Isolation,Identification And Origination Of CD34~+ Liver Cancer Stem Cells

Liver cancer is the fifth most common cancer worldwide and the third leading cause of cancer death.Currently,the treatments of liver cancer include operation,liver transplantation,hepatic artery chemoembolization(TACE)and radiofrequency ablation(RFA).However,in the majority of cases,tumor regrowth and relapse of disease occur on completion of therapy.Over 90%of human liver carcinomas(HLCs)are hepatocellular carcinomas(HCCs).Understanding the pathogenesis and origin of hepatocellular carcinoma can be indispensable for the optimization of current treatment strategies.The concept of cancer stem cells(CSCs)is based primarily on the clinical and experimental observations that indicate the existence of a subpopulation of cells with the capacity to self-renew and differentiate as well as show increased resistance to radiation and chemotherapy.CSCs are proposed to be the origin of relapse and metastasis.CSCs are highly tumorigenic,metastatic,chemotherapy and radiation resistant,responsible for tumor relapse after therapy,and able to divide symmetrically and asymmetrically to orchestrate the tumor mass.Therefore,CSCs are a pivotal target for the eradication of many cancers including liver cancer.The exact origin of cancer stem cells(CSCs)remains unknown,but there are some opinions about the origin of cancer stem cells,including genetic events leading to cell transformation,cell fusion,horizontal gene transfer,and cell microenvironment promoting CSC formation and clonal selection.At present,many studies support that cancer stem cells come from normal adult stem cells.Cancer stem cells and normal adult stem cells have many similar characteristics,and share many pathways.Damaged DNA in normal adult stem cells by exposed to the carcinogenic microenvironment for long time may not be completely repaired timely and effectively,in this case,the key gene of these cells may get abnormal,inactivated or dysfunctional pathway that may cause normal adult stem cells to become cancer stem cells.Generally,current study focused on origin of tumor stem cell are conducted by many different research groups in their own fields that make it difficult to comprehensively understand the mechanism of cancer stem cell formation.The origin of cancer stem cells remains to be further extensive study.Isolating and identifying CSCs of each type cancer is essential for CSCs-targetting.Generally,surface markers of the CSCs have been identified in manycancer types.Therefore,our project focused on isolation and identification of liver cancer cells(LCSCs)by maker CD34,and investigation on origin of LCSCs.This thesis is involved in two study parts:first,we isolatied,identificated and investigated the CD34+ liver cancer stem cells(LCSCs);Second,the origin of CD34+ LCSCs has been studied?Part ?:Isolation and identification of human liver cancer stem cell.PLC/PRF/5 hepatoma cell were isolated from frozen liver cancer cells.Fluorescence activated cell sorting was used to acess cancer stem cell marker expression in PLC hepatocellular cancer cell lines.The sorted CD34+liver cancer stem cells were cultured in vitro.Immun-histochemistry,tumor formation,and in vitro differentiation experiments wstem ere conducted to determine whether sorted cells are asociated with live cancer cells(LCSCs)on the morphological and biological characteristics.1.Sorting and characteristing of CD34+ liver cancer stem cells(LCSCs)Employing flow cytometry,our previous studies showed that population of CD34+ cells in PLC ranged from 3%to 6%,greater than in the other six liver cancer cell lines.Eight CD34+PLC subpopulations and 4 subpopulations of progeny were sorted and injected into the NOD/SCID/IL2RG mice,respectively.Parental PLC and CD34-PLC were also injected into same model mouse.The tumors were formed within 3 months in transplanted mice.Results showed tumor xenografts were formed with injection of only 100 cells of CD34+PLC cells;whereas at least 100000 PLC or CD34-PLC were need to form tumor xenografts.This result indicates that sorted the CD34+PLC like LCSCs has very strong tumorigenicity.Immunohistochemical HE staining showed that the tumor xenografts produced by injection of CD34+PLC had pathological and histological features of human hepatocellular cancer.And immunofluorescence detected that xenografts expressed human liver specific proteins:albumin,alpha-fetoprotein,Alpha-1 antitrypsin and EpCAM.The sorted CD34+PLCs cultured in mouse feeder layer cells grew in clones with self-renewal for many passages that indicates their posses the characteristics of stem cells.HE staining showed cells from Human xenografts formed by CD34+PLCs.Eight subpopulations of CD34+PLCs and 4 progeny subpopulations of CD34+PLCs showed pathological and histological features of HLCs.Three types of HLCs were generated from CD34+PLC(HCC,CHC and CC).This is the first time to confirm that CD34+cells have the self-renew characteristic,like CSCs.These 12 cell subpopulations derived from the same origin,can independently generate tumor,that indicates they may be tumor initiating cells(TICs)Based on the above studies,CD34+PLCs was succesfully isolated from human hepatocellular carcinoma cell,and identified as LCSCs.2.Culture and expansion of the CD34+LCSCHow to apply CD34+LCSCs in vitro?Fresh sorted single CD34+LCSCs were cultured on mouse embryonic fibroblast feeder layer(MEFs)to study cell morphology and function condition after subculture in vitro.After 7-10 days of culture,a round cell clone was formed.CD34+LCSCs cell can not only be cultured in the MEFs,but also grow well in the MEFs after freeze and thaw.Thawed CD34+LCSCs with 22 pasages before frozen have as similar morphology feature as non-frozen cells.CD34+LCSCs also can be cultured on special ECM plate(commercial extracellular matrix).On this plate,cells can be cultured short-term without MEFs,and retain the cell morphology.Nude mice were gave subcutaneous injection with fresh-sorted CD34+LCSCs and CD34+LCSC clone cells(5 and 18 pasages),respectively.The result showed both of cell type injections formed xenografts within same time with same amount of injected cells that indicate that tumorigenic ability of CD34+LCSCs clone cells is same as of fresh-sorted CD34+LCSCs.CD34+LCSC cell clones cultured on MEFs were combined with specific antibody marker by immune staining,the results show that these cells expressed stem cell markers:CD34,CD117(c-kit)and SOX2;normal liver stem cell markers:AFP,CK19 and OV6;and CSC markers:CD44,CD133,EpCAM,CD133 and CD31.Sox2 is associated with stem cell pluripotency and self-renewal,Sox2 expression of CD34+LCSCs cell clone was detected by immune histochemistry.Results suggest that Sox2 is highly expressed in CD34+LCSCs,and plays an important role in the regulation of LCSCs.In order to access in vitro differentiation of the cloned CD34+LCSC,CD34+LCSCs clone cells were harvested after induction at days 0,4,9,and 13,respectively.qPCR measured liver albumin(ALB),alpha fetoprotein(AFP),CK7 and CK19;liver cancer markers CD133,CD44,CD90 and EpCAM,and expression of CD34 and CD31 gene.Expressions of all genes except CD34 were increased over time(p<0.05).The results demonstrated that CD34+PLCs function as an LCSC.Subpopulations showing specific antigenic traits indicate the types of HLC that will form in HLC xenografts,and it support that CD34+LCSCs is pluripotent cells.This study showed we succesfully cultured and amplified the sorted CD34+LCSCs in vitro and identified the clones produced by CD34+LCSCs.CD34+LCSC clone cells can maintain their tumorigenicity and pluripotency in long-term culture under culture conditions of our design,that prove that the CD34+LCSCs can be cultured in vitro like normal stem cells such as ESC and iPSC.Part ?:study on the origin of CD34+LCSCs1.CD34+LCSCs expressed liver stem cell,myelomonocytic cell markers and CD45.The liver stem/progenitor cell(HSPC)marker OV6,monocyte/macrophage markers CD 14,CD68,alpha fetoprotein(AFP),lysozyme and 1-alpha antitrypsin(?1-AT)were detected by flow cytometry and qPCR in CD34+LCSCs.Normally,hematopoietic stem/progenitor cells do not express monocyte gene,but CD34+LCSCs co-expressed CD68,CD14,CD31 and macrophage antigen,which means that the liver stem/progenitor cell markers and myeloid markers co-expressed in CD34+LCSCs.This expression pattern indicate CD34+LCSCs derived from two different cell mixed phenotype of liver and gallbladder stem/progenitor cells(HSPC)and mononuclear cells(monocytes/macrophages).Immunohistochemical showed that coexpression of Hep PARI and CD68,CK19 and CD68,AFP and CD68,?1-AT and CD31 were detected in human xenografts formed by CD34+LCSCs.Hepatic(carcinoma)markers and monocyte markers showing up in the same cell are proposed this two parent cell phenotype show up in a hybrid cell.Immunohistochemical stain showed that CD34+LCSCs cultured on mouse feeder cells were co-stained with antibodies against CD45 and OV6,CD45 and macrophage antigen(Macro Ag);Xenograft cells were co-stained with antibodies against Hep Parl and CD45,CD45 and CK19;This result further suggests that CD34+LCSCs may be a fusion of HSPC and monocyte cells.2.Xenograft cells and PLC expressed macrophage characteristicsExperiments showed CD34+LCSCs xenograft cells and PLC had ability in phagocytosis of Escherichia coli(E.coli)(p<0.05).Phagocytosis is the process by which the cells bind and internalize the particles larger than 0.75 ?m in diameter,and it can be performed only by phagocytes(neutrophils,monocyes,macrophages,dendritic cells,and mast cells).The results of qPCR showed that CD34+LCSCs xenograft cells and PLC expressed cytokine IL-1b,IL-6,IL-12A,IL-18,TNF-?,CSF1(M-CSF),and the chemokine IL-8,CCL2,CCL5(p<0.05).And these expressions could be enhanced by treatment of INF-?,IL-4 or LPS(p<0.05).All of these results suggest the cells have the characteristics of macrophages.3.Xenograft cells and PLC expressed Human liver phenotype and functionXenograft cells and parental PLC also expressed metabolizing phase ? and ?enzymes,CYP3A4,CYP2C9,CYP2C19,UGT1A1,UGT1A3,UGT1A6,UGT1A8,UGT1A10,and phase ? transporter protein,glucose transporter protein 2(Glut2)(p<0.05).The results showed human liver phenotype and function by xenograft cells and parental PLCs.4.Analysis of genomics and geneticsCytogenetic analysis showed that the mean modal number(50-60)of the chromosomes was 82%in CD34+PLC and 76%in parental PLC cells,and 13%of CD34+PLC and 12%of parental PLC cells had a greater chromosome number with a range from 90 to 116 in CD34+PLC and parental PLC.A change in thechromosome number in CD34+LCSCs may indieate whether there was any posibility that the nucleus was extruded from the heterokaryon.Because the PLC integrates with the genome of hepatitis B virus(HBV),we investigated whether CD34+LCSCs also integrates with HBV surface antigen gene(S),core gene(C),polymerase gene(P)and HBV DNA junctions.PCR and sequencing found that they have similar sequence in PLC and CD34+LCSCs,while in Hep G2 cells did not found these DNA fragments.There were 3 base pair differences(TCT instead of CTC)in the human DNA fragment from those in the previous publication.It is identified the three mutations sited in the CD34+LCSCs and PLC by PCR and sequencing.And that suggest hepatocellular carcinoma produced by PLC might come from the CD34+LCSCs.Fusion between two different cell types produces a heterokaryotic hybrid cell that initially contains the genetic elements and organelles of both cell types.So our results suggest that CD34+LCSCs were formed by fusion of HSPC with CD34+hematopoietic-derived myeloid intermediates.Through this comprehensive subject study,we obtained the following accomplishments:1.CD34+PLC was succesfully isolated from human hepatocellular carcinoma cell PLC;isolated CD34+PLC was identified as LCSCs and can differentiate into hepatic cell carcinoma three phenotype,it is the first time to find that HLC may be developed by CD34+ cells,which reveals the diversity of the origins of human liver tumor.The succes in vitro culture and amplification of CD34+LCSCs prove that the CD34+LCSCs can be cultured in vitro and amplification like normal stem cells such as ESC and iPSC.2.It is the first time to find one type hepatoma derived from CD34+hematopoietic precursor stem cells,and also it is the first report that human cancer stem cells may be formed by the fusion of different type cells.The series of experimental results of CD34+LCSCs enriched the theory of liver cancer stem and explored the tumor occurrence and development mechanism,and identified the cancer stem cell specific biological markers which can be used for early diagnosis of hepatocellular carcinoma and the evaluation of drug efficacy in the treatment of CSC and the development of new drugs,provide an important basis for clinical research in the treatment of liver cancer.