Mice and in vivo procedures
KrasLSL−G12D/+; p53fl/fl (KP) mice have been described previously55. KP mice were crossed with Rosa26LSL−tdTomato mice to generate KP; LSL-tdTomato mice. Wild-type C57BL/6 J, Trpv1-cre, Rosa26LSL−DTR (LSL-DTR), Vglut2-cre, Baf53b-cre, Pparγfl/fl and CD11c-cre mice were purchased from The Jackson Laboratory. Trpv1-cre and LSL-DTR mice were crossed to produce Trpv1-cre; LSL-DTR mice. Npy2r-IRES-cre and P2ry1-IRES-cre mice have been previously described14; they were crossed with LSL-DTR mice to produce Npy2r-IRES-cre; LSL-DTR and P2ry1-IRES-cre; LSL-DTR mice, respectively. Cryopreserved sperms of Trpv1-DTR mice were provided by M. Hoon (National Institute of Dental and Craniofacial Research); rederived Trpv1-DTR mice were crossed with KP mice to produce KP; Trpv1-DTR mice. BATF3-knockout mice were generously provided by M. Haldar. ADRB2 knockout mice were a gift from S. Thomas; they were crossed with Trpv1-cre; LSL-DTR mice to produce Trpv1-cre; LSL-DTR; Adrb2−/− mice.
For tumour induction in the autochthonous model, KP mice were intratracheally injected with 2.5 × 108 plaque-forming units of Ad5mSPC-Cre virus (Viral Vector Core, University of Iowa). Tissues were collected 12–16 weeks post tumour initiation. In the orthotopic transplant model, 1.5 × 105 KP cells were intravenously injected into the tail vein. The KP cell line was derived from autochthonously arising lung tumours in KP mice and stocked at low passage; they were cultured in DMEM (Corning) supplemented with fetal bovine serum (FBS, 10%; Gibco), L-glutamine (2 mM; Gibco), Penicillin-Streptomycin (50 U/mL; Gibco) at 37 °C with 5% CO 2 . Before injection, cells were washed 3 times with Hank’s balanced salt solution (HBSS) and resuspended at a density of 1.5 × 106 cells/mL in endotoxin-free HBSS. Cells were tested negative for mycoplasma before use in all experiments. Tissues were collected 24–32 days post tumour cell inoculation.
For bone marrow chimera experiments, Npy2r-IRES-cre; LSL-DTR or Trpv1-cre; LSL-DTR mice were lethally irradiated with a Gammacell-40 Irradiator (5.5 Gy x 2, 3.5 h apart) and then retro-orbitally injected with 4-8 million bone marrow cells from donor mice. Sulfatrim (Pharmaceutical Associates, Inc) water was given for 2 weeks and changed twice weekly. Fecal pellets from unirradiated littermates were transferred to the chimeric mice for a period of 4 weeks after the removal of sulfatrim water to restore normal microbiota. Specifically, for mixed bone marrow chimera experiments, bone marrow cells from wild-type CD45.1 donors or Adrb2−/− donors were mixed in a 1:1 ratio with cells from CD11c-cre; Ppargfl/fl donors and then injected into the recipient mice as described above. Tumour induction was performed 7 weeks post bone marrow reconstitution. Reconstitution efficiency was confirmed by congenic markers to be above 95% in all experiments.
For DT-mediated ablation of VSNs, vagal nodose ganglia were surgically exposed under anaesthesia. Briefly, an incision was made along the ventral surface of the neck, and blunt dissection was applied. A micropipette containing 20 ng DT (Sigma-Aldrich) in 120 nL phosphate-buffered saline (PBS) as well as 0.05% Fast Green (Sigma-Aldrich) was inserted into the nodose/jugular complex. DT solution was injected using a Nanoject II injector (Drummond Scientific Company). The process was repeated for the VNG on the other side of the body. Surgical wounds were closed with CP Medical Sutures (661B, CP Medical). Subcutaneous injection of sustained release Meloxicam was provided as postoperative care. Mice were allowed to recover from surgery for 2-3 weeks before proceeding to downstream experimental procedures.
For chemical denervation of Trpv1+ VSNs, VNG were surgically exposed as described above. Each mouse was injected with 25 ng RTX (Santa Cruz) or vehicle (PBS) as well as 0.05% Fast Green per ganglion. The injection was repeated for the VNG on the other side of the body. Mice were allowed to recover from surgery for 3 to 4 weeks before proceeding to downstream experimental procedures.
For anterograde tracing of VSNs in lung tumours, VNG were surgically exposed as described above. A micropipette containing AAV-flex-tdTomato (Addgene 28306-AAV9) or AAV-eGFP (UNC AV5221), as well as 0.05% Fast Green FCF (Sigma-Aldrich) was inserted into the nodose/jugular complex. The process was repeated for the VNG on the other side of the body. Mice were allowed to recover for 2 weeks from surgery before proceeding to downstream experimental procedures. For retrograde tracing of lung-innervating sensory nerves, 5 μl of AAVretro-hSyn-flex-mCherry (Addgene 50459-AAVrg) was mixed with 45 μl of PBS and intratracheally injected to the lungs of tumour-bearing mice.
For chemogenetic manipulation of RVLM neurons, 200 nl AAV2/9-syn-DIO-hM4Di-mcherry or AAV2/9-CAG-Flex-GFP (Boston Children’s Hospital Viral Core) was injected into the RVLM at anterior–posterior −6.35, medial–lateral ±1.35, dorsal–ventral –6.3 relative to bregma (coordinates were identified using the Paxinos Mouse Brain Atlas). Subcutaneous injection of sustained release Meloxicam was provided as postoperative care. Mice were allowed to recover from the stereotaxic surgery before initiation of any experiments; mice with little or no reporter expression in the brain indicative of technical issues were removed from analyses. CNO (Fisher Scientific, A3317-50) was injected peritoneally (1 mg kg−1) twice daily starting on the same day of tumour inoculation.
For chemogenetic silencing of lung-innervating VSNs, 5 μl of AAVretro-hSyn-flex-mCherry (Addgene 50459-AAVrg), or AAVretro-hSyn-flex-hM4Di-mCherry (Addgene 44362-AAVrg) was mixed with 45 μl of PBS and intratracheally injected to mice. CNO was injected intraperitoneally (1 mg kg−1) twice daily starting 5 days post tumour inoculation and continuing till experimental endpoints. For chemogenetic activation of lung-innervating VSNs in healthy mice, 5 μl of AAVretro-hSyn-flex-mCherry (Addgene 50459-AAVrg), or AAVretro-hSyn-flex-hM3Dq-mCherry (Addgene 44361-AAVrg), was mixed with 45 μl of PBS and intratracheally injected. One dose of CNO was injected intraperitoneally (1 mg kg−1) 90 min before tissue collection.
For systemic CGRP blockade, BIBN4096 (Tocris) was intraperitoneally injected at 2 mg kg−1 daily; for lung local CGRP blockade, BIBN4096 (Tocris) was intratracheally injected at 3 µg per mouse every other day. Treatment was started one week post tumour inoculation or two days before tumour inoculation and lasted until the experimental endpoint.
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