The Associations Between Salivary And Intestinal Microbial Dysbiosis With Non-Small Cell Lung Cancer

Lung cancer,one of the most prevalent cancers globally,is the leading cause of cancer-related deaths among males,with an increased incidence in recent years in China.Lung cancer is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),with NSCLC accounting for about 80%of lung cancers and SCLC accounting for 20%.NSCLC is often asymptomatic or causes only nonspecific symptoms in its early stages.The five-year survival rate for NSCLC is only 4-17%,in part because the disease is usually diagnosed at a late stage and is frequently metastaticand incurable.Early detection is crucial for reducing the morbidity and mortality ofNSCLC.However,it is difficult to promote current approaches to detect NSCLC,due to the high cost and low positive detection rate at the early stages of the disease.Currently,practical and effective approaches to the early diagnosis of NSCLC are desperately needed.The microbiota with a good prospect of clinical application has receivedunprecedented attention internationally recently.More than 1,000 different species,totalling 1014 bacterial cells in the human gastrointestinal tract,play a vital role in the maintenance of normal physiological function in human intestinal assimilation,energy metabolism,adaption of immune system and anti-infection.Accumulating evidence shows that the possible effects of gut bacteria oncarcinogenesis,which primarily involves modulating the immune response,activating Toll-like receptors,producing carcinogenic toxins,and inducing chronic inflammation.Previous studies also have revealed gut bacteria,including Fusobacterium nucleatum,Escherichia coli,Bacteroides fragilis,and Proteobacteria,are associated with carcinogenesis.Further evidence has demonstrated that alterations of gut bacteria play an important role in the development of extraintestinal cancers,such as hepatocellular carcinoma and breast cancer.A series of studies have shown that an altered bacteria community in lung tissue,sputum,bronchoalveolar lavage fluid,or saliva samples is prospectively associated with an increased risk of lung cancer,and one study reported that Lactobacillus shows antitumor effects in the intestinal tract of a Lewis lung cancer mouse model.Furthermore,certain bacteria have been recognized as potential biomarkers for cancer detection and classification.Additionally,gut bacteria influence the efficacy of tumor therapy against epithelial tumors,raising the possibility that the quality of existing therapeutic approaches may be improved in combination with treatment of certain specific bacteria.Studies have also revealed that gut bacteria influence immune and inflammatory responses not only locally at the mucosal level but also systemically,including the pulmonary organs through the gut-lung axis.However,the exact composition of gut bacteria in patients with lung cancer remains poorly understood.Moreover,the correlations between gut bacteria and certain prognostic indexes have not yet been observed.Salivary microbiota,one of the most complex microbial communities in the human,is one of the most important part in Human Microbiology Project.Human saliva has become an attractive medical diagnostic fluid as its collection is convenient and non-invasive.Increasingly,studies have reported an increased risk of certain types of tumours that are related to the dysbiosis of salivary microbiota.Previous studies have identified the core microbiome related to health and shown that shifts in the core microbiome are associated with the immuno-inflammatory response.A number studies have also shown that microbiota markers in saliva are well-known diagnostic and prognostic biomonitors for diverse diseases.In addition,salivary Capnocytophaga and Veillonella have been identified as potential biomarkers for lung cancer detection and classification.Hence,characterizing the salivary microbiota in NSCLC patients could potentially help delineate the pathogenic role of salivary dysbiosis in the progression of NSCLC and direct the management of microbiota-targeted therapies.To better understand the relationship between microbiota and lung cancer,we directly sequenced the 16S rRNA gene in saliva and fecal samples from patients with lung cancer and healthy volunteers using next-generation sequencing technology.Our study also investigated correlations between the microbiota,systemic inflammatory markers,and the predicted metabolites of human microbiota.The identification of specific genera correlated with lung cancer may provide a broader understanding of human microbiota and pave the way for further exciting exploration in this research area.Part I Intestinal Microbial Dysbiosis Is Associated with Systemic Inflammatory Markers and Predicted Gut Metabolites in Non-Small Cell Lung Cancer PatientsObjectiveEmerging evidence suggests that the intestinal microbiota may affect a number of diseases,including lung cancer.The objective of this study is to explore the interactions between altered intestinal microbial dysbiosis and NSCLC and evaluate the identification of specific genera correlated with lung cancer and its value in the prognosis and diagnosis of non-small cell lung cancer.Material and MethodFecal samples were collected from 37 NSCLC patients and 33 matched healthy volunteers.Genomic DNA was extracted and sequenced on Illumina MiSeq platform.The sequencing results include the analysis of the structure and abundance of intestinal microbiota,and the annotation analysis of KEGG database.For the first time,we evaluated the correlations among certain specific bacteria,the predicted KEGG pathways and inflammatory indicators.Result1.There were significant differences in the overall comparative analysis on the structure of intestinal microbiota in the two groups.The metaststs analysis showed that three predominant phyla—Bacteroidetes,Firmicutes,and Fusobacteria—were significantly different between the lung cancer and healthy control group.Despite significant interindividual variation,nine predominant genera were significantly different between the two groups.The lung cancer group had higher levels of Bacteroides,Veillonella,Fusobacterium and Acinetobacter,but lower levels of Escherichia-Shigella,Kluyvera,Fecalibacterium,Enterobacter,and Dialister than the healthy control group2.Significant differences in the comparative analysis of the functional metabolism was also explored in the two groups.The NSCLC group had remarkedly higher levels of replication and repair,energy metabolism,carbohydrate metabolism,enzyme families,and nucleotide metabolism,but dramatically lower levels of cell motility,membrane transport,signal transduction,and transcription3.In the analysis of the correlations among certain specific bacteria and serum inflammatory biomarkers,two identified genera,Escherichia-Shigella and Enterobacter,were positively correlated with serum NLR level,and Dialister was negatively correlated with serum levels of NLR and PLR.Furthermore,correlations were also found between Bacteroides and serum levels of IL-1? and LMR4.In the analysis of the correlations between altered KEGG module and the clinical inflammatory biomarkers,the altered chaperones and folding catalysts,transcription factors,oxidative phosphorylation and transporters were all positively correlated with serum levels of LMR.ConclusionThe community structure and functional structure of intestinal microbiota in NSCLC patients were significantly altered.The significant correlations among microbiota,the predicted KEGG pathways and systemic inflammatory biomarkers suggest that the altered microbiota can be used as an ideal biomarker for the diagnosis of NSCLC.It may also provide a new therapeutic target for clinical treatment of NSCLC through intestinal microbiota.Part II Salivary Microbial Dysbiosis Is Associated with Systemic Inflammatory Markers and Predicted Oral Metabolites in Non-Small Cell Lung Cancer PatientsObjectiveAlthough epidemiological studies of salivary microbiota in carcinogenesis are mounting,no systemic study exists regarding the oral microbiota of NSCLC patients.The objective of this study is to reveal the distinct salivary microbiota composition in patients with NSCLC compared to the healthy controls and explore the identification of specific genera correlated with lung cancer and its value in the prognosis and diagnosis of non-small cell lung cancer.Material and MethodSaliva samples were collected from 39 NSCLC patients and 20 matched healthy volunteers.Genomic DNA was extracted and sequenced on Illumina MiSeq platform.The sequencing results include the analysis of the structure and abundance of intestinal microbiota,and the annotation analysis of KEGG database.For the first time,we evaluated the correlations among certain specific bacteria,the predicted KEGG pathways and inflammatory indicators.Result1.As principal co-ordinates analysis(PCoA)showed,saliva samples clearly differed between the two groups,considering the weighted(p = 0.001,R2 = 0.1 7),and unweighted(p = 0.001,R2 = 0.25)UniFrac distance.Phylum Firmicutes and its two genera Veillonella and Streptococcus were strongly increased in NSCLC group compared to the control.Additionally,the relative abundances of Fusobacterium,Prevotella,Bacteroides,and Faecalibacterium in NSCLC group were generally decreased.2.Significant differences in the comparative analysis of the functional metabolism were also explored in the two groups.KEGG modules inferred by PICRUSt showed that pathways related to xenobiotics biodegradation and metabolism and amino acid metabolism were enriched in the NSCLC group.While folate biosynthesis was significantly decreased in NSCLC group.3.Analysis of the correlations between systemic inflammation markers and salivary microbiota showed that NLR positively correlated with the Veillonella and LMR negatively correlated with Streptococcus.4.In the analysis of the correlations between altered KEGG module and the clinical inflammatory biomarkers,the enriched transporters were negatively correlated with serum levels of PLR.The secretion system was positively correlated with serum levels of AMC,while negatively correlated with serum LMR.Additionally,pyrimidine metabolism and the ribosome involved in transcription and nucleotide metabolism were all positively correlated with serum LMR and negatively correlated with AMC.ConclusionThe community structure and functional structure of salivary microbiota in NSCLC patients were significantly altered.The significant correlations among microbiota,the predicted KEGG pathways and systemic inflammatory biomarkers extended the new sight into salivary microbiota-targeted interventions to clinically improve the therapeutic strategies for salivary dysbiosis in NSCLC patients.Part ? Salivary Microbial Dysbiosis Is Associated with Radiotherapy in IIIA-N2 Stage Non-Small Cell Lung Cancer PatientsObjectivePrevious studies have suggested the dysbiosis of salivary microbiota has been linked to the advancement of NSCLC.However,no systemic study exists regarding the effect of radiotherapy on oral microbiota in NSCLC patients.The obj ective of this study is to explore the altered saliva microbiota community in NSCLC patients receiving radiotherapy.Material and MethodSaliva samples were collected from 16 NSCLC patients before and after radiation for oral microbiota identification.Genomic DNA was extracted and sequenced on Illumina MiSeq platform.The sequencing results include the analysis of the structure and abundance of intestinal microbiota.Result1.After radiotherapy,the incidence of radiation esophagitis was 87.5%(14/16).When comparing the severity of radiation esophagitis with different sexes and pathologic diagnosis,they had no statistical significance.2.We compared the microbial diversity and richness of NSCLC patients before/after radiotherapy using the Chaol,Shannon,and Simpson indices.The NSCLC patients tended to have lower richness and diversity after radiotherapy in our study.3.In the comparative analysis on the structure of saliva microbiota before and after radiotherapy,detection amount of phylum Firmicutes and Actinobacteria both significantly increased after radiotherapy,that of Proteobacteria reduced significantly.Detection amount of genus Bacteroides and Actinomyces after radiation significantly increased,that of Neisseria markedly decreased.ConclusionThe community structure of salivary microbiota in patients receiving radiotherapy for NSCLC had some changes in detection of oral Bacteroides,Actinomyces and Neisseria.The potential associations among salivary dysbiosis and radiation esophagitis extended the new sight into salivary microbiota-targeted interventions to alleviate the side-effect of the radiotherapy in NSCLC patients.