Identification of tumour-infiltrating myeloid subsets associated with overall survival in lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a primary subtype of lung cancer with limited therapeutic options and poor prognosis, and tumour-infiltrating myeloid cells (TIMs) are key regulators of LUSC. However, the correlation between the abundance of TIM subtypes and clinical outcomes of LUSC remains unexplored. This study aimed to develop and validate a prognostic model for low- and high-risk patients with LUSC based on myeloid cell microenvironments. TIM markers in the tumoural (T) and stromal (S) regions were quantified using immunohistochemistry for 502 LUSC patients. L1-penalized Cox regression was used to develop a myeloid survival score (MSS) model based on the training cohort, followed by validation in distinct cohorts from multiple centres. RNA sequencing and immunostaining were used to examine the mechanisms of myeloid cells in LUSC progression and predict potential drug targets and therapeutic agents. Of the 12 myeloid markers, CD163T, CD163S, and S100A12T were highly associated with overall survival (OS) in LUSC patients. The MSS of the three myeloid signatures accurately categorized LUSC patients into risk categories, with an observable difference in OS between the training and validation cohorts. Tumours with high MSS were associated with enhanced antioxidative ability and hedgehog signalling and a shift to a more pro-tumorigenic microenvironment, accompanied by a reduced tumour cell immunogenicity and increased CD8+ T cell exhaustion patterns. Additionally, in high-risk patients, potential drug targets and compounds regulating hedgehog signalling were identified. Our study provides the first prognostic myeloid signature for LUSC, which may help advance precision medicine. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

SLC3A2 promotes tumor-associated macrophage polarization through metabolic reprogramming in lung cancer.

Tumor-associated macrophages (TAMs) are one of the most abundant immunosuppressive cells in the tumor microenvironment and possess crucial functions in facilitating tumor progression. Emerging evidence indicates that altered metabolic properties in cancer cells support the tumorigenic functions of TAMs. However, the mechanisms and mediators the underly the cross-talk between cancer cells and TAMs remain largely unknown. In the present study, we revealed that high solute carrier family 3 member 2 (SLC3A2) expression in lung cancer patients was associated with TAMs and poor prognosis. Knockdown of SLC3A2 in lung adenocarcinoma cells impaired M2 polarization of macrophages in a coculture system. Using metabolome analysis, we identified that SLC3A2 knockdown altered the metabolism of lung cancer cells and changed multiple metabolites, including arachidonic acid, in the tumor microenvironment. More importantly, we showed that arachidonic acid was responsible for SLC3A2-mediated macrophage polarization in the tumor microenvironment to differentiate into M2 type both in vitro and in vivo. Our data illustrate previously undescribed mechanisms responsible for TAM polarization and suggest that SLC3A2 acts as a metabolic switch on lung adenocarcinoma cells to induce macrophage phenotypic reprogramming through arachidonic acid.

Dvl2 facilitates the coordination of NF-κB and Wnt signaling to promote colitis-associated colorectal progression.

Colitis-associated colorectal cancer (CAC) arises due to prolonged inflammation and has distinct molecular events compared with sporadic colorectal cancer (CRC). Although inflammatory NF-κB signaling was activated by pro-inflammatory cytokines (such as TNFα) in early stages of CAC, Wnt/ß-catenin signaling later appears to function as a key regulator of CAC progression. However, the exact mechanism responsible for the cross-regulation between these 2 pathways remains unclear. Here, we found reciprocal inhibition between NF-κB and Wnt/ß-catenin signaling in CAC samples, and the Dvl2, an adaptor protein of Wnt/ß-catenin signaling, is responsible for NF-κB inhibition. Mechanistically, Dvl2 interacts with the C-terminus of tumor necrosis factor receptor 1 (TNFRI) and mediates TNFRI endocytosis, leading to NF-κB signal inhibition. In addition, increased infiltration of the pro-inflammatory cytokine interleukin-13 (IL-13) is responsible for upregulating Dvl2 expression through STAT6. Targeting STAT6 effectively decreases Dvl2 levels and restrains colony formation of cancer cells. These findings demonstrate a unique role for Dvl2 in TNFRI endocytosis, which facilitates the coordination of NF-κB and Wnt to promote CAC progression.

Drebrin promotes lung adenocarcinoma cell migration through inducing integrin ß1 endocytosis.

Lung adenocarcinoma (LUAD) is the most common type of lung cancers, which remains the leading cause of cancer-related death worldwide. Drebrin can promote cell migration and invasion with poor prognosis, but its roes in LUAD tumor progression remains unknown. We showed that the expression of Drebrin was upregulated in clinical LUAD samples. A Kaplan-Meier survival analysis showed that a high expression of Drebrin predicated poor prognosis in LUAD. In vitro, Drebrin promoted anchorage-independent growth and migration of LUAD cells. Drebrin interacted with dynamin through CT domain, and served as an adaptor to promote LUAD cell migration through inducing integrin ß1 endocytosis. Thus, this study demonstrated the critical role of Drebrin in LUAD and associated mechanism.

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

Complex roles of the actin-binding protein Girdin/GIV in DNA damage-induced apoptosis of cancer cells.

The actin-binding protein Girdin is a hub protein that interacts with multiple proteins to regulate motility and Akt and trimeric G protein signaling in cancer cells. Girdin expression correlates with poor outcomes in multiple human cancers. However, those findings are not universal, as they depend on study conditions. Those data suggest that multiple aspects of Girdin function and its role in tumor cell responses to anticancer therapeutics must be reconsidered. In the present study, we found that Girdin is involved in DNA damage-induced cancer cell apoptosis. An esophageal cancer cell line that exhibited high Girdin expression showed a marked sensitivity to UV-mediated DNA damage compared to a line with low Girdin expression. When transcriptional activation of endogenous Girdin was mediated by an engineered CRISPR/Cas9 activation system, sensitivity to DNA damage increased in both stationary and migrating HeLa cancer cells. High Girdin expression was associated with dysregulated cell cycle progression and prolonged G1 and M phases. These features were accompanied by p53 activation, which conceivably increases cancer cell vulnerability to UV exposure. These data highlight the importance of understanding complex Girdin functions that influence cancer cell sensitivity to therapeutics.

CD109 regulates in vivo tumor invasion in lung adenocarcinoma through TGF-ß signaling.

Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-ß binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-ß activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-ß signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.

Dephosphorylation of Girdin by PP2A inhibits breast cancer metastasis.

Dysfunction of Girdin plays a crucial role in the development of a variety of tumors. Phosphorylated regulation of Girdin has been studied extensively. However, how Girdin is dephosphorylated remains unclear. In this study, we report a mechanism of Girdin dephosphorylation and the importance of this mechanism in the migration of breast cancer cells. We show that the protein phosphatase 2A (PP2A) complex can bind to Girdin via the modulating B subunit. Overexpression or knockdown of PP2A inhibits or increases the phosphorylation of Girdin at serine 1416, respectively. PP2Ac-induced Girdin dephosphorylation is involved in the inhibition of breast cancer cell migration. Furthermore, in human breast cancer samples, PP2Ac expression is negatively correlated with the phosphorylation of Girdin, and low expression of PP2Ac is correlated with tumor stage, grade and lymph node metastasis of breast cancer. These data indicate that PP2A regulates Girdin dephosphorylation and highlight the critical role of this pathway in breast cancer metastasis.

Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization.

Pathological observations show that cancer cells frequently invade the surrounding stroma in collective groups rather than through single cell migration. Here, we studied the role of the actin-binding protein Girdin, a specific regulator of collective migration of neuroblasts in the brain, in collective cancer cell migration. We found that Girdin was essential for the collective migration of the skin cancer cell line A431 on collagen gels as well as their fibroblast-led collective invasion in an organotypic culture model. We provide evidence that Girdin binds to ß-catenin that plays important roles in the Wnt signaling pathway and in E-cadherin-mediated cell-cell adhesion. Girdin-depleted cells displayed scattering and impaired E-cadherin-specific cell-cell adhesion. Importantly, Girdin depletion led to impaired cytoskeletal association of the ß-catenin complex, which was accompanied by changes in the supracellular actin cytoskeletal organization of cancer cell cohorts on collagen gels. Although the underlying mechanism is unclear, this observation is consistent with the established role of the actin cytoskeletal system and cell-cell adhesion in the collective behavior of cells. Finally, we showed the correlation of the expression of Girdin with that of the components of the E-cadherin complex and the differentiation of human skin cancer. Collectively, our results suggest that Girdin is an important modulator of the collective behavior of cancer cells.

Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

Girdin/GIV regulates transendothelial permeability by controlling VE-cadherin trafficking through the small GTPase, R-Ras.

Vascular permeability is regulated by intercellular junction organization of endothelial cells, the dysfunction of which is implicated in numerous pathological conditions. Molecular mechanisms of how endothelial cells regulate intercellular junction in response to extracellular signals, however, have so far remained elusive. This study identified that Girdin (also termed GIV), an Akt substrate functioning in post natal angiogenesis, was expressed in a mature endothelial monolayer, where it regulated VE-cadherin trafficking to maintain vascular integrity. Girdin depletion abrogated VEGF-induced VE-cadherin endocytosis and the disassembly of adherens junctions in a monolayer of endothelial cells, thus leading to a significant decrease in the permeability. We also showed that activated R-Ras, a member of the Ras family GTPase, known to be a master regulator of transendothelial permeability, interacts with Girdin, and facilitates the complex formation between Girdin and VE-cadherin in endothelial cells. However, the increased permeability mediated by the loss of R-Ras was rescued by Girdin depletion, thus suggesting that the interaction of Girdin with R-Ras functions in VE-cadherin trafficking pathways distinct from endocytosis. The recycling of VE-cadherin was promoted by the exogenous expression of the active mutant of R-Ras, which was attenuated in the Girdin-depleted endothelial cells. These results show that Girdin regulates transendothelial permeability in synergy with R-Ras and VE-cadherin in an endothelial monolayer.

Potential involvement of kinesin-1 in the regulation of subcellular localization of Girdin.

Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1, a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell-cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain.

Determination of sugar content in honey using LC-Raman and programmable pump-Raman methods.

We combined (i) liquid chromatography and Raman spectrometry (LC-Raman) and (ii) programmable pump and Raman spectrometry (PP-Raman) to separate and identify compounds in a mixture. These techniques were applied to conduct a quantitative analysis of the sugars in honey. The spectral and temporal axes of the LC-Raman data were analyzed using the MCR-ALS analysis procedure, which enabled the separation and identification of four sugars (glucose, fructose, sucrose, and trehalose). The PP-Raman method was employed to examine the sugar concentration dependence of the intensity pattern of the Raman spectrum, and the linear concentration dependence of the intensity was obtained. The sugar contents were quantitatively determined from the integrated area of the elution peaks. The result was consistent with those derived from mass spectrometry and previous studies. The origin of the errors in the derived sugar contents is discussed. Our study presents a novel quantitative LC-Raman spectrometric method that does not rely on resonance or surface enhancement effects.

E3 ligase Trim35 inhibits LSD1 demethylase activity through K63-linked ubiquitination and enhances anti-tumor immunity in NSCLC.

Targeting lysine-specific histone demethylase 1A (LSD1) can improve tumor immunogenicity of poorly immunogenic tumors, such as non-small cell lung cancer (NSCLC), with elevated T cell infiltration and sensitize tumors to anti-PD-1 therapy. However, the lack of reliable biomarkers limits utilization of LSD1 inhibitors in cancer therapy. Here, we identify an E3 ligase, Trim35, as an effective biomarker for high activity of LSD1 to predict prognosis of LSD1-targeted therapy as well as immunotherapy. Mechanistically, Trim35 represses LSD1 demethylase activity by mediating K63 ubiquitination at lysine site 422 of LSD1. Suppressed LSD1 activity facilitates ERGIC1 transcription, followed by autophagy inhibition and IFNGR1 stabilization to activate IFN-γ signaling, leading to increased MHC class I expression and immune surveillance of NSCLC cells. Furthermore, combinational use of an LSD1 inhibitor and anti-PD-1 therapy can significantly eradicate poorly immunogenic lung cancer with low Trim35. These findings strongly suggest that Trim35 is a promising biomarker for prediction of immunotherapy outcome in NSCLC.

Endothelial RIPK1 protects artery bypass graft against arteriosclerosis by regulating SMC growth.

RIPK1 is crucial in the inflammatory response. The process of vascular graft remodeling is also involved in endothelial inflammation, which can influence the behavior of smooth muscle cells. However, the role of endothelial RIPK1 in arterial bypass grafts remains unknown. Here, we established an arterial isograft mouse model in wild-type and endothelial RIPK1 conditional knockout mice. Progressive vascular remodeling and neointima formation occurred in the graft artery, showing SMC accumulation together with endothelial inflammatory adhesion molecule and cytokine expression. Endothelial RIPK1 knockout exacerbated graft stenosis by increasing secretion of N-Shh. Mechanistically, RIPK1 directly phosphorylated EEF1AKMT3 at Ser26, inhibiting its methyltransferase activity and global protein synthesis, which further attenuated N-Shh translation and secretion. Consistently, treatment with the Hedgehog pathway inhibitor GDC0449 markedly alleviated RIPK1 knockout-induced graft stenosis. Our results demonstrated that endothelial RIPK1 played a protective role in arterial bypass graft vascular remodeling, highlighting that targeting Hedgehog pathway may be an attractive strategy for graft failure in the future.

Association between trauma exposure and respiratory disease-A Mendelian randomization study.

Background: Trauma is a well-known risk factor for many disease, but the effect of trauma on respiratory disease is unclarified. In the present study, we aimed to evaluate the association between trauma and respiratory disease. Method: Using both United Kingdom biobank and Finnish biobank genome-wide association study data (GWAS), we performed a two-sample Mendelian randomization (MR) analysis to evaluate the relationship between trauma and respiratory disease. We used four methods including inverse-variance weighted (IVW), weighted median, Maximum likelihood, and MR-Egger in this MR analysis. The IVW MR was selected as the main method. We also performed multivariable Mendelian randomization (MVMR) to simultaneously assess the independent impact of trauma exposure on respiratory disease. Results: In the main two-sample MR analysis, trauma exposure was significantly associated with increased risk of respiratory disease (OR 1.15, 95%CI: 1.05-1.25). Besides, there was no heterogeneity and horizontal pleiotropy observed in the sensitivity analysis. After adjusting for pack years of smoking and body mass index (BMI), trauma exposure retained its association with respiratory disease (OR, 1.13, 95%CI, 1.04-1.23 adjusted by pack years of smoking; and OR, 1.11, 95%CI, 1.04-1.18 adjusted by BMI). Conclusion: Our study discovered the association between trauma exposure and the increased risk of respiratory disease, suggesting the prevention and treatment with trauma to reduce the risk of respiratory disease.

Diagnostic accuracy of lung ultrasonography in childhood pneumonia: a meta-analysis.

This meta-analysis aimed to assess the diagnostic accuracy of lung ultrasonography in pneumonia-affected pediatric patients. Literature search of published articles in Medline, Web of Science, Scopus, Embase and Journal of Web till September 2020 were reviewed for the predescribed accuracy assessors. In compliance with the inclusion and exclusion criteria, two researchers independently screened the literature, collected the results and assessed the risks of bias using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. The pooled sensitivity and specificity, pooled positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio were estimated for the meta-analysis. The overall efficiency of lung ultrasonography (LUS) was evaluated using a summary receiver operating characteristic curve. Q and I2 statistics were used to determine heterogeneity. Meta disc software was used for the analysis of the study. Out of 1182 studies, only 29 articles were chosen; 25 of them were prospective studies and 4 studies were retrospective. The overall pooled sensitivity was 0.83 [95% confidence intervals (CI), 0.81-0.84] and specificity was 0.84 (95% CI, 0.81-0.86), depicting good diagnostic performance. LUS is an efficient imaging technique for detecting childhood pneumonia with a high accuracy rate. It is an appealing alternative to chest X rays to detect and follow-up pneumonia in children because it is simple to do, widely available, comparatively cheap and free of radiation hazards.

Assessment of causal effects of visceral adipose tissue on risk of cancers: a Mendelian randomization study.

BACKGROUND: Despite the established association between obesity and cancer risk, it remains unclear whether visceral obesity is causally related to cancer risk and whether it is more pro-oncogenic than total body fat. METHODS: We conducted two-sample Mendelian randomization (MR) analysis to assess the causal effects of visceral adipose tissue (VAT) on six common cancers. For exposure data, 221 genetic variants associated with the predicted volume of VAT in 325 153 Europeans from UK Biobank were used as instrumental variables. Genetic association data of six common cancers (breast, lung, colorectal, ovarian, pancreatic and prostate cancers) were obtained from large-scale consortia with an average of 19 576 cases and 43 272 controls. We performed univariable MR with five MR methods [inverse-variance weighted (IVW), MR-Egger regression, weighted median, MR-Pleiotropy Residual Sum and Outlier (MR-PRESSO) and Radial MR] and multivariable MR to estimate the effect of VAT independent of body mass index (BMI). Finally, we performed a series of sensitivity analyses as validation of primary MR results. RESULTS: Two associations survived the false discovery rate correction for multiple testing (q-value < 0.05): in IVW, the odds ratios (95% CIs) per unit increase in genetically determined VAT were 1.65 (1.03 to 2.62) for pancreatic cancer and 1.47 (1.20 to 1.82) for lung squamous-cell carcinoma, respectively, which showed the same directions and overlapped confidence intervals with MR-Egger regression and weighted median results. There were no outlier variants identified by MR-PRESSO and no evidence supporting the presence of heterogeneity and pleiotropy in sensitivity analyses, although with wider confidence intervals that included the null, multivariable MR results for these two cancers showed the same directions and similar effect sizes as in IVW, which were independent of the effect from BMI. There was no evidence for a causal effect of VAT on the risk of other types of cancer. CONCLUSION: Our findings suggest that lifelong exposure to elevated volumes of VAT might increase the risk of pancreatic cancer and lung squamous-cell carcinoma, highlighting the importance of revealing the underlying mechanisms for intervention targets.

Girdin Promotes Tumorigenesis and Chemoresistance in Lung Adenocarcinoma by Interacting with PKM2.

Girdin, an Akt substrate, has been reported to promote tumorigenesis in various tumors. However, the role of Girdin in a spontaneous tumor model has not yet been explored. Here, we studied the role of Girdin in lung adenocarcinoma (LUAD) using the autochthonous mouse model and found that Girdin led to LUAD progression and chemoresistance by enhancing the Warburg effect. Mechanistically, Girdin interacted with pyruvate kinase M2 (PKM2), which played a vital role in aerobic glycolysis. Furthermore, Girdin impaired Platelet Derived Growth Factor Receptor Beta (PDGFRß) degradation, which in turn, promoted PKM2 tyrosine residue 105 (Y105) phosphorylation and inhibited PKM2 activity, subsequently promoting aerobic glycolysis in cancer cells. Taken together, our study demonstrates that Girdin is a crucial regulator of tumor growth and may be a potential therapeutic target for overcoming the resistance of LUAD cells to chemotherapy.

Trans-auricular vagus nerve stimulation to reduce perioperative pain and morbidity: protocol for a single-blind analyser-masked randomised controlled trial.

Background: Established or acquired loss of parasympathetic vagal tone is associated with complications, including pain, after noncardiac surgery. We describe a study protocol designed to test the hypothesis that transcutaneous auricular nerve stimulation may preserve efferent parasympathetic activity to reduce pain and morbidity after noncardiac surgery. Methods: Participants aged >18 yr scheduled for urgent/elective orthopaedic surgery (n=86) will be randomly allocated to bilateral transcutaneous auricular nerve stimulation or sham protocol for 50 min at the same time of day, before and 24 h after surgery. Holter monitoring, the analysis of which is masked to allocation, will quantify autonomic modulation of HR. The primary outcome will be pain, quantified by absolute changes in VAS 24 h after surgery following sham or stimulation. Secondary outcomes include presence or absence of >10 mm change in the 100 mm VAS (which defines a minimum clinically important change) and postoperative morbidity (Postoperative Morbidity Survey) before and 24 h after surgery. The relationship between the explanatory variable (HR variability), VAS, and morbidity will be examined using a multilevel (mixed-error component) regression model. Safety and complications of the intervention will also be recorded. The study was approved by the NHS Research Ethics Committee (21/LO/0272). As of 25 December 2021, 34/86 participants (mean [standard deviation] age: 48 [19] yr; 14 females [41.2%]) have been recruited, with complete collection of Holter data. Conclusions: This phase 2b study will explore whether noninvasive autonomic neuromodulation may reduce pain or morbidity using trans-auricular vagus nerve stimulation, providing proof-of-concept data for a non-pharmacological, generalisable approach to improve perioperative outcomes. Clinical trial registration: Researchregistry7566.