| Objective:Lung cancer is one of the leading causes of cancer death worldwide.non-small cell lung cancer（NSCLC）is the most common type of lung cancer.As a mucosal tissue with a large surface area,the lung is chronically exposed to some airborne flora.Therefore,the respiratory system microbiota may affect the occurrence and development of lung cancer.At the same time,in the whole process of lung cancer diagnosis and treatment,there are also many factors that affect the microenvironment of lung tumors at all times,changing the quantity and composition of lung microbiota（LM）.Many studies have shown that the development of lung cancer is closely related to chronic inflammation,but how to create a microenvironment that can promote tumor occurrence,development and drug resistance needs to be solved urgently.Therefore,the purpose of this study was to describe the characteristics of the pulmonary microbiota in patients with NSCLC through multi-angle analysis,to explore the molecular mechanism of some specific bacterial components or metabolites in the malignant process of the occurrence and development of lung cancer.Then we conducted survival follow-up of the enrolled patients and analyzed the potential impact of intratumoral microbiota（ITM）on the occurrence,development and prognosis of lung cancer,so as to provide new ideas for the optimization of clinical treatment strategies for NSCLC in the future.Part One Characteristics of Pulmonary pathogens in patients with stage III and IV non-small cell lung cancer without pulmonary inf-ectionObjective:By analyzing the pulmonary pathogenic in tumor tissue,peripheral blood and bronchoalveolar lavage fluid（BALF）of NSCLC patients with stage III and IV non-small cell lung cancer without pulmonary infection,the distribution characteristics of pathogenic bacteria were preliminarily understood.It will lay a foundation for further exploration of the relationship between pulmonary pathogenic bacteria and NSCLC.Methods:In this study,37 tumor tissues and 6 bloods and 4bronchoalveolar lavage fluid samples from patients with stage III and IV non-small cell lung cancer were screened for Gensizer TMpathogen targeted sequencing,and 2 bronchoalveolar lavage fluid samples for 16S rDNA sequencing for descriptive analysis.Results:1.Venn diagram of pathogen targeted sequencing showed that there were5 common high-frequency pathogenic microorganisms in the three types of samples.Between tumor tissue and BALF samples,11 other common high-frequency pathogens were detected.2.Pairwise comparison of different samples from the same patient showed that among the five pairs of tumor-plasma,the diversity of pathogenic microorganisms（PMs）in plasma was found to be similar to that in tumor tissue,but the specific PM in plasma was not necessarily consistent with that in tumor tissue.In 2 pairs of tumor-BALF samples,PMs diversity in BALF was significantly higher than that in tumor tissue,and PMs detected in BALF could basically cover PMs in tumor tissue.3.16S rDNA sequencing showed that there were 82 common OTUs in16S rDNA sequencing performed in BALF of two male patients,which were similar to those reported in the literature.Conclusion:The amount of intra-tumoral PMs was not necessarily consistent with that in the blood,but there was an obvious correlation between the intra-tumoral microbiota and that in the BALF.Therefore,taxonomic analysis and abundance comparison of microbiota in tumor tissues and bronchoalveolar lavage fluid can be used as a means of NSCLC-related research.Part Two Lung microbiota induces inflammation and induces osteop-ontin expression through TLR2/4 and affects cisplatin sensi-tivityObjective:This vitro study observed the effects of the main bacterial virulence components lipopolysaccharide（LPS）and lipoteichoic acid（LTA）on TLR2/4,CXCL1/6,TNF-αin two types of lung cancer PC9 and H1299cells.It was explored the molecular mechanism of LPS and LTA stimulation alone or in combination on tumor progression and cisplatin sensitivity.Methods:1.Two cell lines,PC9 and H1299,were used to detect the effects of different concentrations of LTA and LPS on the secretion of inflammatory factors in lung cancer cells by ELISA,and the appropriate experimental concentrations were screened.2.PC9 and H1299 lung cancer cells were treated with LPS and LTA,and blank control group,LPS group,LTA group,and LPS+LTA group were set up.CXCL1 and CXCL6 expression was analyzed using qRT-PCR,and the contents of TNF-α,IL-1β,and IL-6 were measured by ELISA.3.CCK-8 assay,transwell and flow cytometry were used to observe and compare the proliferation,apoptosis and migration of lung cancer cells in different treatment groups.4.The effects of different treatments on TLR2/4,inflammasome NLRP3and osteopontin SPP1 expression in lung cancer cells were analyzed at the protein and gene levels by Western blot and qRT-PCR,and each group was treated with NLRP3 inhibitor MCC950 for validation.5.LPS,LTA and Cisplatin were applied to the two types of cell lines,and blank control group,LPS+LTA+Cisplatin group and Cisplatin group were set up.CCK-8 was used to analyze the cell proliferation activity,flow cytometry was used to analyze the apoptosis rate,and Western blot was used to analyze the expression level of caspase-3/9,an apoptotic protein,to evaluate the effect of LPS+LTA on cisplatin resistance.Results:1.ELISA showed that the expression of inflammatory factors in the two cells treated with 1μg/m L LPS and 30μg/m L LTA were significantly increased,so this concentration was selected for subsequent studies.2.The result of qRT-PCR analysis showed that in the PC9 cell line,CXCL1 expression and CXCL6 expression were significantly increased in all three experimental groups compared with the control group,and CXCL1expression and CXCL6 expression were significantly higher in the LPS+LTA group than in the LPS and LTA alone treatment groups（P<0.001）.The result of ELISA showed that all three experimental groups could promote the release of TNF-α,IL-1βand IL-6,while LPS+LTA group significantly increased the expression level of inflammatory factors compared with single treatment group（P<0.001）.The inflammatory effect of LPS was greater than that of LTA（P<0.001）3.The result showed that in PC9 cell line,LPS+LTA significantly inhibited the proliferation of lung cancer cells,increased cell migration,and decreased the apoptosis ratecompared with single treatment（P<0.001）.4.Western blot results showed that LPS significantly activated TLR4expression（P<0.001）and LTA significantly activated TLR2 expression（P<0.001）in PC9 lung cancer cells.All three experimental groups could significantly up-regulate the expression of NLRP3 and SPPI protein in lung cancer cells,and the up-regulation effect was more significant in the combined treatment group alone.The above changes were consistently analyzed by qRT-PCR from the gene level.After treatment of each group using the NLRP3inhibitor MCC950,the m RNA expression of NLRP3 was significantly decreased in the LPS+LTA group（P<0.001）.And the promotion of SPP1 was also decreased（P<0.001）.5.The effect of LPS and LTA on the sensitivity of PC9 lung cancer cells were analyzed.The result showed that LPS+LTA+Cisplatin group could significantly reduce the inhibitory effect of LPS on cell proliferation（P<0.001）,reduce the apoptosis rate（P<0.001）and significantly reduce the expression levels of caspase-3 and caspase-9（P<0.001）compared with Cisplatin group.All of the above experiments in the H1299 cell line showed trends consistent with the PC9 cell line.Conclusion:LPS and LTA,the main components in microorganisms,can induce cancer,activate TLR4 and TLR2,respectively,and then activate the up-regulation of SPP1 by promoting the expression of inflammasome NLRP3.It also reduces the therapeutic sensitivity of lung cancer cells by inducing the up-regulation of caspase-3 and caspase-9 expression.Part Three Lung microbiota promotes osteopontin to induce malignant biological behavior of lung cancer cells through activation of PI3K/AKT pathway by integrinβ3Objective:In vitro studies verified that the pulmonary microbiota regulates integrinβ3 on osteopontin and activates the PI3K/AKT pathway,and preliminarily explored the molecular mechanism that pulmonary microbiota in lung cancer tissue play a role in promoting cancer.Methods:1.The expression of osteopontin SPP1 in PC9 and H1299 cell lines was silenced,transfected with si NC and SPP1si RNA,and LPS+LTA blank control group was set up.The expression levels of SPP1 and Integrinβ3 were analyzed by qRT-PCR.2.After knockdown of Integrinβ3,CCK-8 assay was used to observe the proliferation activity of cells,flow cytometry was used to analyze the apoptosis of cells,scratch assay was used to analyze the migration ability of lung cancer cells.3.After transfection of si NC and Integrinβ3 si RNA in the two lung cancer cells,each group of cells was treated with LPS+LTA,the m RNA expression levels of PI3K,AKT and ERK in the cells were analyzed by qRT-PCR,and the PI3K/AKT/ERK pathway-related protein expression was evaluated by Western blot.4.The PC9 cell line transfected with Integrinβ3 si RNA was used to carry out the in vivo xenograft mouse model to verify that LPS+LTA plays a role in promoting cancer by inducing Integrinβ3 to activate the PI3K/AKT/ERK pathway.Results:1.qRT-PCR results showed that LPS+LTA treatment in the si NC group significantly increased SPP1 and Integrinβ3 expression（P<0.001）,but cells after silencing SPP1 were significantly reduced by LPS+LTA treatment to promote SPP1 and Integrinβ3expression（P<0.001）.2.Experimental results showed that silencing Integrinβ3 decreased the promoting effect of LPS+LTA on lung cancer cell proliferation and promoting apoptosis rate compared with the si NC group（P<0.001）,and the cell migration distance was significantly reduced after silencing Integrinβ3（P<0.01）.3.qRT-PCR results showed that LPS+LTA promoted the m RNA expression levels of PI3K,AKT and ERK in lung cancer cells,which were significantly decreased after silencing Integrinβ3（P<0.01）.Western blot results indicated that p-PI3K,p-AKT,and p-ERK protein expressions were significantly increased in lung cancer cells after LPS+LTA treatment compared with the control group（P<0.001）,and p-PI3K,p-AKT,and p-ERK protein expression levels were significantly decreased after silencing Integrinβ3（P<0.001）,while there were no significant changes in PI3K,AKT and ERK protein expressions regardless of silencing Integrinβ3（P>0.05）.All of the above experiments in the H1299 cell line showed trends consistent with the PC9 cell line.4.Measurement of transplanted tumors transfected with si NC and Integrinβ3 si RNA.PC9 lung cancer cells showed that the weight and volume of transplanted tumors treated with LPS+LTA were significantly increased（P<0.001）,but the weight and volume of transplanted tumors were decreased after transfection with Integrinβ3 si RNA（P<0.001）.Western blot results showed that p-PI3K,p-AKT,and p-ERK protein expression levels were significantly increased in the xenografts in the LPS+LTA-treated group（P<0.01,P<0.05,P<0.001）,and their expressions were significantly inhibited after silencing Integrinβ3.Conclusion:LPS+LTA may play a role in promoting cancer by up-regulating the expression of SPP1 in lung cancer cells,thereby inducing Integrinβ3 and activating the downstream PI3K/AKT/ERK pathway.Part Four Correlation analysis between intratumoral pathogens and first-line treatment effect and survival in patients with non-small cell lung cancer without respiratory tract infectionObjective:We conducted a 5-year follow-up study of screened patients with stage III or IV NSCLC without respiratory infection to analyze the association of pulmonary pathogens with the stage,clinical molecular characteristics,first-line treatment effect,and survival of these patients from the clinical stage.Methods:Patients with NSCLC whose tumor tissues were subjected to pathogen targeted sequencing in our hospital were included.All patients received first-line treatment according to individual conditions.The efficacy of first-line treatment was recorded after a short time of treatment（2 cycles of chemotherapy/immunization,28 days of targeted therapy）,and survival status,progression events,and overall survival（OS）were followed up.Correlation analysis was performed between the detected flora species and clinical characteristics of the disease,including pathological type,clinical stage,gene status,PD-L1 expression,and first-line treatment effect.Besides,survival analysis was performed using Kaplan-Meier survival curve.Results:1.Correlation analysis showed that the abundance of Serratia marce-scens in ASC types of adenosquamous carcinoma was significantly higher than that in ADC and SCC（P<0.05）.In N+Patients,the presence of both A.naeslundii and Haemophilus was negatively correlated with mediastinal lymph node metastasis（P<0.05）.Tumors with S.marcescens were more likely to develop brain metastases（P<0.01）,and tumors with E.cloacae were more likely to develop mediastinal lymph node metastases（P<0.05）.EGFR mutations were negatively correlated with H.parainfluenzae（P<0.05）and positively correlated with Serratia marcescens（P<0.01）.There was a positive correlation between A.agaricus and PD-L1 expression（P<0.05）.2.Stratified analysis of the correlation between the efficacy of first-line treatment showed that Haemophilus parainfluenzae was negatively correlated with the efficacy of first-line treatment in stage IV patients.The disease control rates were poor in patients with H.parainfluenzae present within the tumor tissues（P<0.05）.3.The result of the association between ITM and survival using the Kaplan-Meier method indicate that the presence of H.parainfluenzae was associated with worse progression-free survival PFS in stage IV patients.Haemolytic staphylococcal infections were associated with longer PFS.Serratia marcescens was associated with better overall survival OS and the presence of H.parainfluenzae was associated with worse OS.4.Cox regression model showed that Staphylococcus haemolyticus and Streptococcus cristae were associated with better PFS.In contrast,Haemophilus parainfluenzae and Corynebacterium jeikeium were two risk factors for OS.For patients with stage III or IV non-small cell disease of ADC,SCC,or ASC to predict two-year survival,age,primary stage,pathological type,and seven variables of four bacteria（Haemophilus parainfluenzae,Serratia marcescens,Mobility agar,and Streptococcal constellations）can be used.Conclusion:Above studies showed that Serratia marcescens was more presented in ASC and EGFR mutant types,more prone to brain metastasis,and was associated with better OS;Haemophilus parainfluenzae was more presented in EGFR wild-type patients,and the first-line treatment efficacy,disease control rate,PFS and OS were poor.A.naeslundiiand and Haemophilus were negatively correlated with mediastinal lymph node metastasis,while E.cloacae was inversely correlated;A.agaricus was positively correlated with PD-L1 expression.Staphylococcus haemolyticus and Streptococcus cristae were associated with better PFS,and Haemophilus parainfluenzae and Corynebacterium jeikeium were two risk factors for OS.It indicated that pulmonary pathogens may be related to the pathological type,clinical stage,EGFR mutation,PD-L1 expression and first-line treatment outcome of malignant tumor and survival of patients with NSCLC. |
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