Tumour–brain crosstalk restrains cancer immunity via a sensory–sympathetic axis
Researchers employed a suite of genetically engineered mouse strains to dissect how lung‑tumor growth interacts with sensory and sympathetic nerves, immune cells, and adrenergic signaling.
Mouse models and tumor induction
The study centered on KrasLSL‑G12D/+. p53fl/fl (KP) mice, which were crossed with Rosa26‑LSL‑tdTomato to visualize tumor cells. Additional lines—including Trpv1‑cre, Rosa26‑LSL‑DTR, Npy2r‑IRES‑cre, P2ry1‑IRES‑cre, BATF3‑knockout, and Adrb2‑knockout—were sourced from The Jackson Laboratory or provided by collaborators. Tumors were initiated either by intratracheal delivery of 2.5 × 10⁸ PFU Ad5mSPC‑Cre virus (autochthonous model) or by tail‑vein injection of 1.5 × 10⁵ cultured KP cells (orthotopic transplant). Tissue collection occurred 12–16 weeks after viral induction or 24–32 days after cell inoculation.
Neural manipulation and tracing
Vagal nodose ganglia (VNG) were exposed surgically for diphtheria‑toxin (DT)–mediated ablation of Trpv1⁺ neurons, chemical denervation with resiniferatoxin (RTX), or viral tracing. Anterograde tracing used AAV‑flex‑tdTomato or AAV‑eGFP, while retrograde tracing employed AAVretro‑hSyn‑flex‑mCherry injected intratracheally. Chemogenetic tools (hM4Di, hM3Dq) were delivered to the rostral ventrolateral medulla (RVLM) or lung‑innervating VSNs, and activation or silencing was driven by clozapine‑N‑oxide (CNO) dosing.
Immune and cellular analyses
Flow cytometry involved retro‑orbital injection of PE‑CF594 anti‑CD45 to exclude circulating leukocytes, followed by enzymatic digestion of lungs, filtration, and staining with a comprehensive antibody panel (e.g., CD8a, CD4, PD‑1, ARG1). Single‑cell RNA sequencing of vagal nodose ganglia captured 9,015 healthy and 13,073 tumor‑bearing cells, identifying Phox2b⁺ vagal sensory neurons (VSNs) and lung‑innervating subsets via Kcng1 expression.
Linking nerve activity to tumor outcomes
Noradrenaline measurements employed perchloric‑acid homogenization and HPLC/ECD detection, while RT‑qPCR quantified adrenergic receptors (Adrb1, Adrb2) and Arg1 transcripts. Systemic CGRP blockade (BIBN4096) and aerosolized salbutamol were administered to interrogate neuro‑immune pathways. Alveolar macrophage depletion used clodronate liposomes, and T‑cell depletion employed anti‑CD4/CD8 antibodies.
Clinical relevance
Survival analysis of 518 lung adenocarcinoma patients from TCGA linked combined VSN‑sympathetic gene signatures to overall outcomes, with high‑scoring groups showing distinct CD8⁺ T‑cell signatures. These findings suggest that neural signatures may serve as prognostic biomarkers or therapeutic targets.
Frequently Asked Questions
What mouse strains were used in the experiments?
The study employed KP (KrasLSL‑G12D/+; p53fl/fl) mice, Rosa26‑LSL‑tdTomato, Trpv1‑cre, Rosa26‑LSL‑DTR, Npy2r‑IRES‑cre, P2ry1‑IRES‑cre, BATF3‑knockout, and Adrb2‑knockout lines, among others.
How were lung tumors initiated in the mice?
Autochthonous tumors were induced by intratracheal injection of 2.5 × 10⁸ PFU Ad5mSPC‑Cre virus, while orthotopic tumors were generated by tail‑vein injection of 1.5 × 10⁵ cultured KP cells.
What techniques were used to trace lung‑innervating sensory nerves?
Researchers performed anterograde tracing with AAV‑flex‑tdTomato/eGFP injected into the nodose ganglia and retrograde tracing by delivering AAVretro‑hSyn‑flex‑mCherry intratracheally into tumor‑bearing lungs.
What questions does this line of research raise for future studies on lung cancer and the nervous system?