Genetic Features Of Primary Glioblastoma Cells Outside The Contrast-enhanced Region Of Magnetic Resonance Imaging

Objective:The glioblastoma(GBM)recurrence rate is high,despite multimodal treatment including surgery,radiotherapy,chemotherapy,and immunotherapy.Most recurrences occur at the resection margin that is located outside the GBM contrastenhanced region(C region)of magnetic resonance imaging(MRI)and usually left behind after surgery.However,the nature of GBM cells outside the C region is unclear,which would impede the efficacy of postoperative treatment.Therefore,we sought to investigate the characteristics of GBM cells outside the C region to back up the development of novel treatment against GBM recurrence.Method:Based on the MRI signal,the GBM lesion was divided into three regions,the contrast-enhanced region(C region),peri-enhancement edematous region(P region),and peri-edema normal region(N region).Twelve samples from these three regions of four patients with GBM were obtained under the guidance of MRI during surgery.The C regions were sent to the Department of Pathology for the confirmation of pathological diagnosis as primary isocitrate dehydrogenase 1-wild type(IDH1-wt)GBM.Single cell droplet generation,reverse transcription,and cDNA library preparation were performed according to the protocol of 10×Genomics(Single cell 3’Reagent Kit v2).inferCNV algorithm was used to analyze the copy number variations(CNVs)of GBM cells that were then clustered into different CNV patterns based on the characterizations of CNVs.Monocle2 algorithm was used to perform pseudotime analysis of GBM cells.Transcription factor-transcription cofactor(TF-TCF)interaction networks were constructed to uncover the transcriptional regulatory mechanism of different CNV-subtypes of GBM cells,based on which the core gene sets of networks were figured out.GBM cohorts from The Cancer Genome Atlas(TCGA)and China Glioma Genome Atlas(CGGA)were used for the analysis of the prognostic values of core gene sets in GBM patients.Bioinformatics analysis,primary cell culture,GBM sphere culture,immunofluorescence,immunohistochemistry,optical tissue clearing,and two-photon microscopy were used to analyze the GBM cells in three regions.Result:Based the characteristics of CNVs,GBM cells colleceted in this study were clustered into four CNV patterns,CNV 1,CNV 2,CNV3,and CNV4.CNV 1,CNV 2,and CNV 3 GBM cells showed typical CNVs such as chromosome 7 amplification and chromosome 10 deletion,while CNV 4 GBM cells had only fragmental CNVs in chromosomes such as 1,10,11,12 and 19.More than 90%of CNV 4 GBM cells were distributed in the P and N regions of four patients with GBM,while the other CNV patterns’ GBM cells were found mainly in the C region of particular patients.Pseudotime analysis revealed a transitional trend from CNV pattern 4 to other CNV patterns.Only the core gene set of TF-TCF interaction network of CNV 4 GBM cells(HES1,CEBPD,NOTCH3,JUNB,JUN,EPAS1,ATF3,ACTN1,CEBPB,EGR1,FOS,NFKBIA,MYC)showed prognostic significance in GBM patients,while the core gene sets of other CNV patterns showed little discernible correlation.JUNB,ATF3,ACTN1,CEBPB,EGR1,and NFKBIA belonged to the core gene set of CNV 4 GBM cell.These six genes were upregulated in recurrent GBM tissue when compared to primary GBM tissue,and they were negatively correlated with the disease free survival of patients with GBM.Furthermore,CNV 4 GBM cells showed the potential of tumorigenesis,and a sub-cluster of CNV 4 GBM cells possessed stem cell-like properties.This sub-cluster is mainly distributed in P region(166/173),a few in C region(4/173)and N region(3/173).Conclusion:Our findings indicate that GBM cells exist in C region,P region,as well as N region.CNV 4 GBM cells are mainly located in P and N region,the core gene set of which has prognostic value in patients with GBM.The stem cell-like subset of CNV 4 GBM cells might contribute to GBM recurrence.This study sheds light on a neglected aspect of GBM,and opens new avenues to explore GBM recurrence.