Characterization Of Oligodendrocyte Precursor Cell(OPC)-like Tumor Cells In Human Gliomas

Background:Gliomas are the most common primary tumors of the central nervous system(CNS).Even with advanced clinical practice including surgical resection combined with radio-chemotherapy,the prognosis remains dismal,particularly the most advanced one,glioblastoma multiforme(GBM),the 5-year survival of which is only 5.5%.This lack of progress is largely associated with high inter-and intra-tumoral heterogeneity.Tumor tissues from not only different patients,but also from the same ones,can be stratified into distinct morphopathological groups or molecular subtypes.Such heterogeneity is generally considered as the main reason for drug resistance and high recurrence rate during treatment.The concept of "Cancer Stem Cell" gives hope for solving the challenge of glioma heterogeneity.The cancer stem cell is regarded as the seed of tumorigenesis,progression and migration for its capability to initiate tumor,reconstruct tumor heterogeneity and have resistence to radiotherapy and chemotherapy,therefore becoming the glioma research hotspot.However,the indistinguishable identity of cancer stem cell limit its application in translational medicine and clinical practices.Identifying the cancer cell of origin helps to better understand tumor heterogeneity and therefore giving hope to design novel clinical strategy.Taking advantage of genetic lineage-tracing techniques,performed mainly on genetically engineered mouse models(GEMMs),we and other groups have previously reported a glial cell type termed oligodendrocyte precursor cells(OPCs)can function as the cell of origin of proneural subtype of high-grade glioma and serve as the cancer stem cell(CSC)of this glioma subtype.Our recent work revealed that there exist OPC-like tumor cells in OPC originated glioma model,which were derived from normal OPC and have features of cancer stem cells including proliferation and multiple differentiation potential in vitro and tumor initiation capacity in vivo.With formalin-fixation-paraffin-embedding(FFPE)tissue samples from glioma patients,we found that Olig2 positive tumor cells are prevalent in human gliomas regardless of malignancy and the major proliferating pool in glioma.Considering that Olig2 is a stable cellular marker of normal OPC in brain,it indicated that these Olig2 positive tumor cells are resembling normal OPC to some extent.Therefore,OPC like tumor cells may play an important role in human glioma,and it is of great significance for basic research and clinical translation to deeply dissect the cellular features and biological functions of this cell type.Objective:To characterize OPC-like tumor cells in human gliomas regardless of malignancy including identity,cellular morphology,distribution,biological functions,as well as their relationship with glioma malignancy,heterogeneity,histological and molecular subtypes,cancer stem cells.Methods:(1)?To build a bank of frozen tissue sample of human glioma compatible to high-resolution histochemistry analysis and also to set up a stable protocol of multiplex immunofluorescence staining for human brain(normal or tumor)tissue quenching autofluorescence.(2)?To carry out multiplex immunofluorescence staining with frozen tissue samples from human glioma(WHO ?,?,?).OPC specific markers such as Olig2,PDGFRa,NG2,Sox10 would be co-stained with proliferation marker Ki67,stem cell marker Sox2 and astrocyte specific marker GFAP.Finally,we would quantify the percentage of OPC like tumor cells and proliferating rate in human glioma.(3)?To found the system of primary culture of human glioma cell.To explore the features of OPC-like tumor cells after primary culturing and in secondary tumors of NOD-SCID mice.(4)?To enrich OPC like tumor cells directly from fresh human glioma tissue through immunopanning.The stability and efficiency of immunopanning would be tested by RT-PCT analysis and RNA transcriptome sequencing which would also the analyse the different characteristics of panned cells and supernatant cells.(5)?To investigate the sternness of OPC like tumor cells through sphere formation experiment and proliferation and differentiation experiment in vitro.(6)?To investigate the capability of OPC like tumor cells to initiate secondary tumor in vivo through patient derived xenografting(PDX)mouse model.Results:(1)?We successfully build a bank of frozen tissue sample of human glioma compatible to high-resolution histochemistry analysis and also set up a stable protocol of multiplex immunofluorescence staining for human brain(normal or tumor)tissue quenching autofluorescence.(2)?Multiplex immunofluorescence staining revealed that 40?50%tumor cells are Olig2 positive in human glioma regardless of malignancy,among which about 30?40%are co-stained with PDGFRa,a highly specific marker to OPC,therefore resembling normal OPC.Approximate 30?50%proliferating cells(Ki67 positive)are Olig2 and PDGFRa co-stained.Further,almost all PDGFRa positive cells are also Olig2 positive and Sox2 co-stained.Sox2 is regarded as a cellular marker of stem cells.(3)?We successfully found the system of primary culture of human glioma cell.Proliferating OPC-like tumor cells exist in primary human glioma cell lines and secondary tumors of NOD-SCID mice.(4)?We successfully enriched OPC like tumor cells from fresh glioma tissue through immunopanning.And the RT-PCR analysis revealed that compared with the crude cells before enrichment and non-OPC like cells in the supernatant,the enriched OPC like cells express higher level of specific genes of normal OPC,including PDGFRa,Olig2,NG2,NKX2.2.Besides,some other OPC specific genes such as SOX10,DLL3,PLP1,CLDN11,MBP were also enriched in OPC like tumor cells rather than non-OPC like cells according to RNA sequencing analysis.Single sample Gene Set Enrichment Analysis(ssGSEA)at the transcriptome level further revealed that the enriched OPC like tumor cells were consistently enriched for the proneural signature compared to the non-OPC like cells.(5)?The results of in vitro experiments revealed that OPC like tumor cells enriched from fresh human glioma tissue have capacity of sphere formation and proliferating in vitro.Conclusion:OPC-like tumor cells are prevalent in human gliomas regardless of malignancy and encompass a significant proliferating pool with features of cancer stem cell,therefore may play an important role in human glioma.