Ambient Mass Spectrometry For The Molecular Diagnosis Of Lung Cancer And Liver Cancer

Lung cancer and liver cancer are the most common malignancies and are the leading cause of cancer-related death worldwide.Although the widely acknowledged method for the diagnosis of lung cancer and liver cancer continues to be histopathology,molecular analysis has gained more attention in recent years.This is largely because molecular analysis provides more information that may be unavailable in histopathology,and offers a new opportunity to incorporate cancer-related biomarkers into clinical decision-making.Mass spectrometry(MS)is one of the most versatile technologies for the analysis of various clinical specimens(incuding tissue,serum,urine,sputum as well as exhaled breath),which plays an paramount role in the medical research.Though conventional technologies(such as liquid/gas chromatography-mass spectrometry(LC/GC-MS)could achieve the molecular information within various clinical specimens,which generally accompanied with a series of tedious sample pretreatment procedures(e.g.,gradation,extraction,enrichment,separation,etc.).Particularly,these methods may not be suitable for the direct and high-throughput molecular analysis of real clinical samples in clinical applications.Therefore,it is urgent demand to develop efficient and powerful tools for the study of various types of clinical samples in both research lab and clinical diagnosis applications.Among the technologies suitable for the rapid and accurate molecular diagnosis of lung cancer and liver cancer,ambient mass spectrometry has gained increasing interest as it allows the direct profiling of molecular information from various biological samples(e.g.,tissue,serum,urine and sputum)in real-time and with minimal or no sample pretreatment.With the aim at achieving the rapid and accurate molecular diagnosis of lung cancer and liver cancer,this paper is on the basis of the advantages of a novel ambient mass spectrometry technique,termed as internal extractive electrospray ionization mass spectrometry(iEESI-MS),which is with the unique feature of direct characterization of “internal” chemicals within bulk samples,with high sensitivity and specificity,low sample consumption and simple operations.Moreover,combining multiple statistical analysis methods and mouse cancers models,the stuides of iEESI-MS in molecular diagnosis of lung cancer and liver cancer were performed,including integrative omics analysis for improved molecular differentiation accuracy of lung cancer,tracing molecular margins of lung cancer,and comparative studies of phospholipids alterations upon liver cancer between mice and humans.The main contents of this thesis are as follows:(1)Integrative omics analysis for improved molecular differentiation accuracy of lung cancer.Traditionally,molecular information on small metabolites,lipids and proteins is collected from separate individual tissue samples using different analytical approaches.Herein a novel strategy to minimize the potential material losses and the mismatch between metabolomics,lipidomics and proteomics data has been demonstrated based on iEESI-MS.Sequential detection of lipids,small metabolites,and proteins from the same tissue sample was achieved without sample re-loading and hardware alteration to MS instrument by sequentially using extraction solutions with different chemical composition.With respect to the individual compound class analysis,the sensitivity,specificity,and accuracy obtained with the integrative information on small metabolites,lipids and proteins from 57 samples of 13 patients for lung cancer prediction was substantially improved from 54.0%,51.0% and 76.0% to 100.0%,respectively.The established method is featured by low sample consumption(ca.2.0 mg)and easy operation,which is important to minimize systematic errors in precision molecular diagnosis and systems biology studies.(2)Tracing molecular margins of lung cancer using iEESI-MS.Accurate determination of lung cancer margins at the molecular level is of significance for the maximal beneficial effects of surgical resection and the minimal risk of recurrence.In this study,internal extractive electrospray ionization mass spectrometry(iEESI-MS)was used to profile phospholipid composition in tissue samples from 10 nude mice with lung cancer.Molecular differential model of lung cancer established via partial least-squares discriminant analysis of iEESI-MS data collected from lung tissue pieces within cancer tumor area and iEESI-MS data collected from lung tissue pieces outside cancer tumor area allows determination of lung cancer with the accuracy of 95.83%.Importantly,the developed molecular differential model yields ca.1-2 mm wider margin of a lung cancer tumor compared to the conventional histological analysis.These results indicate the potential of iEESI-MS to increase the accuracy of tumor margins determination during lung cancer surgery.(3)Comparative study of alterations in phospholipid profiles upon liver cancer in humans and mice.Comparative studies of molecular alterations upon cancer between mice and humans are of great importance in order to explore the usability range of mouse cancer models for the development of diagnostic and therapy approaches in clinical practice as well as for the mechanistic studies of pathology.Herein,using molecular fingerprinting by iEESI-MS,we identified 50 differential metabolites in mouse liver tissue and 62 differential metabolites in human liver tissue that undergo significant intensity alterations(VIP > 1.0)upon liver cancer,out of which only 27 were common in both mouse and human tissues.Out of the 27 common differential metabolites,six types of phospholipids were also identified to undergo significant alterations in human serum upon liver cancer,including PC(34:2),PC(36:4),PC(38:6),PC(36:2),PC(38:4)and PC(42:9).Statistical analysis of the relative intensity distribution of these six identified phospholipids in serum allowed confident determination of liver cancer in humans(sensitivity 91.0 %,specificity 88.0 %,and accuracy 90.0 %).Thus,our results indicate that,despite the significant difference in the overall alterations of phospholipid profiles upon liver cancer between humans and mice,the identified six ‘core' differential phospholipids of liver cancer found in the liver tissues of both humans and mice as well as in human serum show high potential as a minimal panel for the rapid targeted diagnosis of liver cancer with high accuracy,sensitivity and specificity using direct MS analysis.