Proteomic Research Of Exosomes Derived From Cerebrospinal Fluid Of Leptomeningeal Carcinomatosis

Leptomeningeal carcinomatosis(LC)happens when tumor cells reach the leptomeninges,subarachnoid space,and other cerebrospinal fluid compartments.The most common origin is the lung.The prognosis of LC is poor and the median survival period is very short.Besides classical radiotherapy and chemotherapy,treatments of LC include emerging immunotherapy and molecular targeted therapy.The clinical application of radiotherapy and chemotherapy is sometimes limited due to their side effects while molecular targeted therapy and immunotherapy have shown bright prospects in the treatment of LC.Therefore,it is in great need to clarify the pathogenesis and to provide more therapeutic targets of this disease in clinical practice.Tumor microenvironment is a local stable environment composed of extracellular matrix,tumor cells and stromal cells.Tumor microenvironment is related to the proliferation and invasion of tumor cells.Exosomes are vesicles released to the outside by different cells.Exosomes contain information and substance related to their sources,such as m RNA,non-coding RNA,protein,etc.Exosome is an important way of cell-cell information transmission.Exosomes exist in the extracellular matrix which is part of the tumor microenvironment.Exosomes participate in a variety of physiological and pathological processes,especially for tumors.Cerebrospinal fluid is a kind of special tumor microenvironment in LC patients.However,the role of exosomes in cerebrospinal fluid of LC patients is not clear.In this study,we collected cerebrospinal fluid in subjects without tumor(normal group)and in patients suffered from non-small cell lung cancer(NSCLC)patients with/without LC(LC/NSCLC group).Exosomes in the cerebrospinal fluid were seperated by ultracentrifugation and verified thereafter.Proteomic characteristics of exosomes were analyzed by label-free method and bioinformatics tools.Candidate proteins related to LC were selected and verified by parallel reaction monitoring or Western blotting.This study aimed to provide insights for the molecular mechanism of LC.Results showed that exosomes could be successfully extracted from cerebrospinal fluid in LC patients.There were differentially expressed proteins in exosomes which might play important roles in the occurrence and development of LC.Part One Separation and verification of exosomes derived from cerebrospinal fluid in patients with leptomeningeal carcinomatosisObjective: To separate and verify exosomes in cerebrospinal fluid(CSF)and provide basis for subsequent proteomics analysis.Methods: 22 subjects who were admitted to the Second Hospital of Hebei Medical University were included.They were divided into three groups named LC group,NSCLC group and normal group according to their diagnosis.The enrolled subjects were male or female,aged 18-80 years old.Informed consent forms were signed in each patient.CSF samples were obtained by lumbar puncture.Exosomes in CSF were separated by ultracentrifugation.The size,morphology and molecular markers of exosomes were characterized by nanoparticle tracking analysis,transmission electron microscopy and Western blotting.Results:1.10 patients in LC group,4 patients in NSCLC group and 8 patients in normal group were enrolled in this study.Subjects in LC group were aged 43 to 82.4 of them were males and 6 were females.Tumor cells were found in CSF of all the patients in LC group by cytology.2.The size distribution of exosomes was 42.5-337.5 nm and the peak concentration was at 121.5 nm in normal group;the size distribution of exosomes was 22.5-382.5 nm and the peak concentration was at 118.6 nm in NSCLC group;the size distribution of exosomes was 62.5-397.5 nm and the peak concentration was at 119.3 nm in LC group.3.Vesicle structures were observed by transmission electron microscopy and the expressions of CD63 was verified by Western blotting in normal,NSCLC and LC groups.Conclusions: Vesicles,accordding with the characteristics of exosomes,could be isolated successfully from CSF of subjects in LC,NSCLC and normal groups.There was no difference of surface characteristics among groups and further research was needed on their contents.Part Two Label-free proteomic of exosomes derived from cerebrospinal fluid in patients with leptomeningeal carcinomatosisObjective: To explore the proteomic characteristics of CSF exosomes in patients with LC by label-free proteomic.Methods: After BCA quantification and SDS-PAGE,CSF exosome proteins extracted in part one were resolved by FASP method.Then samples after enzymolysis were separated by the Easy n LC system with nano-rise flow rate.Subsequently,the Q Exactive Plus mass spectrometer was applied.Proteins were identified when two peptide segments or at least one unique peptide were detected.The mass spectrometry data was searched by Max Quant software,and LFQ algorithm was used for quantitative analysis.Proteins were regarded as differential expression proteins when differential expression was greater than 2.0 times(up or down)and P < 0.05.Results:1.In this study,a total of 7314 peptides and 814 proteins were identified among which 523 proteins were overlapped with the exosome library.There were 441 common proteins in the three groups.82 proteins existed only in the CSF of the normal group.104 proteins existed only in the NSCLC group.69 proteins existed only in the LC group.186 proteins were detected only in tumor patients.2.In this study,384 different expression proteins were identified.There were 158 different expression proteins in LC group compared to normal group with 51 up-regulated and 107 down-regulated.Compared with NSCLC group,there were 72 different expression proteins in LC group with 39 up-regulated and 33 down-regulated.There were 154 different expression proteins in the NSCLC group compared with the normal group with 60 up-regulated and 94down-regulated.Conclusions: Proteomic analysis of CSF exosomes from patients with LC was successfully progressed by label-free method.The differentially expressed proteins in CSF exosomes of LC patients might be related to LC.Part Three Bioinformatics analysis and differentially expressed proteins identification of cerebrospinal fluid exosome proteins in patients with leptomeningeal carcinomatosisObjective: To identify candidate proteins related to LC by bioinformatics analysis for further validation.Methods: Bioinformatics analysis was performed for LC,NSCLC and normal group respectively through GO annotation and KEGG pathway analysis.Meanwhile,differentially expressed proteins were analyzed by GO annotation and KEGG pathway analysis,trend analysis,cluster analysis and protein-protein interaction(PPI)network analysis.Results:1.GO annotation and KEGG pathway analysis of CSF exosome proteins in LC,NSCLC and normal group showed that protein expression patterns of the three groups were similar.2.After trend analysis of differentially expressed proteins,PPI analysis was performed in candidate proteins related to LC.It was revealed that there were possible links between TKT,MSN,CD9,TIMP1,MRC1,LBP,ENO1,GSTP1,ACTA1,COL3A1,ACTB,SPP1,RTN4 R and SEMA7 A.3.Bioinformatics analysis comparing LC & NSCLC group and LC &normal group was performed.GO annotation was similar and functional enrichment analysis was not the same.4.Differentially expressed proteins between LC and NSCLC group and between LC and normal group were analyzed by PPI network analysis.Proteins that played important roles in both comparison included ACTB,APOB,SPP1 and TIMP1.Conclusions: Bioinformatics analysis combined with proteomics results showed that ENO1,TIMP1,SPP1,p Ig R,FN1 and MRC1 might be related to LC.They were referred as candidate proteins for subsequent validation.Part Four Parallel reaction monitoring and Western blotting verification of candidate proteins in cerebrospinal fluid exosomes in patients with leptomeningeal carcinomatosisObjective: To clarify the correlation between candidate proteins in CSF exosomes and LC by parallel reaction monitoring(PRM)and Western blotting.Methods: CSF from LC,NSCLC and normal group was collected respectively.Exosome proteins were extracted.Part of exosome proteins were used for Western blotting.The other part of exosome proteins were used for PRM.Exosome proteins were enzymatically hydrolyzed by FASP method,separated by the Ultimate 3000 chromatography system and analyzed by Q Exactive Plus spectrometer for mass spectrometry.T-test was used for inter-group comparison,and SPSS 25.0 software was applied for statistical analysis.P < 0.05 was recognized as statistically significant.Results:1.FN1 in LC group was significantly up-regulated after PRM validation,consistent with the results of mass spectrometry.2.No significant difference of CSF exosome proteins TIMP1,ENO1,MRC1 and SPP1 between groups was found after PRM.3.Role of CSF exosome protein p Ig R in LC needed to be further clarified.Conclusions: CSF exosomes and their differentially expressed proteins have potential value in the diagnosis and treatment of LC.