Effects Of Mental Stress On Immune Microenvironment And Its Mechanism In Patients With Non-Small Cell Lung Cancer

1 BackgroundWith the gradually accelerating pace of life today,the gradually widening income gap and the COVID-19 pandemic have caused anxiety and stress in people around the world.Mental stress can lead to a variety of well-known adaptive physiological changes,including abnormalities in blood pressure,heart rate,endocrine and neural activity,which severely disrupt human health and social routines.In recent years,more and more evidence has shown that mental stress is closely related to the occurrence and development of tumors.After undergoing tumor diagnosis,surgery,chemotherapy and suffering from various tumor-related complications,most tumor patients will suffer from severe mental stress,which not only affects the enthusiasm of tumor patients for treatment,but also promotes tumor progression.Mental stress can affect a variety of tumor phenotypes,including proliferation,metastasis,genomic instability,and angiogenesis.Recent preclinical experiments have demonstrated that mental stress enhances autophagy in tumor cells,thereby enhancing the proliferation and metastasis of gastric cancer.Mental stress can lead to various metabolic disorders and suppress the patient’s immune system,causing the failure of antitumor therapy.In addition,the changes of gut microbiota caused by mental stress are also closely related to the deterioration of physiological system diseases and tumors.Therefore,mental stress is one of the important factors affecting tumor progression,and it is necessary to dig deep into its specific mechanism,which provides an important theoretical basis for formulating intervention strategies to alleviate the effect of mental stress on tumor promotion.Lung cancer is one of the most common malignancies in the world and the leading cause of cancer-related deaths,with an estimated 2.2 million new cases of lung cancer and more than 1 million deaths each year.As one of the subtypes of lung cancer,non-small-cell lung cancer(NSCLC)accounts for about 85%of all lung cancers,and about 70%of patients are found in the middle and late stages,with a 5-year survival rate.still lower.In the past two decades,with the in-depth understanding of tumor biology,the acceleration of new drug research and development,and the promotion of early diagnosis of lung cancer,the survival rate of lung cancer patients has improved significantly.Especially in recent years,the clinical application of targeted therapy and immunotherapy has greatly enhanced the therapeutic effect of lung cancer.At the same time,due to the strong specificity of treatment and few side effects,the quality of life of patients has also been greatly improved.improve.However,most NSCLCs will develop resistance to current treatments and progress,so more mechanisms that affect the development of tumors need to be explored.At present,the intervention of NSCLC patients mainly focuses on the tumor cells themselves,while the mental stress of patients caused by the diagnosis and treatment of NSCLC is often ignored.More and more studies have shown that mental stress is one of the important risk factors for the progression of NSCLC.Elucidating the mechanism of this phenomenon will provide new ideas for the precise treatment of NSCLC.In this paper,the mental stress of NSCLC patients was evaluated by the international standard general mental stress scale,and the relationship between mental stress and clinical parameters and immune function of patients was analyzed.Through 16SrDNA sequencing,mass spectrometry,and transcriptome sequencing,it was found that stress can change the composition of intestinal flora in NSCLC patients,leading to intestinal epithelial Serotonin Transporter(SERT).And the increased expression of Monoamine oxidase A(MAOA)resulted in the disorder of chromoic acid metabolic pathway and the increase of 5-hydroxyindole-3-acetic acid(5-HIAA)as the terminal product of this metabolic pathway.At the same time,5-HIAA can bind to Aryl hydrocarbon receptor(AHR)in CD8~+T cells,promote the expression of PD-1 protein in CD8~+T cells,and inhibit anti-tumor immunity in NSCLC patients.Then,in animal experiments,when intestinal flora was removed,the promoting effect of mental stress on tumor was weakened,suggesting that for patients with NSCLC under mental stress,the development of therapeutic strategies targeting intestinal flora may be beneficial to inhibit tumor progression.Meanwhile,there was a negative correlation between mental stress and the treatment effect of PD-1 inhibitor in non-small cell lung cancer.Through quantitative analysis of various neurotransmitters,it was found that norepinephrine played a key role.Further in vivo and in vitro experiments showed that mental stress promoted the production of norepinephrine.However,norepinephrine can activate the Wnt7A/β-catenin signaling pathway in NSCLC cells,inhibit the production of CXCL9 in tumor tissues,reduce the invasion of CD8~+T cells in tumor tissues,and thus produce resistance to PD-1 inhibitors.2 Methods2.1 Part 1 Effects of mental stress on clinical parameters and prognosis of patients with non-small cell lung cancer2.1.1 200 NSCLC patients were evaluated for mental stress based on the internationally accepted mental stress scale,and their clinical data were collected and prognosis was followed up.2.1.2 Integrated analysis of the collected patient scales to study the correlation of various indicators of mental stress with TNM stage(Tumor,Node,Metastasis),clinical stage(Ⅰ,Ⅱ,Ⅲ,Ⅳ),EGFR and ALK mutation status,PD-1 expression and other clinical indicators of NSCLC patients.NSCLC patients were divided into two groups with high and low mental stress according to the mental stress scale score,and the composition of tumor stages in different groups was analyzed and the survival curve was drawn.2.1.3 The data analysis method of multivariable linear regression model was used to explore the mediating factors of mental stress affecting the progression of NSCLC;2.1.4 Transcriptome was used to explore the influence of mental stress on immune signaling pathway in tumor patients.2.1.5 Flow cytometry was used to compare the function of peripheral blood CD8~+T cells of NSCLC patients under different mental stress.2.1.6 The tumor-bearing model of mice under mental stress was established by binding method,and peripheral blood,small intestine and feces of mice were collected for use to verify the influence of mental stress on the progression of NSCLC,and the function of CD8~+T cells in spleen and tumor tissues of mice was detected.2.2 Part 2 Mental stress mediates the regulation of gut microbiota on tumor immunity in non-small cell lung cancerThe patients were divided into high stress group and low stress group according to the mental stress scale score,and the mice were divided into normal group and stress group based on the establishment of the mouse pressure model.The collected peripheral blood and feces of the patients and mice were analyzed by multiple omics.2.2.1 16SrDNA sequencing technology was used to detect the difference in the composition of intestinal flora in feces of mice in different pressure groups.2.2.2 Mass spectrometry was used to explore the differences of metabolites in serum and feces of patients/mice in different stress groups.2.2.3 Metabolic pathway enrichment analysis was conducted based on differential metabolites to explore the changes of main metabolic pathways.2.2.4 Explore the effects of metabolic pathway disturbances on stage prognosis and immune function of NSCLC patients.2.2.5 In vitro experiments were conducted to study the mechanism of metabolic pathway disturbances affecting immune function and the influence of corresponding genes on clinical parameters and prognosis of NSCLC patients.2.2.6 Animal experimental verification:A mouse stress model and a mouse intestinal microflora removal model were established,and LLC cells were subcutaneously inoculated to verify the changes of the above-mentioned differential genes and signal pathways and their mechanisms.2.3 Part 3 Mental stress-mediated neurotransmitter effects on immunotherapy of non-small cell lung cancer2.2.1 Analysis of the correlation between mental stress and the effect of PD-1 inhibitor on NSCLC patients.2.2.2 ELISA was used to detect the changes of major neurotransmitters in patients with pD-1 inhibitor treatment sensitivity and resistance.2.2.3 Animal experiments were conducted to explore the effects of different neurotransmitters on tumor immune microenvironment.2.2.4 In vitro and in vivo experiments and transcriptome profiling to explore the possible mechanism of differential neurotransmitters affecting the immune microenvironment.2.2.5 Use gene editing technology to verify the molecular biological mechanism of differential neurotransmitters affecting the immune microenvironment.3 Results3.1 Part 1 Effects of mental stress on clinical parameters and prognosis of patients with non-small cell lung cancer3.1.1 Mental stress was positively correlated with the clinical stage and TNM stage of patients with NSCLC,while negatively correlated with the prognosis of patients.3.1.2 Mental stress can promote the expression of CD8~+T cells PD-1,inhibit the production of Granzyme B,and thus suppress the anti-tumor immunity,promoting the progression of tumors.3.2 Part 2 Mental stress mediates the regulation of gut microbiota on tumor immunity in non-small cell lung cancer3.2.1 Mental stress can cause changes in the composition of intestinal flora in patients with NSCLC.3.2.2 Changes in intestinal microenvironment can promote the disorder of tryptophan metabolic pathway.3.2.3 The disorder of tryptophan metabolism leads to the increase of 5-HIAA production,which can inhibit the function of CD8~+T cells and is negatively correlated with the prognosis of patients.3.2.4 5-HIAA can bind to AhR protein of CD8~+T cells and promote the expression of PD-1,while deletion of AhR gene can inhibit the promoting effect of 5-HIAA on THE expression of PD-1 in T cells.3.2.5 Removal of intestinal flora can inhibit the progression of tumors caused by mental stress3.2.6 Removal of intestinal flora can inhibit tryptophan disorder caused by mental stress,thus reversing the inhibition of anti-tumor immunity.3.3 Part 3 Mental stress-mediated neurotransmitter effects on immunotherapy of non-small cell lung cancer3.3.1 Norepinephrine produced by patients with mental stress leads to resistance to PD-1 inhibitor therapy,while propranolol can reverse the effects of norepinephrine.3.3.2 Norepinephrine can inhibit the infiltration of CD8~+T cells in tumor tissues.3.3.3 Norepinephrine can activate the Wnt7A/β-catenin pathway of tumor cells and inhibit CXCL9 expression.4 Conclusion4.1 Mental stress is significantly correlated with the stage,prognosis and PD-1 expression of patients,and mental stress is an important risk factor for the progression of NSCLC.4.2 Mental stress can change the composition of intestinal flora and cause disorder of tryptophan metabolic pathway,resulting in increased production of 5-HIAA,the final product of tryptophan metabolism.5-HIAA can induce pD-1 expression of CD8~+T cells,inhibit anti-tumor immunity and promote tumor progression.4.3 Therapeutic strategies for intestinal flora can inhibit the promoting effect of mental stress on the progression of NSCLC.4.4 Norepinephrine induced by mental stress can activate the Wnt7A/β-catenine signaling pathway in NON-small cell lung cancer cells,inhibit the production of CXCL9 in tumor tissues,reduce the invasion of CD8~+T cells in tumor tissues,and thus produce resistance to PD-1 inhibitors.Propranolol reverses the effects of norepinephrine.To sum up,our study elucidated the molecular mechanism of lung cancer occurrence and development from a new dimension,laying a theoretical foundation for the development of new anticancer therapies in the later stage.