Construction And Preliminary Clinical Verification Of A Circulating Tumor Cell Separation System For Pediatric Brain Tumors

Background: Brain tumors are of the highest morbidity among solid tumors in children.The early symptoms of the patients are concealed.The disease has become severe when obvious clinical symptoms appear.At present,clinical imaging examinations will affect the accurate judgment of reading due to bleeding,inflammation and other factors.In addition,the lack of specific serum markers in pediatric brain tumors has limited the diagnosis and evaluation of the efficacy of children with brain tumors,resulting in the inability to adjust the treatment plan in time and delay in treatment.Therefore,finding more suitable detection methods in clinic to judge and evaluate the conditions of children with brain tumors has gradually become an important topic to improve the level of diagnosis and treatment of children with brain tumors.Objective: This study was based on Epidermal Growth Factor Receptor(EGFR)and Glial Fibrillary Acidic Protein(GFAP)antibody immunolipid magnetic sphere technology and immunofluorescence identification technology to construct an immunomagnetic spherical / immunofluorescent children's brain tumor circulating tumor cell(CTC)separation and identification system,used to efficiently capture and identify CTC in peripheral blood and cerebrospinal fluid of children with brain tumors.The stability,security and effectiveness of the CTC isolation and identification system for children's brain tumors are evaluated through cell experiments,animal experiments,clinical validation and genetic testing.It was verified that the cells obtained by this system were circulating tumor cells.Methods: In this paper,EGFR magnetic liposome(EG-ML)and GFAP magnetic liposome(G-ML)were prepared by reverse evaporation.We used particle size potential analyzer,protein electrophoresis instrument,atomic force microscope,ultraviolet spectrophotometer,magnetic analyzer,and X-ray diffraction to characterize and study its molecular structure and crystalline properties,and to establish a brain tumor CTC isolation and identification system.We make evaluations through cell experiments,animal experiments,and clinical validations.Cells obtained by EG-ML and G-ML magnetic sorting were used to extract DNA and RNA respectively,and then performed quantitative PCR ?RNA-seq ?Sanger sequencing and WES.Combined with the mutant genes identified in solid tumor samples,the differences between the mutant gene and RNA expressions in CTC were analyzed.We also followed up the children to analyze the factors affecting survival time.Results:(1)Preparation and characterization of immunolipid magnetic spheres.EG-ML and G-ML immunolipid magnetic spheres were successfully prepared.The prepared EG-ML and G-ML have a particle diameter of about 122 nm and a potential of about 30 m V.They are relatively stable in aqueous solution,uniformly dispersed,basically monodisperse and difficult to agglomerate.The antibody on the surface of the immunolipid magnetic sphere has high purity,closed hysteresis curve,superparamagnetism,saturation magnetization of 32.0 Am2/Kg,and crystalline properties of magnetic particles.Cytotoxicity experiments show that EG-ML and GML have low cytotoxicity and good biocompatibility,and can meet the requirements of clinical use.(2)Construction and research of a new CTC isolation and identification system for children's brain tumors.By gradually optimizing the parameters such as the amount of reagents and reaction conditions during the construction of the separation and identification process,a set of CTC detection systems was initially constructed.Then immunofluorescence was used to identify the cells,DAPI for intact cells,CD45-PE for leukocytes,and EGFR-FITC/GFAP-FITC for brain tumor cells.In addition,cell experiments and animal experiments have shown that the two magnetic spheres have a high ability to capture children's brain tumor cells,and their recovery rate of capture can reach 95%.(3)Clinical verification shows that EG-ML and G-ML can effectively capture CTC in peripheral blood and cerebrospinal fluid of children with brain tumors,and the staining effect is clear and discernible.According to the analysis of clinical data,in cerebrospinal fluid or blood of children with brain tumors,there was no significant correlation between CTC quantity and WHO grade of tumors and age of children.(4)Follow-up of the children,15 children died,and the 1-year survival rate was 82%.The results showed no significant differences in age,gender,and WHO classification with survival time.However,the amount of CTC in CSF was significantly correlated with survival time.With the increase of CTC,the survival time is shorter and the prognosis is worse.(5)We screened 2,551 RNAs with significant differences in CTC,of which1,238 were up-regulated and 1,313 were down-regulated.(6)The expression of NPR3 gene in the blood and cerebrospinal fluid CTC of some children with medullary tumor(group 3)is highly expressed,and the NPR3 cytoplasmic nucleus of tumor cells in the corresponding tumor specimen tissue is positive.(7)We performed Sanger sequencing on some children with diffuse endogenous pontine glioma.Mutations in H3K27 M were detected in cerebrospinal fluid and tissue.The results showed that genetic testing may be more effective in cerebrospinal fluid samples.Patients with specific genetic changes need to undergo CTC single-cell sequencing.(8)We also performed WES sequencing on glioma patients.The results showed that KMT2 A and TMPRSS2 mutations in blood and tissue.This indicated that the cells selected by the self-made immunolipid magnetic spheres are brain tumor cells.TMB was lower than the average TMB,and the response to immunotherapy was not obvious.Conclusion: In this study,the lipid material DOPC with low cytotoxicity and high biocompatibility was used as the matrix of magnetic microspheres.The amino group on the surface of GHDC was combined with GFAP and EGFR antibodies to prepare EG-ML and G-ML.It is stable,the surface antibody content is controllable,and it has the crystallization properties of magnetic particles.It can react with children's brain tumor cells to form antigen-antibody specific binding,and has high capture efficiency.A high-efficiency and accurate CTC detection system for children's brain tumors was formed.This system has the advantages of non-invasiveness,convenience and dynamic monitoring.Combined with genetic testing,it has important clinical reference value for early screening and diagnosis,curative effect detection and prognostic evaluation of brain tumors in children.