Mice and in vivo procedures
The KrasLSL−G12D/+; p53fl/fl (KP) mouse model has been covered in earlier studies. In this research, KP mice were bred with Rosa26LSL−tdTomato mice, resulting in a new KP; LSL-tdTomato line. Various wild-type strains, including C57BL/6 J, Trpv1-cre, and others were sourced from The Jackson Laboratory. The Trpv1-cre and LSL-DTR strains were crossed to create Trpv1-cre; LSL-DTR mice. Previously characterized Npy2r-IRES-cre and P2ry1-IRES-cre strains were also crossed with LSL-DTR for further experimentation. Cryopreserved sperm from Trpv1-DTR mice was obtained from M. Hoon, and rederived mice were interbred with KP strains to form KP; Trpv1-DTR mice. Additionally, BATF3 knockout strains were kindly provided by M. Haldar, and ADRB2 knockout mice came from S. Thomas, who helped generate Trpv1-cre; LSL-DTR; Adrb2−/− mice.
To induce tumors in the autochthonous model, KP mice received an intratracheal injection of 2.5 × 108 plaque-forming units of Ad5mSPC-Cre virus. Tissues were harvested between 12 and 16 weeks post-induction. In the orthotopic transplant model, 1.5 × 105 KP cells were injected via the tail vein. These cells originated from lung tumors in KP mice and were maintained at low passage in a specific media before use. They were cultured in DMEM supplemented with fetal bovine serum, L-glutamine, and Penicillin-Streptomycin at 37 °C with 5% CO2. Prior to usage, the cells were washed multiple times and resuspended in a balanced salt solution, ensuring no mycoplasma contamination. Tissues were taken 24 to 32 days after tumor cell inoculation.
For bone marrow chimeras, either Npy2r-IRES-cre; LSL-DTR or Trpv1-cre; LSL-DTR mice underwent lethal irradiation. They were then injected with bone marrow cells from donors. Over a two-week period, Sulfatrim was administered, along with fecal pellets from unaffected littermates to help restore microbiome diversity. For mixed chimeras, donor cells were combined in equal parts before injection into
the recipient mice. Tumor induction occurred seven weeks post bone marrow reconstitution, with reconstitution efficiency checked to remain above 95% in trials.
For the selective ablation of vagal sensory neurons (VSNs), ganglia were exposed and treated under anesthesia with a micropipette containing DT in PBS. Surgical wounds were treated and healed in a recovery period of 2-3 weeks. Similarly, to chemically denervate Trpv1+ VSNs, injections were made in each ganglion using either RTX or a vehicle; recovery was also set at about 3 to 4 weeks before proceeding with experiments.
Anterograde tracing involved the injection of viral vectors into VNGs, while retrograde tracing utilized a different viral construct injected into the lungs. Chemogenetic manipulation of RVLM neurons was conducted, followed by recovery and experimental initiation. This included ongoing injections of CNO amidst tumor inoculation procedures.
For manipulating VSNs in tumors, different constructs were injected intratracheally before subsequent analysis. Systemic CGRP blockade and specific local CGRP blockade followed established protocols, starting before or soon after tumor inoculation. Aerosolized treatments and macrophage depletions were managed through set schedules prior to concluding experiments, ensuring a high level of rigor throughout.
Male and female mice aged between 6 to 24 weeks were used, with groups randomly assigned. Ethical consideration was paramount, as all practices were vetted by relevant committees, maintaining humane standards.
Flow cytometry and FACS sorting
For the analysis of immune cells, retro-orbital injections of PE-CF594 conjugated anti-CD45 were performedjust before euthanasia. Lungs were harvested and processed into a single-cell suspension before staining and analysis using a series of antibodies. Various cell-specific markers were utilized in order to achieve a clear profile of the immune cells involved.
Post-staining, flow cytometry was employed, and for sorting, tissues were once again digested. The relevant cells were filtered, stained, and separated via FACS, with the intention of isolating cells for further RNA analyses.
Alveolar macrophage culture
For macrophage differentiation, bone marrow samples were grown in a specific medium over nine days, with certain growth factors added. The differentiated cells underwent stimulation with noradrenaline and tumor extracts, followed by washing and collection for RNA extraction.
VSN culture
Nodose ganglia neurons were meticulously dissected and prepared before analysis. They were cultured along with tumor cells in different setups to investigate their interactions, especially under varying conditions of external factors like NGF.
Each experiment involved pooling neurons from multiple mice, ensuring replicates for clarity in results. Neurite growth was then quantified through rigorous imaging and analysis processes, all conducted blinded to ensure data integrity.
Noradrenaline treatment of tumor cells
Frozen stocks of tumor cells were thawed and cultured briefly. Continuous treatment with noradrenaline was given, and adherent cells were counted afterward to assess proliferation and exposure effects.
Histology and immunofluorescence staining of lung tissue
Lung samples were fixed and stained for various assessments. Scanned images were analyzed to quantify tumor burden across lung sections, while specific antibodies were used to visualize cellular components and differentiation markers.
Immunofluorescence staining of brain tissue
Brains underwent fixation before dehydration and embedding. Similar staining techniques were employed to assess relevant markers within brain sections, with imaging performed under specified conditions to yield high-quality visuals.
Immunofluorescence staining of ganglia
Ganglia were processed akin to brain samples, with comprehensive antibody panels assessing multiple neuronal markers for further understanding their functional roles.
RNAscope in situ hybridization, imaging and quantification
RNAscope assays were deployed to visualize gene expression at the cellular level in various tissues. This involved meticulous preparation and imaging, following established protocols for clarity of the respective markers used.
Whole-mount clearing and immunofluorescence staining
Using advanced clearing techniques allowed for visualization of nerve fibers within the lung tissue, with subsequent staining amplifying the signals for targeted markers.
Multiplex volumetric imaging with clearing-enhanced 3D
After a careful fixation and embedding process, samples were treated for imaging at high resolutions. This methodology emphasizes the reliability of the observed interactions present within the tissues sampled.
Noradrenaline measurement
Lung tissues were processed and analyzed for noradrenaline concentrations through an established HPLC protocol, ensuring accurate biochemical profiling.
RT–qPCR
Total RNA was isolated for subsequent reverse transcription and quantitative PCR. A standardized set of primers was used to assess gene expression across different conditions.
scRNA-seq and analysis
Vagal nodose ganglia were harvested and processed from groups of healthy and tumor-bearing mice. Single-cell sequencing was performed, with the data processed to yield insights into cell type distributions and gene expression profiles, especially focusing on lung-innervating VSN populations.
Clinical data analysis
For survival outcomes in LUAD patients, gene expression profiles were correlated with clinical data. External validation sets and statistical methods were employed to derive meaningful insights between nervous system activity and immune cell interactions.
Statistics
Statistical assessments were rigorously conducted using appropriate software, ensuring data significance was accurately reported alongside relevant metrics throughout the research.
Reporting summary
Extended information concerning research design and methodology can be found in linked resources.
