Evaluation Of NK Cell Cytotoxicity Using Single-cell Metabolites By Mass Spectrometry

Natural killer cells(NK cells)are an important type of immune cells.In recent years,NK cell therapy has been gradually applied in tumor immunotherapy,and NK cell cytotoxicity evaluation has become an important topic in clinical medicine.At present,the methods to evaluate the cytotoxicity of NK cells are mostly based on the analysis of DNA,RNA,protein,and metabolites of population cells.Such analysis methods can only obtain the average results in a statistical sense,but mask the heterogeneity of single cells,making it difficult to detect specific anti-killer cells.With the development of single-cell analysis technology,new technologies such as single-cell RNA sequencing and protein analysis have emerged.However,these methods are complicated to operate,showing a deficiency in meeting the needs of rapid clinical detection and testing.At the same time,the synthesis and degradation rates of RNA and protein are relatively slow,experiencing a certain lag in reaction to cell physiological activity,which fails to synchronically reflect the active state of cells.Metabolites directly reflect cellular physiological activity,which is suitable for rapid and accurate determination of NK cell cytotoxicity.This thesis proposes a method to obtain the metabolite information of tumor single cells by applying mass spectrometry and use the information to evaluate NK cell cytotoxicity.The main research contents are as follows:1.Methods for extracting and analyzing circulating tumor cells(CTC)and identifying metabolite types are established.Breast cancer cells are extracted from peripheral blood through magnetic spheres labelled by epithelial cell adhesion molecule(Ep CAM).The extracted breast cancer cells are detected by droplet microextraction and picoliter electrospray mass spectrometry in order to obtain single-cell metabolite data,thereby differentiating normal breast cells from breast cancer cells and identifying different subtypes of breast cancer cells.This method can rapidly extract and identify tumor cells from peripheral blood with simple and convenient operation,which lays the foundation for evaluating NK cell cytotoxicity based on changes in tumor single-cell metabolites.2.A model of NK cell immunotherapy is constructed and a method for evaluating the cytotoxicity of NK cells based on the changes in tumor single-cell metabolites is developed.The model of NK cell immunotherapy is established by co-culturing hepatoma cells and NK cells.The single-cell metabolite profile of hepatoma cells before and after being killed is obtained by label-free flow cytometry.It is found that the profile changed dramatically with a significant decrease in the content of small molecular metabolites such as glutathione,glutamic acid and glutamine.According to the content and distribution of metabolites,the cytotoxicity of NK cells can be evaluated,and the tumor single cells can be further divided into two categories,namely resistant type and sensitive type.3.Based on the above model,a method is established to evaluate NK cell cytotoxicity according to the content changes of lipid and lipid peroxide in tumor cells.It is discovered that the content of lipids and their peroxide in hepatoma cells changed significantly after being killed by NK cells,with a decrease in the content of polyunsaturated lipids and an increase in the content of lipid peroxides.Based on the ratio of lipids to their peroxides in single cells,the cytotoxicity of NK cells can be evaluated and the level of cellular oxidative stress can be judged.