotated, including PNI. The extent of viable tumour or tumour bed and uninvolved tissue was drawn or designated on whole-slide scans. The respective tumour and surrounding tissue were obtained using the annotations in Aperio files. The nerve density was calculated as the ratio between total number of nerves and the tumour or normal area (mm2), and the nerve invasion index was calculated as the number of invaded nerves divided by the tumour area (mm2).<\/p>\n Next, two consecutive 5-\u03bcm-thick tissue sections from samples obtained before and after anti-PD-1 therapy were stained according to the semiautomated GeoMx DSP standard protein protocol62<\/a> using the BOND-RX system to profile TANs and their perineural microenvironments (neural niches). Both sections were stained for the following morphology biomarkers: panCK, SYTO 13 (GeoMx Solid Tumour TME Morphology Kit, NanoString, 121300301), \u03b2-III-tubulin (EP1569Y, AF, Abcam, ab52623; 1:2,000\u2009mg\u2009ml\u22121) and neurofilament (EPR20020, AF, Abcam, ab207176; 1:1,000\u2009mg\u2009ml\u22121; Extended Data Fig. 6<\/a>). Optimization of IF biomarkers was previously performed with different antibody dilutions using normal colon tissue to achieve the highest signal-to-noise ratio. One tumour section was used to profile TANs with the DSP neural cell profiling core (GMX-PROCO-NCT-HNCP-12) and GeoMx Parkinson\u2019s pathology panel (GMX-PROMOD-NCT-HPDP-12). A serial tissue section was used to profile the perineural microenvironment with the following DSP human immuno-oncology protein core panel and modules: GeoMx Immune Cell Profiling (121300101), GeoMx IO Drug Target Assay (121300102), GeoMx Immune Activation Status Assay (121300103) and GeoMx Immune Cell Typing Assay (121300104; 49 protein targets). The full list of targets is described in Supplementary Table 10<\/a>.<\/p>\n After scanning using the GeoMx DSP device, mIF imaging slides were visualized with the adjustment of channel thresholds for each fluorophore. ROIs were selected after pathological evaluation of sequential sections of H&E-stained nerve fibres in tumour tissue and tumour bed tissue identified by an expert pathologist (P.N.). Using a two-step strategy, a polygon and rectangle selection tool was applied to select ROIs of up to 660\u2009\u00d7\u2009785\u2009mm. (1) Nerve profiling: a slide labelled with a DSP neuroprotein panel was used to select up to 12 intratumoural ROIs containing tumour-associated nerve fibres. Neural niches were identified and segmented using the \u03b2-III-tubulin (+), Neurofilament Heavy (NF-H) (+) and panCK (\u2212) phenotype. (2) Perineural niche immune profiling: a consecutive section labelled with a DSP human immuno-oncology protein panel was used to select up to 12 matching ROIs with TANs. The matching perineural microenvironment compartment, that is, neural niches, were identified using the same morphology markers (\u03b2-III-tubulin (+), neurofilament heavy (NF-H) (+) and panCK (\u2212) phenotype). Segmented areas (also known as areas of illumination) were illuminated individually using ultraviolet light with the GeoMx DSP device. Oligonucleotide tags conjugated with antibodies present within each area of illumination were photocleaved. Released tags were quantified using nCounter, and tag counts of the various markers were mapped back to their corresponding tissue locations, yielding a spatially resolved digital profile of analyte abundance. Digital counts were normalized using background correction. DSP data analysis software was used to visualize protein expression patterns and perform statistical analysis62<\/a>. These data are available in Supplementary Table 8<\/a>.<\/p>\n Bulk RNA-sequencing of cSCC anti-PD-1 clinical trial tumour samples<\/p>\n Nucleic acid extraction, library preparation and sequencing: DNA\/RNA extraction was performed using the Mag-Bind FFPE DNA\/RNA 96 Kit (Omega Bio-Tek) according to the manufacturer\u2019s protocol. Isolated RNA sample quality was assessed using the High Sensitivity RNA Tapestation (Agilent Technologies) and quantified by Qubit 2.0 RNA HS assay (Thermo Fisher Scientific). Libraries were constructed with KAPA RNA HyperPrep with RiboErase (Roche) and performed based on the manufacturer\u2019s recommendations. The final library quantity was measured by KAPA SYBR FAST qPCR, and library quality was evaluated by TapeStation D1000 ScreenTape (Agilent Technologies). The final library size was about 430\u2009bp with an insert size of about 200\u2009bp. Illumina 8-nucleotide dual-indices were used. Equimolar pooling of libraries was performed based on quality-control values and sequenced on the Illumina NovaSeq system (Illumina) with a read length configuration of 150 PE for 80\u2009million paired-end reads per sample (40\u2009million in each direction). Extracted genomic DNA was then quantified using the Qubit 2.0 DNA HS Assay (Thermo Fisher Scientific), and the quality was assessed using the Tapestation genomic DNA Assay (Agilent Technologies). Library preparation was performed using SureSelectXT Low Input Reagent Kits (Agilent Technologies) according to the manufacturer\u2019s recommendations. Exome capture was performed with IDT xGen Exome Research Panel v2.0. Library quality and quantity were assessed using the Qubit 2.0 DNA HS Assay (Thermo Fisher Scientific), Tapestation High Sensitivity D1000 Assay (Agilent Technologies) and QuantStudio 5 System (Applied Biosystems). Illumina 8-nucleotide dual-indices were used. Equimolar pooling of libraries was performed based on quality-control values and sequenced on the Illumina NovaSeq (Illumina) system with a read length configuration of 150 PE for 130\u2009million paired-end reads (65\u2009million in each direction) or 26\u2009million paired-end reads (13\u2009million in each direction). RNA-seq data processing and analysis: raw paired-end reads in FASTQ format were checked for read quality using FastQC (v.0.11.8; http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/<\/a>). Illumina TruSeq adapters were trimmed from the paired-end reads using cutadapt (v.1.18). The trimmed reads were aligned to the GENCODE human reference genome GRCh38 using STAR software (v.2.7.0f). FeatureCounts (subread 1.6.3) was applied to count reads mapped to each gene. Genes were annotated using the gene transfer format file for GRCh38. Read counts were normalized using the trimmed mean of M method implemented in the R Bioconductor package edgeR to determine the abundance of each gene. The generalized linear model likelihood ratio test from edgeR was used to identify DEGs between groups. The Benjamini\u2013Hochberg correction method was applied to the P values for multiple testing adjustments. These data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession umber GSE289743<\/a>.<\/p>\n PNI and nerve injury signature<\/p>\n The PNI signature was based on an independent and previously published gene expression signature associated with PNI in cSCC of the head and neck30<\/a>. The initial dataset consisted of DEGs from tumours lacking PNI compared with those exhibiting incidental PNI, identified solely through histopathological evaluation (n\u2009=\u20092,412). The second dataset included DEGs from tumours with no PNI versus those with clinical PNI, detected using imaging techniques (n\u2009=\u20097,793). To curate a gene list that captures the molecular features associated with any form of PNI (both incidental and clinical), we focused on DEGs that were upregulated in both datasets. Further refinement involved excluding genes exhibiting a fold change\u2009<\u20092 and a FDR\u2009>\u20090.001 across both comparisons (no PNI versus incidental PNI and no PNI versus clinical PNI). In our analysis, DEGs represented by multiple microarray probes were excluded if one or more probes failed to satisfy our inclusion criteria. Specifically, cases in which one probe suggested significant upregulation of a gene in PNI-positive cases, while another indicated either downregulation in PNI or no significant difference, were not considered. Ultimately, the final gene set consisted of 46 genes enriched in PNI-positive cases of cSCC of the head and neck (Supplementary Table 1<\/a>).<\/p>\n This 46-gene PNI\/nerve injury signature was first tested on our anti-PD-1 neoadjuvant cSCC clinical trial bulk RNA-seq data using GSEA. GSEA was conducted to investigate the enrichment the signature in the pretreatment trial tumour samples performing 1,000 permutations63<\/a>. GSEA calculates an enrichment score (ES) for gene sets by ranking genes based on their overrepresentation in two groups. A positive ES indicates enrichment in one group, while a negative ES indicates enrichment in the other. The ES is normalized to a NES to enable comparisons. We considered the gene sets to be significantly enriched at a threshold of P\u2009<\u20090.05. We did not apply corrections for multiple-hypothesis testing in this case, as only a single gene signature was assessed. The 46-gene PNI\/nerve injury signature was later validated in external, publicly available cohorts of patients who had PNI-associated tumours and were treated with anti-PD-1 therapy. The bulk RNA-seq data of three patient cohorts were available (two melanoma32<\/a>,33<\/a> and one gastric cancer34<\/a>; Supplementary Table 2<\/a>):<\/p>\n (1)<\/p>\n Reference 33<\/a>: this study included 88 patients with metastatic melanoma, receiving either nivolumab (n\u2009=\u200939) or pembrolizumab (n\u2009=\u200949). The response rates were as follows: complete response (CR)\u2009=\u200914 (15.9%), partial response (PR)\u2009=\u200927 (30.7%), and progressive disease (PD)\u2009=\u200947 (53.4%). For GSEA, patients with CR and PR were classified as responders (n\u2009=\u200941, 46.6%), while those with PD were categorized as non-responders (n\u2009=\u200947, 53.4%).<\/p>\n (2)<\/p>\n Reference 32<\/a>: this study evaluated 41 samples from patients treated with nivolumab (n\u2009=\u20099) or pembrolizumab (n\u2009=\u200932). The response distribution was: CR\u2009=\u20094 (9.7%), PR\u2009=\u200915 (36.6%), stable disease (SD)\u2009=\u20096 (14.6%) and PD\u2009=\u200916 (39%). In GSEA analysis, the CR and PR patients were grouped as responders (n\u2009=\u200919, 46.3%), while SD and PD patients were grouped as non-responders (n\u2009=\u200922, 53.7%).<\/p>\n (3)<\/p>\n Reference 34<\/a>: this study comprised 45 patients with metastatic gastric cancer who received pembrolizumab as salvage therapy, with available RNA-seq data. Patients were treated with pembrolizumab in second-line or third-line settings for metastatic disease. The response rates recorded were: CR\u2009=\u20093 (6.6%), PR\u2009=\u20099 (20%), SD\u2009=\u200915 (33.3%) and PD\u2009=\u200918 (40%). For GSEA, those with CR and PR were considered responders (n\u2009=\u200912, 26.7%), while patients with SD and PD were classified as non-responders (n\u2009=\u200933, 73.3%).<\/p>\n NanoString gene expression analysis of the cSCC anti-PD-1 clinical trial tumour samples<\/p>\n RNA was isolated from FFPE tumour sections by dewaxing using a deparaffinization solution (QIAGEN), and total RNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion) according to the manufacturer\u2019s instructions. The RNA purity and quantity were assessed using a NanoDrop ND-1000 spectrometer (Thermo Fisher Scientific). For a NanoString assay, 100\u2009ng of RNA was used to detect immune gene expression using a nCounter PanCancer Immune Profiling Panel along with a custom CodeSet (NanoString). Counts of the reporter probes were tabulated for each sample using a nCounter Digital Analyzer, and raw data output was imported into the nSolver software program (v.4.0; NanoString). Moreover, the nSolver (Advanced Analysis 2.0) data analysis package was used for the normalization of expression levels, cell type and differential gene expression analyses. Gene set enrichment analysis was performed with Qlucore Omics Explorer software (v.3.7). Data were plotted using Prism software (v.9; GraphPad Software) and two-tailed Mann\u2013Whitney U-tests were performed to compare groups. P\u2009<\u20090.05 was considered to be significant. These data are provided in Supplementary Table 9<\/a>.<\/p>\n Non-trial original clinical cohorts<\/p>\n Non-trial original clinical cohorts were used in this Article: (1) 32 treatment-naive patients with stage I\u2013II cSCC whose tumours were excised at MDACC (The University of Texas MD Anderson Cancer Center) before February 2023. The archived tumour samples were used for the construction of an 86-core tissue microarray (TMA). The TMA was used to characterize immune infiltration in perineural niches using mIF. (2) 187 treatment-naive patients with stage I\u2013II PDAC whose tumours were resected at MDACC between 2004 to 2015. Archived tumours from these patients were used for the construction of another TMA, as described before64<\/a>. This TMA was also used to characterize immune infiltration in perineural niches using mIF as a means of external validation. (3) 11 treatment-naive patients with stage I\u2013II cSCC whose primary tumours were excised by Mohs surgery at MDACC between 2020 and 2022 (Supplementary Table 4<\/a>). Their fresh-frozen collected tumours were used for spatial transcriptomic analysis. (4) 7 treatment-naive patients with PDAC whose tumours were resected at Massachusetts General Hospital, provided to this study by W. Hwang. This cohort has been described previously45<\/a>. In brief, these patients with locally advanced PDAC underwent surgical resection of the primary tumour with or without previous neoadjuvant chemotherapy and radiotherapy. The tumours were FFPE.<\/p>\n Visium spatial transcriptome sequencing (non-trial cSCC tumour samples)<\/p>\n Fresh-frozen collected and FFPE archived\u00a0cSCC primary tumours were used to demonstrate the spatial relationship between nerve injury and different immune phenotypes. The tumours were prepared for sequencing according to the manufacturer\u2019s instructions (10x Genomics, Visium Spatial) using the following kits: Visium Spatial Tissue Optimization Slide & Reagent Kit, 4 slides PN-1000193; Visium Gateway Tissue Optimization Slide & Reagent Kits, 1 slide (PN-1000313, PN-1000314); Visium Accessory Kit, PN-1000194. The prepared libraries were pooled and sequenced on the NovaSeq 6000 (Illumina) system, generating ~40\u2009million 2\u2009\u00d7\u2009150 base paired-end reads per sample. The raw spatial sequencing data were processed in the Space Ranger workflow (https:\/\/support.10xgenomics.com\/spatial-gene-expression\/software\/pipelines\/latest\/choosing-how-to-run<\/a>).<\/p>\n The spaceranger (v.1.3.0) mkfastq pipeline was used to convert Illumina sequencer\u2019s binary base call (BCL) files into FASTQ format. The samples were then run through the spaceranger count pipeline, which performs alignment, tissue detection, fiducial detection and barcode\/unique molecular identifier counting. The RNA-seq aligner STAR was used as the samples were fresh-frozen. Reference-sequence alignment of human samples was performed using the GRCh38 Reference 2020-A (23 June 2020). Images were processed using Fiji to divide them into individual capture areas and then rotated to align the fiducial spot patterns65<\/a>. The pipeline uses Visium spatial barcodes to generate feature-spot matrices, determine clusters and perform gene expression analysis. Functions in the R package Seurat (v.4.1.1) were used for downstream analysis66<\/a>. Spots that did not overlap with the tissue sections or had \u226520% mitochondrial reads (10% for humanized mouse) or had <200\/500 detected genes were removed from the downstream expression analysis. Expression counts were normalized using Seurat\u2019s NormalizeData. The 3,000 most variable genes were identified using the FindVariableFeatures function. The normalized expression data were further scaled to mean 0 and variance 1 using the function ScaleData. RunPCA and RunUMAP were used for dimensionality reduction. Sample batch effect correction was performed using RunHarmony. Clustering was done using the functions FindNeighbors and FindClusters. We performed transcriptional phenotyping for each sequenced tissue section based on the molecular characteristics of TANs and tumour immune infiltrate using gene signatures obtained from the literature67<\/a>. Nerves were scored according to the CINI signature that included the following genes: TAGAP, KCNJ8, COL1A1, PECAM1, TMEM119, ATF3, JUN, KLF6, NOCT, LMO7, CSF1, ENTPD1, UCHL1, PINK1, BHLHE41, ITGAM, CHL1, SNCA, SCPEP1 and VEGFA. The immune infiltrate was scored according to two signatures: antitumoural immunity (CD8A, PRF1, GZMB, IL12A, IFNG, IRF8, CD86, NOS2, TNF and IL2) and immunosuppression (IDO1, CTLA4, FOXP3, IL10, IL6, CD163, MSR1, MRC1, PDCD1, PDCD1LG2 and CD274) signatures. The gene signature scores were calculated for all spots using the Seurat AddModuleScore function68<\/a>. Spatial feature expression plots were generated using the SpatialFeaturePlot function. One-way ANOVA with Tukey\u2019s post hoc test was used to assess the significance of the group difference. Pearson correlation coefficients were calculated to assess the magnitude of the association between the CINI and the immune signatures. These data have been deposited in the NCBI GEO under accession number GSE289745<\/a>.<\/p>\n Nanostring CosMx spatial molecular imaging (MDACC-external PDAC cohort): this spatial molecular imaging analysis was conducted on human PDAC primary tumours provided by W. Hwang (see above for a description of the clinical cohort). FFPE tissue sections (5\u2009\u00b5m) were prepared and mounted onto Superfrost Plus Micro Slides (VWR) under RNase-free conditions. The samples underwent deparaffinization, proteinase K digestion and heat-induced epitope retrieval using the Leica Bond RX system. After preparation, Nanostring CosMx spatial transcriptomics imaging was performed on the sections. TANs were categorized into three groups based on ATF3 or JUN expression (healthy: no ATF3 or JUN expression; intermediated: ATF3 or JUN expression; injured: ATF3 and JUN concomitant expression). Perineural niches were defined as regions within 150\u2009\u03bcm of TANs in the TME. RNA in situ hybridization probes targeting a 960-plex base panel, along with 30 additional custom-selected probes, were hybridized overnight at 37\u2009\u00b0C. After hybridization, the samples were washed and blocked and protein staining was performed using a fluorophore-conjugated antibody cocktail targeting CD298, B2M, PanCK, CD45 and CD3 proteins, along with DAPI for nuclear staining. Data acquisition and image processing were conducted using an in-house spatial molecular imaging data-processing pipeline45<\/a>. z-stack images were collected, and cell segmentation was performed using Cellpose (v.2.0.5), with robustness evaluated through comparison to Baysor (v.0.6.0). Transcripts were assigned to individual cells based on location and cell segmentation boundaries. Expression profiles for individual cells were normalized and log-transformed. Cell types were annotated using Insitutype (v.1.0.0) based on protein and RNA expression profiles, with malignant and nonmalignant cells identified using PanCK expression and cytokeratin RNA markers. Batch correction across tissue slides was performed using ComBat (sva, v.3.46.0). These data have been deposited in the NCBI GEO under accession number GSE199102<\/a>.<\/p>\n In vitro studies<\/p>\n The different cell types used for in vitro assays in this study, along with the figure panel presenting the results of their relevant analysis, are summarized in Supplementary Table 10<\/a>. For in vitro experiments in which there were replicate samples, we repeated the experiments at least three times to confirm the findings.<\/p>\n Human neurons<\/p>\n Human neurons used in this study were obtained from human donors or were derived from commercially available human iPS (hiPS) cells. RealDRG cryopreserved hPS-cell-derived sensory neurons (nociceptors). Using the Senso-DM directed differentiation process on internal hiPS cell lines, we can generate billions of neurons at scale and at 99% purity\u2014without the need for mitotic inhibitors. Principal component analysis shows that RealDRG neurons mature in the shortest time and are closer to primary human DRG tissue compared with other hiPS-cell-derived sensory neurons (https:\/\/www.anatomic.com\/realdrg<\/a>). Donors provided written informed consent to donate tissue samples for the study, for which the protocol was reviewed and approved by The University of Texas MD Anderson Cancer Center Institutional Review Board. In brief, each donor was undergoing surgical treatment that necessitated ligation of spinal nerve roots to facilitate tumour resection or spinal reconstruction. Spinal roots were ligated proximal to the DRG, spinal roots were sharply cut both proximal and distal to the DRG and excised DRGs were transferred immediately into a cold (\u223c4\u2009\u00b0C) and sterile balanced salt solution containing nutrients. DRGs were transported to the laboratory on ice in a sterile, sealed 50\u2009ml centrifuge tube. After arrival at the laboratory, each ganglion was carefully dissected from the surrounding connective tissues and sectioned into several 1\u20132\u2009mm pieces, digested in 2\u2009ml of a mixed enzyme solution (0.1% trypsin (Sigma-Aldrich, T9201), 0.1% collagenase (Sigma-Aldrich, C1764; w\/v, final concentration) and 0.01% DNase (Sigma-Aldrich, D5025) diluted in DMEM\/F-12), and transferred to a 37\u2009\u00b0C rotator to shake at a speed of 124\u2013128\u2009rpm. Every 20\u2009min, tissue fragments were allowed to settle, and the supernatant\/dissociated cells were collected and transferred to DMEM\/F-12 with an enzyme inhibitor. The supernatant was replaced with 2\u2009ml of fresh digestion solution. The tissue was returned to the 37\u2009\u00b0C rotator, and this process was repeated until tissue fragments were completely digested. Dissociated cells were centrifuged at 180\u2009rpm for 5\u2009min, the supernatant was removed and the cells were gently resuspended in culture medium with DMEM\/F-12 supplemented with EV-free 10% serum and 2\u2009mM glutamine. Cells were plated onto laminin-coated \u00b5-Slide 8 Well (Ibidi) and cultured at 37\u2009\u00b0C with 5% CO2 for 24\u201372\u2009h. hIPS-cell-derived motor (RealMOTO) and sensory neurons (RealDRG) were purchased from Anatomic.<\/p>\n Mouse neurons<\/p>\n Primary sensory neurons were isolated from the TG dissected from 6\u20138-week-old mice as previously described69<\/a>. After TG dissection, tissue was enzymatically digested with papain (40\u2009U\u2009ml\u22121, EMD Millipore) for 20\u2009min at 37\u2009\u00b0C followed by 20\u2009min of digestion with collagenase II (4\u2009mg\u2009ml\u22121)\/dispase II (4.6\u2009mg\u2009ml\u22121) solution. Using the Percoll gradient, comprising 12.5% and 28% Percoll in complete L-15 medium (L-15 with 5% fetal calf serum, penicillin\u2013streptomycin, HEPES), we separated the myelin and nerve debris from trigeminal neurons. Neurons were labelled with NeuroFluor NeuO (01801) membrane-permeable fluorescent probe for detecting live neurons according to the manufacturer\u2019s protocol (StemCell Technologies) and sorted by flow cytometry using the LSR II flow cytometer running FACSDiva 8.0 software and FACSAria (all BD Biosciences).<\/p>\n Co-culture<\/p>\n The co-culture system using neurons and cancer cells is based on a technique initially described for prostate cancer cells70<\/a> and modified by our group12<\/a>. Neurons were prepared as described previously12<\/a> and cocultured with cancer cells or normal keratinocytes. Human neurons were co-cultured with the human cSCC IC8 cell line (obtained from the laboratory of I.L. and C.H.37<\/a>, or with the human epidermal keratinocyte HEK cells (ATCC, PCS-200-011). Mouse neurons were co-cultured with the mouse oral squamous cell carcinoma MOC1 cell line (Sigma-Aldrich, scc469) and B6-10K (obtained from the laboratory of K.Y.T.). Sensory or motor neurons were cultured to assess the impact of nerves\u2019 degenerative\/regenerative status on immunotherapy in vitro. Neurons were plated on laminin-coated (Life Technologies, 23017015) round glass coverslips set in 12-well plates. Then, 2\u2009h after plating, the wells were flooded with 1\u2009ml of warm (37\u2009\u00b0C) Ham\u2019s F12 culture medium (Sigma-Aldrich) supplemented with 10% EV-depleted fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin\u2013streptomycin and incubated at 37\u2009\u00b0C in 5% CO2 and 95% air. The carcinoma cells were plated (1:10 neuron: cancer cells ratio), and cultures were grown in RPMI 1640 medium containing 10% EV-depleted fetal bovine serum in 37\u2009\u00b0C and 5% CO2 incubation conditions, for 72\u2009h, with or without anti-PD-1 antibody (cemiplimab, 1,000\u2009\u03bcg\u2009ml\u22121, or RMP1-14, 100\u2009\u03bcg\u2009ml\u22121, InVivoMAb Antibodies, BioXCell, for human and mouse, respectively), before neuron sorting and RNA extraction.<\/p>\n Neuron viability assay<\/p>\n TG neurons were obtained from C57BL\/6J mice (n\u2009=\u20098), processed using the above protocol, seeded to 12-well culture plates (3\u2009\u00d7\u2009105 total cells per well) and cultured for 72\u2009h. The cultured neurons were then divided into four experimental groups (two wells per group) in which each group was exposed to different culture conditions for an additional 72\u2009h. The first group (control) received no medium change; the second group received fresh medium (20% FBS in Ham\u2019s 12\u2009+\u2009penicillin\u2013streptomycin\u2009+\u20091% vitamin); the third group received conditioned medium from the MOC1 mouse oral squamous cell carcinoma cell line previously cultured (10\u2009\u00d7\u2009103 seeded cells in 20% FBS in Ham\u2019s 12\u2009+\u2009penicillin\u2013streptomycin\u2009+\u20091% vitamin) for 3 days. The fourth group of neurons was co-cultured with the MOC1 (10\u2009\u00d7\u2009103) cells. After 72\u2009h of exposure, neurons were collected by trypsinization, medium was added (F-15\u2009+\u200930% FBS) and then live neuro cells were stained with NeuO (membrane-permeable fluorescent dye) dye for 1\u2009h and washed. NeuO-positive cells were sorted by flow cytometry using the CytoFLEX SRT Cell Sorter. RealDRG cryopreserved hPS-cell-derived sensory neurons were obtained from Anatomic. RealDRG neurons were seeded on 0.01% poly-l-ornithine-coated (Millipore Sigma, A-004-C) \u00b5-slide 8-well ibidi plates (80827) at a density of 0.15\u2009\u00d7\u2009105 total cells per well. The poly-l-ornithine coating was performed overnight at room temperature in the tissue culture hood, followed by three washes with sterile water. Subsequently, iMatrix-511 silk (Takara, T304) diluted 1:50 in Dulbecco\u2019s PBS (dPBS) was added as a secondary coating and incubated at 37\u2009\u00b0C for 2\u2009h after washing off the poly-l-ornithine. The RealDRG cells were cultured for 10 days to allow for maturation, with half of the Senso MM medium (Anatomic) being replaced every alternate day. After the initial 10-day culture period, the neurons were divided into four experimental groups (two wells per group) and exposed to different culture conditions for an additional 4\u2009days (96\u2009h), aligning with the timeline in the provided PDF. The experimental groups were as follows: fresh medium: cells received fresh Senso MM medium (Anatomic). No medium change: cells were cultured for the entire 14-day period without any medium change. Conditioned medium: cells received conditioned media from B16-F10 (melanoma) and Mia PaCa2 (PDMC) cells. To obtain conditioned medium, B16-F10 and Mia PaCa2 cells were seeded at 10\u2009\u00d7\u2009103 cells in Senso MM medium and cultured for 24\u2009h. The medium was then replaced, and the supernatant was collected after another 24\u2009h, filtered with a 0.22\u2009\u00b5m filter and stored at 4\u2009\u00b0C. This process was repeated, and the two collections were mixed at a 1:1 ratio. Co-culture: cells were co-cultured with 2\u2009\u00d7\u2009103 seeded B16-F10 (melanoma) and Mia PaCa2 (PDMC) cells.<\/p>\n Immunofluorescence staining and imaging<\/p>\n After the 4-day exposure period, the mature neurons from \u00b5-slide 8-well ibidi plates were fixed with 4% buffered formalin. Neurons were then stained with NFH antibody (ab4680, Abcam) followed by a secondary Texas Red-conjugated antibody. Images were acquired using the Cytation 7 imaging reader.<\/p>\n Cell counts and neurite morphology were assessed using the neurite outgrowth module of the Gen5 Cytation 7 software. The experiment included four conditions (fresh medium, no medium change, conditioned medium and co-culture), with two wells per condition.<\/p>\n Neurite quantification<\/p>\n rDRGs were plated in duplicate wells in an 8-well Ibidi plate and assigned to one of four conditions: (1) fresh medium; (2) no medium change; (3) conditioned medium; or (4) direct co-culture with either B16-F10 or Mia PaCa-2 cells. Cell viability was confirmed using NeuO staining in the GFP channel before fixation. Cells were then fixed and stained with neurofilament heavy chain (NFH, Texas Red channel) to assess neuronal morphology and with DAPI to label nuclei. Using a \u00d720 magnification, multichannel fluorescence images were taken as a 12\u2009\u00d7\u200912 image montage from the centre of each well in an 8-well Ibidi plate. The Agilent BioTek Cytation 5 cell imaging multimode reader was used for all image acquisition. A minimum of ten ROIs per condition was selected based on consistent cell density. ROIs were excluded if they exhibited low cell density or imaging artifacts. Morphological analysis and thresholding for the selection of neurons and assessment of neuronal morphology were completed using the neurite outgrowth module in Gen5 Cytation 7 software. We assessed neurite length and thickness across the four conditions, with two wells per condition. The Gen5 neurite outgrowth module analysis automatically provides results for both image-level metrics, such as total outgrowth length per image and cell-level average values, like the per-cell average neurite length. Default thresholding for soma selection was applied to exclude weak NFH signal in the TexasRed channel, and neurons were selected and distinguished from cancer cells by using the \u2018optimize soma using nuclear signal\u2019 setting for DAPI staining and by selecting for cells with NFH+ protrusions extending more than 20\u2009\u00b5m from the cell, which represent neurites (dendrites and axons). \u2018Only keep neurites connected to a soma\u2019, \u2018Discard short neurites\u2019 and \u2018Discard short ending branches\u2019 were selected in the \u2018Neurite\u2019 settings window, with short neurites under 20\u2009\u00b5m excluded and short ending branches under 5\u2009\u00b5m excluded to ensure that only the true intact neurite signal was studied. From these 8\u201310 final ROIs, we exported morphology data from the manually programmed neurite outgrowth module. The metrics reported from each ROI were the average neurite length (\u00b5m) and the average neurite thickness (calculated as NFH+ area divided by neurite length in \u00b5m).<\/p>\n MEA for cell lines<\/p>\n The 24-well CytoView MEA plate (Axion, M384-tMEA-24W) electrodes were prepared for culturing realDRG according to the Anatomic protocol (https:\/\/www.anatomic.com\/_files\/ugd\/a239f4_97800271c1964458aa21c9a9008849a3.pdf<\/a>). In brief, around 7\u2009ml cell-culture-grade water with penicillin\u2013streptomycin was added to the outer rim of the plate. Then, electrodes were coated with 50\u2009\u03bcl per well of 0.01% PLO for 1\u2009h at room temperature. PLO was washed off four times with 500\u2009\u03bcl cell-culture-grade water, ensuring complete removal between washes. Then, 50\u2009\u03bcl of iMatrix-511 SILK (Takara, T304) diluted 1:50 in dPBS per well was added as a secondary coating and incubated at 37\u2009\u00b0C for 2\u2009h. The human realDRG cells were quickly thawed in a water bath, collected in fresh F12 medium (20% FBS, 1% penicillin\u2013streptomycin), centrifuged at 300\u2009rpm for 4\u2009min and resuspended in Senso MM media (Anatomic). The realDRGs were plated at 20,000 and 50,000 cells per well. Basal spontaneous firing rate of iPS-cell-derived sensory neurons assessed by MEA. After 10 days in normal culture, mature, firing neurons were cultured for 6\u2009days under control conditions, stressed conditions (no medium exchange), in MOC1 conditioned medium or in MOC1-co-culture. Spontaneous firing rates were recorded at the baseline, 24 and 72\u2009h. The mean firing rate of iPS-cell-derived sensory neurons co-cultured with MIA PaCa-2 cells (20,000 or 50,000 cells per well) over 5 days was measured by MEA. RealDRGs were plated at 20,000 and 50,000 cells per well on 24-well CytoView MEA plates, with medium changes every 48\u2009h. After 8\u20139\u2009days in culture, mature neurons started firing spontaneously and, at day 10, all neurons established stable spontaneous activity. After 10\u2009days in normal culture, mature firing neurons were cultured for 6\u2009days under control conditions, stressed conditions (no medium exchange), in Mia-PaCa2-conditioned medium or in Mia-PaCa2 co-culture. The spontaneous functional activity of the realDRGs was recorded for a minimum of 10\u2009min and every 24\u2009h for 6 days (days 10, 11, 12, 13, 14 and 15). Statistical analysis was performed using GraphPad Prism. Error bars in the figures represent the s.e.m. One-way ANOVA with Tukey\u2019s multiple -comparisons test was used to determine statistical significance between groups, with a significance level of P\u2009<\u20090.05.<\/p>\n Neuron mRNA library preparation and sequencing<\/p>\n RNA was isolated from cultured neurons using total RNA was extracted using the RecoverALL Total Nucleic Acid Isolation kit (Ambion) according to the manufacturer\u2019s instructions. RNA libraries were prepared and sequenced at the University of Houston Sequencing and Editing Core according to standard protocols. Enrichment for mRNA from total RNA was performed by coding-region-specific biotinylated capture probes, which were selected using streptavidin magnetic beads. mRNA-enriched libraries were prepared using the QIAseq Stranded Total RNA Kit (Qiagen) with 200\u2009ng of input RNA. RNA was fragmented, reverse-transcribed into cDNA, ligated with sequence adaptors and amplified by PCR. Size selection for libraries was performed using SPRIselect beads (Beckman Coulter). The library purity was analysed using the DNA HS1000 tape with a 4200 TapeStation system (Agilent) and the Qubit fluorometer (Thermo Fisher Scientific). The prepared libraries were pooled and sequenced on the NovaSeq 6000 system (Illumina), using an S4 flow cell at paired-end 150\u2009bp configuration at 20\u201330\u2009million read depth for each sample.<\/p>\n Neuron transcriptome analyses<\/p>\n The raw sequencing data (FASTQ files) were imported into Qiagen CLC Genomics Workbench 20.0.4. Initial quality control of the sequencing reads was performed using the built-in tools, including the Quality Control function, to evaluate read quality metrics, such as read length distribution, quality scores and potential adapter contamination. Low-quality reads and contaminants were trimmed or filtered out. The cleaned sequencing reads were mapped to the GRCh38.p14 reference genome using the Map Reads to Reference tool in the CLC Genomics Workbench. Gene expression levels were quantified using the Count Features tool. This step involved counting the number of reads mapped to each gene based on the annotation file corresponding to the reference genome. The output generated a count table indicating the raw read counts for each gene across all samples. Read counts were normalized using the trimmed mean of M method implemented in the R Bioconductor package edgeR (v.4.2.1)71<\/a> to determine the abundance of each gene. Next, we fit a negative binomial generalized log-linear model to estimate the quasi-dispersions with empirical Bayes moderation (glmQLFit function), followed by hypothesis testing with empirical Bayes quasi-likelihood F-tests. P-value adjustment was performed using the Bonferroni correction method.\u00a0These data have been deposited in the NCBI GEO under accession number GSE289744<\/a>.<\/p>\n GSEA of neurons<\/p>\n GSEA was conducted to investigate the enrichment of gene sets of mouse neurons cultivated in vitro. GSEA analysis was performed considering the Hallmark gene set (MH) from the Mouse MSigDB Collections (mh.all.v2024.1.Mm.symbols.gmt) and performing 1,000 permutations63<\/a>,72<\/a>.<\/p>\n SEM analysis<\/p>\n Fixed samples containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1\u2009M cacodylate buffer, pH\u20097.3, were washed with 0.1\u2009M cacodylate buffer, pH\u20097.3, post fixed with 1% cacodylate-buffered osmium tetroxide, washed with 0.1\u2009M cacodylate buffer, then in distilled water. Next, the samples were sequentially treated with Millipore-filtered 1% aqueous tannic acid, washed in distilled water, treated with Millipore-filtered 1% aqueous uranyl acetate, then rinsed thoroughly with distilled water. The samples were dehydrated with a graded series of increasing concentrations of ethanol, then transferred to a graded series of increasing concentrations of hexamethyldisilazane (HMDS) and air-dried overnight. The samples were mounted on double-stick carbon tabs (Ted Pella), previously mounted onto glass microscope slides. The samples were then coated under vacuum using a Balzer MED 010 evaporator (Technotrade) with platinum alloy for a thickness of 25\u2009nm, then immediately flash-carbon-coated under vacuum. The samples were transferred to a desiccator for examination at a later date. The samples were examined\/imaged in a JSM-5900 scanning electron microscope (JEOL) at an accelerating voltage of 5\u2009kV.<\/p>\n TEM<\/p>\n Samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1\u2009M cacodylate buffer, pH\u20097.3, then washed in 0.1\u2009M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate buffered tannic acid, post fixed with 1% buffered osmium tetroxide and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated and embedded in LX-112 medium. The samples were polymerized in a 60\u2009\u00b0C oven for approximately 3\u2009days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica), stained with uranyl acetate and lead citrate and examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80\u2009kV. Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques).<\/p>\n Luminex assay<\/p>\n Cytokine, chemokine and growth factor profiling of the cell culture supernatants was performed using the Luminex Mouse Immune Monitoring 48-Plex (ProcartaPlex; Invitrogen) according to the instructions provided by the manufacturer. The samples were not diluted before use in the assay and were assessed in triplicate. Then, 50\u2009\u00b5l of capture beads were added per well to the assay plate, followed by 50\u2009\u03bcl of cell culture supernatant. The assay plate was incubated overnight at 4\u2009\u00b0C and then at room temperature for 30\u2009min with shaking at 500\u2009rpm. After three washes, 25\u2009\u00b5l of detection antibody was added per well and incubated for 1\u2009h at room temperature with shaking at 500\u2009rpm. Then, 50\u2009\u00b5l of streptavidin-PE solution was added per well and incubated for 30\u2009min at room temperature with shaking at 500\u2009rpm. After three washes, 120\u2009\u00b5l of reading buffer was added per well and incubated for 5\u2009min. Data were acquired using the Luminex 200 analyzer and xPONENT v.4.2 software. Analysis was performed using Bio-Plex Manager v.6.1 software.<\/p>\n Animals and in vivo procedures<\/p>\n The different cell types used for in vivo experiments in this study, along with the figure panel presenting the results of their relevant analysis, are summarized in Supplementary Table 10<\/a>. A minimum of five mice per experimental group was used to ensure that appropriate statistical analyses could be conducted. This determination was made based on our preliminary data, the implementation of internal controls and the observed variability within the experimental groups. In each experiment conducted, all efforts to replicate the results were successful. All animal studies were carried out according to protocols approved by The University of Texas MD Anderson Cancer Center\u00a0(protocols 00000950-RN03, 00001522-RN01, 00001522-RN02, 00002342-RN00), NYU, Queen\u2019s University (protocols 2380 and 2393), and Universit\u00e9 de Montr\u00e9al (protocols 21046 and 21047) Institutional Animal Care and Use Committee. Mouse housing, husbandry and care practices met or exceeded the minimum requirements outlined in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (8th Edition). Disease development and progression were closely monitored. According to our approved protocol, mice with metastases were euthanized as soon as we noticed signs of discomfort in the mice or when the largest dimension of a tumour reached 20\u2009mm. In none of the experiments were these limits exceeded.<\/p>\n Animal housing<\/p>\n Mice were housed in standard environmental conditions (12\u2009h\u201312\u2009h light\u2013dark cycle; 23\u2009\u00b0C; food and water ad libitum) at facilities accredited by the Canadian Council of Animal Care (UdeM and Queen\u2019s University).<\/p>\n IACUC end points<\/p>\n As per our IACUC-approved protocol, the following end points have been used in all the experiments and have not been exceeded. Along with excessive body weight loss (maximum of 10%), the end points include excessive tumour volume (10% of animal\u2019s body weight; ~20\u2009mm\u2009\u00d7\u200920\u2009mm, not exceeded in any experiment), skin ulceration, necrosis, bleeding, infection and self-inflicted injury, prostration, lethargy, unresponsiveness to stimulation and\/or lack of grooming.<\/p>\n B6 UV-induced skin SCC cell lines<\/p>\n To generate murine skin SCC cell lines, p53-deficient (K14Cre; p53R172H\/flox) C57BL\/6 mice (4\u20136-week-old, both males and females) were exposed to 7\u2009kJ\u2009m\u22122\u2009s\u22121 of ultraviolet B light, 3 doses per week for 100 days. The hair on the mice\u2019s dorsal surface was shaved twice or thrice a week before UV exposure. This model has been shown to effectively simulate human cuSCC development, producing tumours with a genomic profile similar to those found in humans with cuSCC73<\/a>. Skin tumours (minimum 5\u2009mm in diameter and persistent for at least 2 weeks) started to develop 9\u2009months after the end of the UV-exposure protocol. Each tumour was split in half. One section was processed for FFPE, H&E stained and IF staining with anti-pan-keratin (type I) was performed for confirmation of cSCC diagnosis. The remaining tissue section was macrodissected and incubated in cell culture medium for 7\u201310 days. The cultured cells were sorted using the CD326 (EPCAM) antibody, and the resulting isolated epithelial cells were again incubated in cell culture medium, IF stained with anti-pan-keratin (type I) (E6S1S) antibody and DAPI to confirm their epithelial origin. The isolated malignant keratinocytes were injected intradermally (200,000 cells per 60\u2009\u03bcl) into C57BL\/6 WT mice, and their tumorigenicity was recorded. The newly developed tumours were assessed using H&E staining to confirm the histopathological diagnosis of cSCC. This process resulted in five distinct UVSCC cell lines (M2, M3, M4, M5 and M6) (Extended Data Fig. 3b<\/a>).<\/p>\n In vivo nerve injury modulation<\/p>\n We used denervation and surgical axotomy to assess in vivo nerve injury modulation of the immune response. For denervation, we used 6\u20138-week-old\u00a0male and female SKH1-Elite mice (SKH1-Hrhr, Charles River). For axotomy, we used 6\u20138-week-old male and female C57BL\/6J\u00a0mice (strain 000664, Jackson Laboratory) and 4\u20136-week-old female HU-NCG CD34+ humanized mice (Charles River). Denervation was performed under microscopy guidance, and an incision was made along the dorsal midline from the base of the neck to approximately 0.5\u2009cm above the tail. Using blunt forceps, we gently reflected the skin on the right side away from the flank to visualize the underlying tissue from the scapular fat pads near the neck to just above the hind limb. Next, we removed the nerves exclusively from the animal\u2019s right side at anatomical sites T3\u201312 by plucking from where the segments bend at the trunk wall to their entry sites into the skin. Then, we removed any nerves from the skin flap. Next, 7\u2009days after denervation, the mice were injected with the mouse (SKH1-Hrhr derived) ultraviolet-induced cSCC B610K cells (mouse cells generated by primary culture of UV-induced skin tumours in SKH1-Elite, hairless, euthymic and immunocompetent mice (Charles River; https:\/\/www.criver.com\/products-services\/find-model\/skh1-elite-mouse?region=3646<\/a>)74<\/a>. For axotomy, we made an incision along the dorsal midline from the base of the neck to roughly 0.5\u2009cm above the tail. Using blunt forceps, we gently reflected the skin on the right side away from the flank to visualize the underlying tissue from the scapular fat pads near the neck to just above the hind limb. Next, we used a scalpel to sever the nerves innervating the skin at anatomical sites T3\u201312, where the segments bend at the trunk wall to their entry sites into the skin. We did not pluck the nerves or remove any nerves from the skin flap. Then, 7\u2009days after axotomy, C57BL mice were injected with the mouse ultraviolet-induced cSCC M4 cells (Extended Data Fig. 3b<\/a> and Supplementary Table 3<\/a>). Hu-NCG CD34+ Humanized Mice were injected with the human IC8 cuSCC cell line (C.H.)37<\/a>. The human cell line HLA was tested at the Histocompatibility Typing Laboratory at the Hematopoietic Stem Cell Transplant program of MD Anderson Cancer Center. For the sham-operated control, we used blunt dissection to gently reflect the skin on the right side of the dorsal midline incision without removing or cutting the nerves. Then, 1\u2009week after tumour implantation, the mice were treated biweekly with either murine (intraperitoneal (i.p.), 250\u2009\u03bcg, RMP1-14) or human (for experiments with humanized mice) (i.p., 10\u2009mg per kg, cemiplimab-rwlc, NDC 61755-008-01) anti-PD-1 for 4 weeks75<\/a>,76<\/a>. We used the rat IgG2a (BE0089, 2A3, BioXCell) as a control.<\/p>\n IFN\u03b1 receptor blockade and anti-PD-1 antitumour efficacy<\/p>\n An Ifnar1-KO mouse model (B6(Cg)-Ifnar1^tm1.2Ees\/J, 028288, Jackson Laboratory) and WT C57BL\/6J mice (000664, Jackson Laboratory), aged 10\u201312 weeks,\u00a0both male and female, were used in this experiment. On day 0, UVSCC-M4 cells (200,000 cells in 30\u2009\u00b5l PBS) were inoculated into the footpads of Ifnar1-KO and WT mice. Then, 17\u2009days after inoculation (day 17), the mice underwent either injection of ethidium bromide (25\u2009\u00b5l of 0.5% solution) into the tibial nerve at the tumour\u2013normal skin interface to induce demyelination or a sham procedure involving PBS injection into the same location. Anti-PD-1 treatment (BioXCell Clone RMP1-14, BP0146; 250\u2009\u00b5g per mouse, i.p. in sterile PBS) was initiated 2\u2009days after demyelination (day 19) and administered twice weekly for a total of 6 doses. WT mouse groups, both with and without demyelination, were treated with anti-PD-1 alone or in combination with the STING agonist c-di-GMP (CDG; Invivogen, 25\u2009\u00b5g per mouse, intratumoural), administered according to the same schedule to enhance IFN type I signalling. An InVivoPlus rat IgG2a isotype control (anti-trinitrophenol, 2A3, BioXCell, BP0089) was used as a control. The tumour volumes were measured every 3\u2009days using callipers, and after euthanizing the mice, the footpads were excised, weighed and fixed in 10% buffered formalin for histopathological viability analysis. Pathologic assessment of treatment response was performed by histopathologic evaluation of H&E-stained sections of tumours\/tumour bed, as previously described19<\/a>. In brief, the tumour bed was evaluated, and the average percentages of viable tumour, necrosis, anucleate keratin, inflammatory infiltrate or fibrosis were estimated for each tumour sample.<\/p>\n IL-6 signalling blockade on anti-PD-1 efficacy<\/p>\n We evaluated the effect of IL-6 signalling blockade on anti-PD-1 efficacy in 6\u20138-week-old male C57BL mice (000664, Jackson Laboratory). One week after the orthotopic inoculation of mouse UV-induced cSCC M4 cells into the skin, mice received either an injection of ethidium bromide into the tumour periphery at the tumour-normal skin interface to induce demyelination or a sham injection. Anti-PD-1 treatment (BioXCell, RMP1-14, BP0146; 250\u2009\u00b5g per mouse, i.p.) was initiated 2\u2009days after demyelination and administered twice weekly for 8 doses over 4 weeks. To allow for the priming of immune cells against the tumour, the first two doses of anti-PD-1 were given alone. Subsequent anti-PD-1 doses were administered either alone or in combination with an anti-IL-6 receptor antibody (InVivoMAb anti-mouse IL-6, BioXCell, MP5-20F3, BE0046; 200\u2009\u00b5g per mouse, twice weekly for 3\u2009weeks). Tumour volumes and immune response outcomes were monitored throughout the experiment. Pathologic assessment of treatment response was performed by histopathologic evaluation of H&E-stained sections of tumours\/tumour bed, as previously described19<\/a>. In brief, the tumour bed was evaluated, and the average percentages of viable tumour, necrosis, anucleate keratin, inflammatory infiltrate or fibrosis were estimated for each tumour sample.<\/p>\n PNI over time (whiskers pad model): 8\u201310-week-old female C57BL mice (000664, Jackson Laboratory) were used. All procedures were approved by the New York University Institutional Animal Care. Animals were housed in a temperature-controlled, pathogen-free room under a 12\u2009h\u201312\u2009h light\u2013dark cycle (06:00\u201318:00) with ad libitum access to food and water. The MOC2 mouse oral squamous cell carcinoma cell lines (Sigma Chemicals) were cultured in IMDM\/F12 (2:1 mixture) with 5% FBS (Thermo Fisher Scientific), 1% penicillin\u2013streptomycin, 1% amphotericin, 5\u2009ng\u2009ml\u22121 EGF (Millipore), 400\u2009ng\u2009ml\u22121 hydrocortisone and 5\u2009mg\u2009ml\u22121 insulin. Under 3% isoflurane anaesthesia, 20\u2009\u03bcl of MOC2 cells (5,000 cells total) in serum-free culture medium was injected into the left whisker pad using a 10\u2009\u03bcl Hamilton syringe. The mice were randomly allocated to four groups. The experimental groups were defined by the interval between cancer cell injection and the subsequent euthanasia of the animals, resulting in tumour development periods of 7, 9, 11 and 13 days. The tumours were processed for histological assessment and mIF staining, focusing on identifying PNI by tumour cells and characterizing the perineural immune infiltrate.<\/p>\n MEA analysis of mouse tissue<\/p>\n Mouse tumours were quickly dissected from euthanized animals (orthotopic B610K intradermal model in SKH1-Elite animals and normal skin controls) and analysed by P.D.V.\u2019s\u00a0laboratory, as previously described77<\/a>. Tissues were immediately sectioned to generate tissue slices. One tumour slice\/sample was formalin-fixed, paraffin-embedded and processed for histological staining (H&E) to assess tumour presence. Electrical recordings were captured using a MEA1060-Inv-BC microelectrode array system (Multichannel Systems) with a perforated microelectrode array, 60pMEA100\/30iR-Ti MEAs (890335, Harvard Apparatus). The MEAs used contained 60 electrodes, of which one is a reference electrode. These electrodes are spaced 100\u2009\u00b5m apart in a 6\u2009\u00d7\u200910 grid (ten electrodes per row arranged in six columns generating a rectangle of electrodes onto which tissue slice is placed); each electrode is 30\u2009\u00b5m in diameter. The titanium nitride electrodes are contained within a glass ring, enabling us to maintain tissue slices under oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (119\u2009mM NaCl, 2.5\u2009mM KCl, 1\u2009mM NaH2PO4, 26.2\u2009mM NaHCO3, 11\u2009mM glucose, 1.3\u2009mM MgSO4, 1.5\u2009mM CaCl2); this buffer preserves neuronal functions. The perforated MEAs allow for a gentle suction (applied by a vacuum pump) to keep the tissue slice in contact with electrodes for the duration of recordings. This enables us to capture the electrical activity from every electrode for every second of each recording. As such, recordings from 59 electrodes per second per slice were collected (one electrode is a reference electrode). Recordings occurred at room temperature using a 25-kHz sampling frequency and a Butterworth second-order digital filter set to high-pass with a cut-off frequency of 10\u2009Hz (to eliminate slow FPs). A STG4000 stimulus generator (Multichannel Systems) was used for stimulation, which consisted of biphasic voltage, \u22120.5\u2009V and +0.5\u2009V each for 100\u2009\u00b5s and repeated after a 23\u2009ms interval. Stimulation was applied to selected electrodes; evoked spike responses were recorded. Two independent types of stimulations were used on each slice. The first consisted of 14 electrodes while the second consisted of 20 electrodes. The same electrode sets were stimulated for all slices analysed and the stimulation parameters were identical. Recordings continued after electrical stimulations were turned off to allow for capturing of activity back to baseline. In all cases, continuous electrical activity per second per electrode recordings were captured and analysed using the MC_Rach4.6.2 software (Multichannel Systems).<\/p>\n TEM analysis of mouse samples<\/p>\n TEM analysis was conducted on mouse tissue samples from a previously established model of PNI in the sciatic nerve78<\/a>. In brief, mice were perfused with 4% paraformaldehyde in 0.1\u2009M phosphate buffer (pH\u20097.4). Sciatic nerves were dissected and fixed for 2\u2009h. Fixation was performed using a modified reduced osmium impregnation method. The samples were embedded in fresh 100% durcupan. The processed samples were sectioned (1\u2009\u00b5m) and stained with 1% toluidine blue to identify the areas of interest. Thinner sections (70\u2009nm) were mounted onto slot grids and loaded onto the Zeiss grid holder for EM imaging. Images were taken with a beam acceleration of 20.0\u2009kV and a working distance of approximately 2.0\u2009mm, capturing 6,144\u2009px by 4,608\u2009px images with a pixel size of 100\u2009nm to 200\u2009nm. A Talos120C transmission electron microscope with the Gatan (4k \u00d7 4k) OneView Camera was used for high-resolution EM imaging. Ultrathin sections (50\u201360\u2009nm) were imaged at \u00d71,250.<\/p>\n Bulk RNA-seq analysis of lung metastasis-innervating neurons<\/p>\n Bulk RNA-seq analysis of lung-metastasis-innervating neurons was performed after intravenous injection of 5\u2009\u00d7\u2009105 B16F10-OVA melanoma cells or PBS vehicle into nociceptor neuron reporter mice (Trpv1cre::TdTomatofl\/WT). Then, 2\u2009weeks later, the mice were euthanized, and lung metastases were visually confirmed in the B16F10-eGFP-inoculated group. Jugular nodose ganglia (JNC) were dissected into ice-cold HEPES-buffered DMEM (Thermo Fisher Scientific, 12430062). For each biological replicate, JNC from four mice (two male, two female) were pooled and transferred into HEPES-buffered DMEM containing 1\u2009mg\u2009ml\u22121 collagenase IV (Sigma-Aldrich, C5138) and 2.4\u2009U\u2009ml\u22121 dispase II (Sigma-Aldrich, 04942078001), then incubated at 37\u2009\u00b0C for 70\u2009min. After washing with DMEM, ganglia were gently triturated using glass Pasteur pipettes of decreasing diameter. Cells were centrifuged at 200g over a 15% BSA gradient in PBS to remove debris, stained with SYTO 40 (10\u2009\u03bcM, Thermo Fisher Scientific, S11351) for 5\u2009min at room temperature to distinguish cells from axonal debris, washed with PBS and resuspended in sterile flow cytometry buffer (PBS with 2% FBS and 1\u2009mM EDTA). The cell suspension was filtered through a 70\u2009\u00b5m mesh (VWR, 10204-924) and nociceptor neurons (tdTomato+) were FACS-enriched on a BD FACSAria cell sorter, collected directly into 500\u2009\u00b5l TRIzol reagent (Invitrogen, 15596026), and stored at \u221280\u2009\u00b0C until RNA extraction, according to established protocols79<\/a>. Library preparation was conducted at the Institut de Recherche en Canc\u00e9rologie et en Immunologie (IRIC), Universit\u00e9 de Montr\u00e9al. The RNA quality was confirmed on an Agilent Bioanalyzer, with all samples achieving an RNA integrity number of \u22657.5. Libraries were prepared using a poly(A)-enrichment, single-stranded RNA-seq method (KapaBiosystems, KAPA RNA Hyperprep Kit, KR1352) and sequenced on the Illumina NextSeq500 platform with 75-cycle single-end reads. Basecalling was performed using Illumina RTA v.2.4.11, and demultiplexing was done using bcl2fastq v.2.20 (allowing for one mismatch in the index). Trimmomatic was used to remove adapter sequences and low-quality bases from the 3\u2032 end of each read, and the resulting high-quality reads were aligned to the GRCm38 mouse genome using STAR v.2.5.11, which also generated gene-level read counts. Differential expression analysis was performed using edgeR. The generalized linear model likelihood ratio test from edgeR was used to identify significant DEGs between groups. Genes were considered to be differentially expressed if they had an FDR-adjusted P\u2009<\u20090.05. log2-transformed fold changes and \u2212log10-transformed P values were calculated from the normalized data, and additional data analysis and visualization were carried out in RStudio. These data have been deposited in the NCBI GEO under accession number GSE292089<\/a>.<\/p>\n Mouse lines<\/p>\n Six- to ten-week-old male and female C57BL6J (000664); Trpv1cre (017769)80<\/a>, td-tomatofl\/fl (007908)81<\/a>, Dtafl\/fl (Jax, 009669)82<\/a> and NaV1.8cre (036564) mice were purchased from Jackson Laboratory. Atf3fl\/fl and Atf3GFP\/GFP were supplied by C. J. Woolf. All lines were backcrossed >6 generations on the C57BL6\/J background (H2-Kb). We used the cre\/lox toolbox to engineer the various mice lines used (Trpv1cre::tdTomatofl\/WT, NaV1.8cre::Atf3fl\/fl and littermate controls) by crossing heterozygote cre mice with homozygous loxP mice. Littermates with no Cre (that is, Trpv1WT::tdTomatofl\/WT, NaV1.8WT::Atf3fl\/fl) were used as controls referred to as WT for the Atf3\u00a0gene in the relevant figures. Mice of both sexes were used for these crosses. All Cre driver lines used were viable and fertile, and abnormal phenotypes were not detected. Offspring were tail-clipped; tissue was used to assess the presence of transgene by standard PCR, as described by Jackson Laboratory or the donating investigators. Offspring of both sexes were used at 6\u201310 weeks of age.<\/p>\n Cell lines<\/p>\n B16F10-mCherry-OVA83<\/a> (M. F. Krummel), B16F10-OVA (Biocytogen, 311537), B16F10-eGFP (Imanis, CL053) or non-tumorigenic keratinocytes (CellnTEC, MPEK-BL6100) were cultured in complete Dulbecco\u2019s modified Eagle\u2019s medium high glucose (DMEM, Corning, 10-013-CV) supplemented with 10% FBS (Seradigm, 3100) and 1% penicillin\u2013streptomycin (Corning, MT-3001-Cl), and maintained at 37\u2009\u00b0C in a humidified incubator under 5% CO2. All of the cell lines tested negative for mycoplasma, and none are listed by the International Cell Line Authentication Committee registry (v.11). Non-commercial cell lines (B16F10-OVA-mCherry) were authenticated using antibody (against OVA, mCherry) and\/or imaging as well as morphology and growth property. Commercial cell lines have not been further authenticated.<\/p>\n Cancer inoculation and volume measurement<\/p>\n Cancer cells were resuspended in PBS (Corning 21040CV) and injected into the mouse skin right flank (2.5\u2009\u00d7\u2009105 cells or 5\u2009\u00d7\u2009105 cells; intradermal, 100\u2009\u03bcl) or hindpaw (2\u2009\u00d7\u2009105 cells intradermal, 50\u2009\u03bcl). Growth was assessed daily using handheld digital calipers and the tumour volume was determined using the formula L\u2009\u00d7\u2009W2\u2009\u00d7\u20090.52 (ref. 84<\/a>), where L is the length and W is the width.<\/p>\n scRNA-seq analysis of tumour-innervating neurons<\/p>\n Six-week-old male and female littermate control (Trpv1WT::Diptheria-Toxinfl\/WT) mice were intradermally injected into the left paw with 2\u2009\u00d7\u2009105 B16F10-OVA cells or non-tumorigenic keratinocytes (MPEK-BL6). Then, 7\u2009days later, tumour-infiltrating neurons (or neurons in MPEK-injected skin) were retrogradely labelled by an acute intratumour injection of AAV9-CAG-mCherry-WPRE-SV40p (10\u2009\u00b5l, 5.5\u2009\u00d7\u2009109). Then, 14\u2009days after inoculation, the mice were euthanized, and the L3\u2013L5 DRG was collected and digested. Most CD45+ cells were depleted using the MagniSort mouse CD45 depletion kit (Invitrogen, 8804-6864). The remaining neurons were resuspended in FACS buffer (PBS with 2% FCS and EDTA) and stained with Viability Dye eFluor 780 (eBioscience, 65-0865-14) at 4\u2009\u00b0C for 15\u2009min. Subsequently, nuclei were stained with SYTO 40 (10\u2009\u00b5M, Thermo Fisher Scientific, S11351) for 5\u2009min at room temperature to distinguish cells from axonal debris. Cells were then purified by FACS on the BD FACSAria IIu cell sorter and subjected to single-cell barcoding using the Chromium Next GEM Single Cell 3\u2032 GEM, Library and Gene Expression v3.1 system (10x Genomics). Libraries were sequenced on an Illumina NovaSeq X platform, generating 120\u2009million total reads. Reads were aligned to the mouse reference genome using Cell Ranger (10x Genomics) and analysed with Seurat. Low-RNA-content and dead cells were excluded (nFeature_RNA\u2009>\u2009100, percent.mt\u2009<\u20095%). AAV9 expression was not detected and therefore subsequent analysis was performed using principal component analysis, which identified 19 clusters, which were assigned cell types using known markers and the Tabula Muris reference atlas. Clusters were grouped into major cell types, and cells that were not classified as neurons were excluded from the analysis. Four biological replicates per group were analysed to compare B16F10-injected mice and keratinocyte-injected mice. These data have been deposited in the NCBI GEO under accession number GSE292522<\/a>.<\/p>\n Single-cell GSEA<\/p>\n Enrichment of gene signatures at the single-cell level was evaluated by performing single-cell GSEA using the R package irGSEA85<\/a>. Enrichment calculation was performed using the ssGSEA86<\/a> method using a rank-normalized approach and generating an empirical cumulative distribution function for each cell. For this analysis, we considered the 50 gene expression signatures from the Mouse MSigDB MH collection: hallmark gene sets72<\/a>.<\/p>\n scRNA-seq\u00a0analysis of melanoma tumours in the\u00a0Atf3\u00a0conditional knockout model<\/p>\n B16F10-mCherry-OVA melanoma cells (5\u2009\u00d7\u2009105) were inoculated intradermally into the flank of 8-week-old male and female mice of which the nociceptor neurons were either permissive (NaV1.8WT::Atf3fl\/fl) or resistant (NaV1.8cre::Atf3fl\/fl) to injury. Then, 10\u2009days after tumour inoculation, the tumours were excised, minced with a razor blade and digested for 30\u2009min in DMEM containing 10\u2009mM HEPES, 1.6\u2009mg\u2009ml\u22121 collagenase IV (Sigma-Aldrich, C5138) and 10\u2009\u03bcg\u2009ml\u22121 DNase I (Sigma-Aldrich, 4942078001). The resulting cell suspensions were filtered through a 40\u2009\u03bcm mesh, and samples from two mice were pooled for each experiment. After red blood cell lysis (Gibco, A10492-01), cells were stained with Zombie Aqua viability dye (BioLegend, 423105) and a FITC anti-mouse CD45 antibody (BioLegend, 103107). Approximately 500,000 live immune cells, which included around 80% CD45+ cells, were sorted on the BD FACSAria III cell sorter into a collection buffer (PBS, 0.04% BSA and 50% FBS; Sigma-Aldrich, F4135), then fixed and prepared for single-cell library construction using the Chromium 10x Fixed RNA kits (PN-1000414 and PN-1000497). Libraries were sequenced on the Illumina NovaSeq X system and reads were aligned to the mouse reference genome using Cell Ranger (10x Genomics). Count matrices were generated and analysed in Seurat. Next, low-quality cells, potential doublets and multiplets were removed. Batch effect was corrected using canonical correlation analysis in Seurat. The threshold for dead cells was percent.mt\u2009<\u200910. The threshold to remove low-quality cells, potential doublets and multiplets were nFeature_RNA\u2009>\u2009200 and nFeature_RNA\u2009<\u20092500. Identification of the major immune phenotypes was performed based on the expression of key phenotypical markers (Extended Data Fig. 9i<\/a>). Differences in the frequency of tumour-associated leukocytes between groups were assessed using the Wilcoxon rank-sum test. Over-representation analysis was performed using the Enrichr R package87<\/a>. These data have been deposited in the NCBI GEO under accession number GSE292090<\/a>.<\/p>\n Immunophenotyping of melanoma tumours in the Atf3 conditional knockout model<\/p>\n Mice were euthanized 14 days post-tumour inoculation. Tumours were harvested and enzymatically digested in DMEM + 5% FBS (Seradigm, 3100) + 2 mg ml\u22121 collagenase D (Sigma, 11088866001) + 1 mg ml\u22121 Collagenase IV (Sigma, C5138-1G) + 40 \u00b5g ml\u22121 DNAse I (Sigma, 10104159001) under constant shaking (40 min, 37 \u00b0C). The cell suspension was centrifuged at 400g for 5 min. The pellet was resuspended in 70% Percoll gradient (GE Healthcare), overlaid with 40% Percoll and centrifuged at 500g for 20 min at room temperature with acceleration and deceleration at 1. The cells were aspirated from the Percoll interface and passed through a 70-\u03bcm cell strainer. Single-cell suspensions were prepared in FACS buffer (PBS containing 2% FCS and EDTA) and stained with either Zombie Aqua (BioLegend, 423102) for 15 min at room temperature or Viability Dye eFluor 780 (eBioscience, 65-0865-14) for 15 min at 4 \u00b0C. After washing, cells were incubated with Fc Block (0.5 mg ml\u22121; BD Biosciences, 553141) for 20 min at 4 \u00b0C, then stained for 30 min at 4 \u00b0C with anti-CD45-FITC (1:100; BioLegend, 103107) and anti-CD8-PerCP\/Cyanine5.5 (1:100; BioLegend, 100733). For cytokine expression analysis, cells were stimulated in vitro for 3 h with PMA (50 ng ml\u22121; Sigma-Aldrich, P1585), ionomycin (1 \u03bcg ml\u22121; Sigma-Aldrich, I3909) and GolgiStop (1:100; BD Biosciences, 554724). They were then fixed and permeabilized (1:100; BD Biosciences, 554714), stained with anti-IFN-\u03b3-APC (1:100; BioLegend, 505810) and analysed on a Cytoflex (Beckman) flow cytometer.<\/p>\n Immunofluorescence analysis of ATF3 expression in melanoma-innervating neurons<\/p>\n A total of 2\u2009\u00d7\u2009105 B16F10-OVA-mCherry melanoma cells were injected intradermally into the right hindpaw of C57BL\/6 mice or Atf3GFP\/GFP mice. Then, 14\u2009days after inoculation, the mice were anaesthetized with urethane (Sigma-Aldrich, U2500, 20%, 250\u2009\u03bcl, i.p.) and perfused with 10\u2009ml of PBS followed by 10\u2009ml of 4% paraformaldehyde (PFA, Sigma-Aldrich, 158127). The ipsilateral (tumour-bearing paw) and contralateral (control paw) L3\u2013L5 DRGs were collected, post-fixed in 4% PFA for 24\u2009h at 4\u2009\u00b0C and then immersed sequentially in 10%, 20% and 30% sucrose (Sigma-Aldrich, S8501) for 24\u2009h each. The tissues were embedded in Fisher Healthcare Tissue-Plus O.C.T. Compound (Thermo Fisher Scientific, 23-730-571), frozen at \u201380\u2009\u00b0C and cryosectioned into 15\u201330\u2009\u03bcm sections. The sections were blocked in PBS containing 0.2% Triton X-100, 5% BSA and 5% goat serum (Sigma-Aldrich, G9023) for 2\u2009h at room temperature, then incubated at 4\u2009\u00b0C for 48\u2009h with rabbit anti-mouse ATF3 (1:100, Abcam, ab207434) and chicken anti-mouse TUBB3 (1:500, Abcam, ab41489) in staining solution (PBS with 0.2% Triton X-100 and 3% goat serum). After three 15-minute washes in PBS containing 0.2% Triton X-100, the sections were incubated at 4\u2009\u00b0C for 16\u2009h with AF488 anti-chicken (1:1,000, Abcam, ab150169) and AF647 anti-rabbit (1:1,000, Invitrogen, A-21245) antibodies. The samples were then washed and stained with DAPI (2\u2009\u03bcg\u2009ml\u22121, Invitrogen, D1306) for 10\u2009min at room temperature. Images were acquired using the Nikon Ti2 microscope equipped with a Photometrics Prime 95B camera. Using ImageJ or Nikon Elements software, circular ROIs were drawn around each neuron and nucleus. The mean grey values for ATF3 and TUBB3 fluorescence (in a.u.) within each ROI were exported for further analysis in Microsoft Excel. The ratio of ATF3+ neurons to TUBB3+ neurons was determined, indicating increased ATF3 expression in ipsilateral (tumour-bearing) L3\u2013L5 DRGs compared with the contralateral side. In parallel, a threshold-based method was also applied to define the relative number of ATF3+ nuclei, which also show increased relative expression in ipsilateral (tumour-bearing) L3\u2013L5 DRGs neurons. Values from Atf3GFP\/GFP mice independently validated these findings and are not shown.<\/p>\n Statistical analysis<\/p>\n Unpaired Wilcoxon rank-sum tests were conducted to analyse immunohistochemical, in vitro and mouse data. Survival was analysed using the Kaplan\u2013Meier method and compared using the log-rank test. P values of less than 0.05 were considered to indicate nominal statistical significance. Owing to the variance of xenograft growth in control mice, we used at least three mice per genotype to give 80% power to detect an effect size of 20% with a significance level of 0.05. The number of independent mice used is listed in the figure legend for all mouse experiments. No statistical methods were used to predetermine the sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. To assess differences in cytokine levels between groups, we used a nested one-way analysis of variance (ANOVA) to account for both technical and biological replicates, followed by Tukey\u2019s post hoc test. The cytokine levels were standardized by calculating z scores based on the mean for plotting purposes. To compare tumour growth in mouse models under different interventions, tumour volume was recorded at multiple timepoints. In this way, we used a mixed-effects model (REML) to compare tumour growth curves and account for repeated tumour measures. Intervention groups, time and their interaction were considered to be fixed effects, and individual animals were considered to be random factors. Differences among growth curves were considered to be significant if the P value from the fixed-effects test for the interaction between the intervention group and time was lower than 0.05. Moreover, post hoc Tukey\u2019s multiple-comparison test was conducted for pairwise comparisons between groups at specific timepoints. Analyses were performed using JMP Pro v.17.0 software and R.<\/p>\n Reporting summary<\/p>\n