The Study On The Mechanism Of Malignant Transformation Of Neural Stem Cells Induced By Extracellular Vesicles Derived From Glioblastoma

Objective:Glioblastoma multiform(GBM)is a common malignant tumor in central nervous system(CNS),the mortality and recurrence rate stay at a high level for years with a 5-year survival less than 10%.Most patients have to suffer from recurrent GBM in a short period even after surgical excision,radiotherapy and chemotheropy.The mechanisms of GBM recurrence are still unclear.Neural stem cells(NSCs)are a cluster of cells with multidirectional differentiation and infinite proliferation potential that reside in hippocampus,corpus striatum,cerebral cortex and subventricular zone(SVZ)of CNS in mammalian embryos,whereas in adults,NSCs are only found in hippocampus and SVZ.NSCs participate in injury repair,neural cell regeneration,neural degenerative disorders and neural inflamation.Through clinical observation and literature review,we found that recurrent GBM usually had close relationship with SVZ,and it 1s less than 12 months of overall survival for patients that suffered from GBM derived from SVZ or adjacent place.The results suggest that NSCs in SVZ may take part m the relapse of GBM.Extracellular vesieles(EVs)are particles with diameters in 30-1000 nm and are secreted into extracellular space by almost each kinds of human cells,furthermore,tumor cells secreted more EVs than normal cells.It has been confirmed that EVs are involved in tumorogenesis,invasion,metastasis,neoangiogenesis and drug resistance.In GBM,EVs mediated transportation of important proteins and RNAs,which realized signal transduction among cells.This study explored whether EVs derived from GBM play an important role through NSCs in the process of GBM recurrence.Methods:Select proper GBM cell lines by tumor formation in nude miee.The selected cell lines were cultured with serum-free medium,and EVs were extracted from these culture medium.The EVs were identified by Transmission electron microscope(TEM),western-blotting and flow cytometry.The NSCs gifted by other labs were identified through immunocytochemistry.In vitro induction experiments were performed by adding EVs into culture medium of NSCs,a series of controls were set to exclude disturbing factors.The differentiated cells were tested by proliferation assay,migration assay,clone formation assay,invasion assay and tumor formation assay.Single-cell sequencings were performed to reveal the transcriptome changes that happened in the transformation of NSCs into differentiated cells mediated by EVs from GBM.Results:Ln229 cell line was selected by tumor formation assay.The EVs derived from Ln229 cells were extracted and identified.NSCs began to differentiate into spindle-like cells and grew around the agar surface in radial pattern after EVs were added into the culture medium.There was no significant difference between the differentiated cells and Ln229 cells in proliferation rate,Ki67 expression and migration;the differentiated cells could form clones in soft agar,invade into adjacent aeras and obtain obvious invasion ability compared to NSCs in Transwell assay;the differentiated cells could significantly accelerate tumor formation of Ln229 cells in nude mice.Single-cell sequencings were performed and 10172 of these cells were divided into 9 clusters with differential gene expression,among which cluster 8 had close relationship with glioma.CXCL14,EFEMP1,S100B and SCRG1 played an important role in the transformation revealed by WGCNA analysis in cluster 8,PPAR signaling pathway was enriched in cluster 8 by KEGG analysis.Joint-tSNE and marker gene analysis revealed HMGA1,LGALS3,LGALS1,PDPN,EMP1,GLIPR1 and CD44 were also important in the process.Conclusions:EVs derived from GBM can induce malignant transformation of NSCs in vitro,EVs have the ability of performing signal transduction.The differentiated cells possess tumor-like properties,express differentiation markers,and have enhanced abilities in proliferation,clone formation,migration,invasion and acceleration of tumor formation in vivo.CXCL14,EFEMP1,SIOOB and SCRG1 may play an important role in NSCs transformation,the activation of PPAR signaling pathway and expression of HMGA1,LGALS3,LGALS1,PDPN,EMP1,GLIPR1 and CD44 may also be involved.