| Background and objectiveNasopharyngeai carcinoma?NPC?is a rare form of epithelial cancer accounting for 78.08%of head and neck tumor.China is the highest incidence of NPC,its occurrence is particularly high in Guangdong and Guangxi.Its incidence rate is 25-fold higher than that in Western cotmtries.Surgical approaches to treat NPC are limited by the inaccessibility of the anatomic location.therefore,treatments primarily rely on radiotherapy.Radiotherapy technique is advanced,however,approximately 30%-60%of patients presenting with loealized tumors develop recurrent disease or metastasis within 5 years.In recent years,the concept of cancer stem cell?CSC?has been proposed in 2001,which are defined as tumor cells that possess the self-renewal ability and can produce the heterogeneous lineages of cancer cells.Cancer stem cells have four characteristics including:?1?the self-renew capacity,self-renewal capacity means the ability cancer stem cells occupied to differentiate into precursor cells;?2?multiple differentiational potential,which means the ability stem cells possessed to produce offspring with varying degrees of differentiation of tumor cells and to form new tumors in the body.In tumor tissue,differentiated mature tumor cells have low malignancy,while poorly differentiated tumor cells have high malignancy;?3?high proliferative capacity,CSCs have high proliferative capacity than tumor cells;?4?drug resistance,multidrug resistance is a major cause of tumor fail to treatment.Therefore,cancer stem cells are the critical cause of tumor metastasis and recurrence.Only eradicate cancer stem cells,it will be possible to cure cancer.In order to eliminate tumor effectively,it is important to isolate the CSCs.Separation of CSCs from solid tumors has been successfully done through three different methods on the basis of the characteristics of CSCs.First of all,CSCs are isolated by flow cytometry according to CSC surface markers such as CD44,CD24 or CD 133.Second,with Hoechst 33342 staining,CSCs are enriched in the side population of cancer cells,excluding intracellular Hoechst 33342 in vitro and can be isolated by flow cytometry.Third,CSCs are enriched in a population of cells that can form tumor spheres under the cultivation of defined serum-free medium with basic fibroblast growth factor?bFGF?,epidermal growth factor?EGF?and B27 where the serum-free culture condition can maintain the CSCs undifferentiated state[1].Unlike other tumors,nasopharyngeal carcinoma has no explicit surface markers.It's difficult to sort CSCs of NPC by CD133.Side population method for sorting is not only complicated,but also unable to realize the isolation of CSCs in many tumors.The method of tumor spheres culture only can obtain limited tumor spheres,and it is not favor to the animal experiment in vivo.Although Wen-Jun Wang et.al studies verified the capacity of PKH26 dye retention to successfully identify slow cycling PKH26 +cells in NPC,where these cells possessed CSCs characteristics.In my experiment,we found that PKH26 labeled cells required repeated markers,and it was hard to trace in vivo due to the gradually weakened cell fluorescence intensity after many times subculture.It is necessary for us to seek new approach to separate CSCs in NPC.Nanog is a homeodomain-containing transcription factor that is crucial for maintaining the pluripotency of the inner cell mass[2],and is specifically expressed in human ES cells,primordial germ cells and fetal testis.The expression of Nanog has been shown to be regulated by Oct4/Sox2 heterodimers,in which Oct4/Sox2 binds to the octamer/sox elements within the Nanog proximal promoter region and induces Nanog transcription.It has been reported that downregulation of Nanog expression induced ES cells differentiation,whereas overexpression of Nanog inhibited differentiation of ES cells.Many studies showed that Nanog was expressed in many human tumors,including breast carcinomas,colorectal cancer,renal carcinomas oral cavity carcinomas and cervical cancer.The expression level of Nanog has been shown to be higher in cancer stem cells than non-stemness cancer cells in many types of tumors.Functional studies have demonstrated that Nanog is not only a CSC marker,but also promotes CSC-like characteristics in many tumors.Cheng Qian et.al and CR Jeteret.al studies utilized the Nanog promoter as a reporter to drive the expression of green fluorescence protein?GFP?.They could successfully isolate a small subpopulation of Nanog-GFP+ cells.They demonstrated that Nanog-GFP+ cells showed cancer stem cells?CSCs?characteristics such as increased self-renewal ability,clone formation ability and initiation of tumors and enhanced the expression of many CSC-associated molecules,including Oct4,Sox2,CD133,ABCG2 and CD44.Therefore,we can utilize pLV-Nanog-GFP vector to label CSCs in NPC.Through this method,CSCs can be visualized so as to test the therapeutic efficacy of targeting CSCs in animals.Cancer immunotherapy is the fourth method of cancer treatment after surgery,radiotherapy,chemotherapy.Its efficacy has been proven in a variety of tumors.The overall advantages of cancer immunotherapy including raising human immune system;improving the quality of life of patients;killing tumor cells and preventing tumor recurrence and metastasis.Adoptive immunotherapy is an important method of tumor immunotherapy,mainly including:CIK cells,LAK cells,CTL cells,TIL cells.Cytokine-Induced Killer?CIK?cells are ex-vivo expanded T-NK lymphocytes potentially able to address some of the issues currently limiting clinical application of other immunotherapies.CIK cells can be massively expanded from peripheral blood mononuclear cells?PBMC?cultured with the timed addition of IFN-gamma,Ab anti CD3,and IL-2 through simple standardized culture conditions.CIK cells are known to have several attractive advantages;1)CIK cells have strong anti-tumor effects;2)Cytotoxicity is MHC-unrestricted;3)More importantly,the toxicity is minimal and no graft-versus host reaction is observed.Tumor killing activity of CIK cells is mainly mediated by the interaction of their NKG2D membrane receptor with MICA/B ligands on tumor targets.More importantly,CIK cells have shown promising antitumor effects on melanoma stem cells and bone and soft tissue sarcomas stem cells.At present,whether CIK cells can kill cancer stem cells of NPC has not been reported.The aim of this experiment was to explore whether CIK cells can kill CSCs of NPC and its mechanism;whether Using Nanog promoter driving GFP and Luc gene expression to label CSCs of NPC was feasible.MethodsPart ? Killing activity of CIK cells against NPC cells and cancer stem cells in vitro and vivo1.CIK cells culture?1?Extracte 20 ml peripheral blood from healthy people,with 1640 solution according to the proportion of 1:1 dilution.?2?Add about 5 ml lymphocyte separation medium to 15 ml centrifuge tube,then join the diluted blood in the lymphocyte separation medium along the wall slowly,be careful not to blending.?3?After balancing,1800 rpm centrifugal for 15 minutes.?4?Sucked out lymph cell layer,with 1640 liquid washing again,1500 rpm centrifugal for 5 minutes,then throw the supernatant away,put the cells into the plate liquid culture.?5?Add CIK working liquid after 2-3days,the total cultured time is 3-4 weeks.?6?plate liquid:1640 100ml IL-2 100000 U Human serum 5%PHA 1mg Anti-CD3Ab 50 ? g?-IFN 10000 U?7?CIK working liquid:1640 100ml.IL-2 100000U Human serum 5%Anti-CD3Ab 50? g2.Expression of CD3+/CD56+,CD4+,CD8+,NKG2D+ cells of CIK cells by flow cytometry.3.To examine the killing activity of CIK cells against NPC cells?CNE2,SUNE1?by CCK8 and colony formation assay.4.To examine the migration and invasion ability of NPC cells after treated with CIK cells with Transwell and Boyden assay.5.To examine the ratio of SP cells of NPC cells after treated with CIK cells by flow cytometry.6.To examine the tumorsphere formation ability of NPC cells after treated with CIK cells by tumorsphere formation assay.7.Stable cell lines constructionLentivirus was produced by cotransfection of expressing plasmid along with packaging plasmids psPAX2 and pMD2.G into 293T cells using Lipofectamine 2000 reagent.Using the produced lentivirus to infect NPC cells by observing the EGFP expression efficient we determined the establishment of PNanog-GFP-T2A-Luc stably expressing NPC cell lines.8.Sorting Nanog-GFP?+?CSCs and Nanog-GFP?-?NPC cells,then to examine the killing activity of CIK cells against Nanog-GFP?+?CSCs and Nanog-GFP?-?NPC cells by CCK8.9.Using PKH26 dye to label CSCs,sorting PKH26+ CSCs and PKH26-NPC cells,then to examine the killing activity of CIK cells against PKH26+ CSCs and PKH26-NPC cells by CCK8.10.To observe CM-Dil labeled CIK cells gather with tumorsphere with GFP fluorescence in vitro.11.To observe the interaction between CIK cells and NPC cells or CSCs by Time-lapse.12.To observe PKH26 labeled CIK cells gather with Nanog-GFP?+?CSCs at tissue level by confocal microscope.13.To examine the ratio of SP cells,clone formation ability and tumor sphere ability of NPC cells after blocking of NKG2D receptor with neutralizing monoclonal antibody.14.To examine the clone formation ability and tumor sphere ability of Nanog-GFP?+?CSCs after blocking of NKG2D receptor with neutralizing monoclonal antibody.15.To examine apoptoic of NPC cells and Nanog-GFP?+?CSCs after treated with CIK cells and examine secretion of immune cytokines of CIK cells.16.In vivo activity of CIK cells against CNE2 cells in nude mice.17.In vivo activity of CIK cells against CNE2 cells transduced with LV-Nanog-GFP-Luc in NOD/SCID mice.18.To examine bioluminescent light signals intensity from whole body imaging after treated with CIK cells by I VIS imaging system.19.To examine cell apoptosis and necrosis of tumor CIK cells treatment by HE.20.To analyze cell proliferation with BrdU,Ki67 staining and expression of GFP after CIK treatment by IHC.21.Statistical analysisStatistical analysis was performed using a SPSS 13.0 software package and Graphpad 5.0 software.Data were presented as mean+SD?X±S?.Two-tailed Student' S t test was used for comparisons of 2 independent groups for Tranwell and Boyden assay,TUNEL and ELISA assay.One-way ANOVA was used for comparisons of more than two groups for CIK cells phenotype analysis,CCK8 assay,clone formation assay,tumorsphere formation assay,SP assay,imunohistochemistry assay.If homogeneity of equal variance assumed,multiple comparison were performed using LSD method,if homogeneity of equal variance not assumed,Welch was used for comparisons of more than two groups,multiple comparison were performed using Dunnett T3 method.The comparison of tumor growth curve and Luc signaling curve were analysed using repeated measures.P<0.05 was considered as statistically significant.Part ? Identify the sternness of Nanog-GFP?+?CSCs1.To construct stable NPC cell lines carrying PNanog-GFP-T2A-Luc.2.To examine the proportion of Nanog-GFP?+?cells.3.To examine the correlation between the number of tumor cells and bioluminescent light signals intensity by I VIS imaging system.4.To detect protein expression of stem markers in Nanog-GFP?+?cells and Nanog-GFP?-?cells by Western blot.5.To detect tumor sphere formation ability in Nanog-GFP?+?cells and Nanog-GFP?-?cells by tumor sphere formation assay.6.To detect colony formation ability in Nanog-GFP?+?cells and Nanog-GFP?-?cells by the plate colony formation assay.7.To detect migration and invasion ability in Nanog-GFP?+?cells and Nanog-GFP?-?cells by Transwell and Boyden assay.8.To detect protein expression of EMT markers in Nanog-GFP?+?cells and Nanog-GFP?-?cells by Western blot.9.Statistical analysisStatistical analysis was performed using a SPSS 13.0 software package and Graphpad 50 software.Data were presented as mean+SD?X±S?.Two-tailed Student'S t test was used for comparisons of 2 independent groups for clone formation assay,Tranwell and Boyden assay,tumorsphere formation assay.P<0.05 was considered as statistically significant.ResultsPart ? Killing activity of CIK cells against NPC cells and cancer stem cells in vitro and vivo1.Phenotype analysis of CIK cellsCIK cells were expanded from fresh PBMCs cultured with the timed addition of IFN-gamma,Ab-anti-CD3,and IL-2.CD3+ CD56+ cells are negligible in fresh human PBMCs,but they will markedly expand from T cell precursors,the absolute number of immune cells increased by more than 200-fold for 14-21 days.Most CIK cells were CD3+ CD8+ CD56+ cells.When we examined the phenotypes of the cultured cell population with fluorescence-activated cell sorting analyses,the cell population was composed of?4.7±2.5?%CD3+/CD56+ at 7 days,?41.4±7.5?%at 14 days,?61.9±7.9?%at 21 days,the proporation at different culture time were statistically significant difference?F=42.888,P<0.001?;?67.5±8.9?%CD8+ at 7 days,?79.3±10.4?%at 14 days,?94.8±1.5?%at 21 days,the proporation at different culture time were statistically significant difference?F=8.892,P=0.016?;?24.8±4.4?CD4+ at 7 days,?10.2±2.5?%at 14 days,?2.9±0.8?%at 21 days,the proporation at different culture time were statistically significant difference?F=42.888,P<0.001?;The median membrane expression of the NKG2D receptor,main responsible for tumor recognition,on expanded CIK cells was?85.4±6.9?%at 7 days,?85.4±6.9?%at 14 days,?92.8±2.7?%at 21 days,the proporation at different culture time were not statistically significant difference.2.Killing activity of CIK cells against NPC cell lineThe cytotoxic effect was obviously increased with the increase in effector cells/target cells ratio?E/T?.The average specific tumor killing was?CNE2:74.1%,67.7%,59.5%,and 58.4;SUNE1:88.3%,81%,70.3%,and 62.3%?at 50:1,30:1,10:1,and 5:1 effector/target ratio,respectively?n=6?utilizing an CCK8 assay.The inhibition rate in different treatment groups were statistically significant difference?CNE2:F=38.375,P<0.001;SUNE1:F=55.754,P<0.001?.Colony formation assay showed that when NPC cells treated with CIK cells,with the increase of effector/target ratio,NPC cells formed colonies gradually decreased.The colony formation number in different treatment groups were statistically significant difference?CNE2:F=229.642,P<0.001;SUNE1:F-99.121,P<0.001?.Transwell and Boyden assay showed NPC cells treated with CIK cells significantly reduced cell migration and invasion.Compared with control group,the mobility was reduced by 61.2%and 77.8%respectively in CIK cells treatment group,invasion ability was decreased by 88.8%and 69.5%respectively.The mobility at different groups was statistically significant difference?CNE2:t = 7.992,P = 0.001;SUNE1:t = 9.95,P =0.001?,the invasion ability at different groups was statistically significant difference?CNE2:t = 8.66,P = 0.001;SUNE1:F= 11.27,P = 0.007?.In summary,CIK cells can significantly inhibit the proliferation of NPC cells in vitro.3.CIK cells effectively kill CSCs of NPCIn order to validate whether CIK cells can kill CSCs of NPC,first,we used SP assay to detect the ratio of side population cells of NPC cells after CIK cells treated.SP assay showed with the increase of effector/target ratio,the ratio of SP cells was decreased gradually.In CNE2 cells,the ratio of SP cells was decreased from 8.8%to 3.3%;In SUNE1 cells,the ratio of SP cells was decreased from 4.6%to 0.2%.The ratio at different groups was statistically significant difference?CNE2:F=8.614,P=0.017;SUNE1:F=156.069,P<0.001?.Tumor sphere is a method of identification and isolation of cancer stem cell.To evaluate the tumorigenic capability of NPC cells after treated with CIK cells.We cultured cells under serum-free medium with bFGF,EGF and B27.We found that with the increasing of effector/target ratio,NPC cells formed tumor spheres gradually reduced.Compared with control group,tumorsphere formation number was decreased by?CNE2:53.1%,65.8%,76.4%,88%;SUNE1:35.5%,68.1%,90%,99.6%?respectively.The tumorsphere formation number at different groups was statistically significant difference?CNE2:F=1020.851,P<0.001;SUNE1:F=495.1,P<0.001?.We concluded that CIK cells could impair stem cell self-renewal properties.4.CIK cells can directly kill Nanog-GFP?+?CSCs and PKH26+ CSCsTo identify putative CSCs,we transduced NPC cells with a lentiviral vector encoding the GFP controled by the human Nanog promoter.The average GFP expression,10 days after transduction,was 5.4%in CNE2 cells line and 1.4%in SUNE1 cells line.Based on GFP expression,transduced NPC cells were sorted into two fractions?Nanog-GFP?+?and Nanog-GFP?-??that served as targets to assess separately the antitumor activity of CIK cells.We assessed the ability of CIK cells to kill Nanog-GFP?+?and Nanog-GFP?-?cells.Result showed CIK cells could both kill Nanog-GFP?+?and Nanog-GFP?-?cells.The cytotoxic effect was obviously increased with the increase in effector cells/target cells ratio?E/T?.The average specific killing was?CNE2-GFP+:91.3%,82.3%,67.9%,and 63.4%;CNE2-GFP-:94.2%,82.1%,67.9%,and 62,8%;SUNE1-GFP+:94%,83.5%,67.6%,and 62.7%;SUNE1-GFP-:92.2%,81.6%,67.9%,and 64.1%??n=4?at 50:1,30:1,10:1,and 5:1 effectors/target ratio,respectively.The inhibition rate in different treatment groups were statistically significant difference?CNE2:FGFP+=68.367,P<0.001,FGFP=71.722,P<0.001;SUNE1:FGFP+=71.454,P<0.001,FGFP =111.127,.P<0.001?.In addition,we used the PKH26 fluorescent dye to identify slow cycling PKH26+ cells,which have many stem cell properties.PKH26+ and PKH26-NPC cells were sorted by flow cytometry.The data showed CIK cells could efficiently killed PKH26+cells and PKH26-cells;the cytotoxic effect was obviously increased with the increase in effector cells/target cells ratio?E/T?,The average specific killing was?CNE2-PKH26+:93.4%,90%,85.9%,76.1%;SUNE1-PKH26+:92.8%,86.6%,82.2%,and 78.6%??n=6?at 50:1,30:1,10:1,and 5:1 effectors/target ratio,respectively.The inhibition rate in different treatment groups were statistically significant difference?CNE2:FPKH26+=47.849,P<0.001,FPKH26-=43.69,P<0.001;SUNE1:FPKH26+=8.827,P=0.006,FPKH26-=21.565,P<0.001?.In order to visualize the killing activity of CIK cells against NPC cells and cancer stem cells,we applied Time-lapse to observation the interaction of CIK cells with nasopharyngeal carcinoma tumor cells and cancer stem cells.The results showed CIK cells can directly kill NPC cells,Nanog-GFP?+?CSCs and tumorsphere.At the same time,in vitro,we used red fluorescent dye CM-Dil to labele CIK cells,then concultured with tumorsphere carrying GFP fluorescent,we observed that CIK cells gradually gathered in the tumorsphere surface,then gradually killed tumorsphere.At tissue level,we labeled CIK cells with PKH26 dye,we also observed that CIK cells gradually gathered in Nanog-GFP?+?CSCs,then gradually killed Nanog-GFP?+?CSCs by confocal microscopy.5.CIK cells kill NPC cells and CSCs via the NKG2D receptor recognition and the induction of apoptosis and secretion of immune cytokinesIn order to define whether CIK cells kill NPC cells and cancer stem cells by NKG2D receptor recognition,when NPC cells treated with CIK cells,the ratio of SP cells decreased from 5.6%to 1.7%in CNE2 cells and 2.5%to 0.4%inSUNE1 cells.But after blocking with anti-NKG2D neutralizing antibody?20mg/mL?,the ratio of SP cells increased from 1.7%to 5.0%in CNE2 cells and 0.4%to 2.1%in SUNE1 cells.The ratio of SP cells at different groups was statistically significant difference?CNE2:F=11.872,P=0.008;SUNE1:F=36.963,P<0.001?.Clone formation assay showed that when NPC cells treated with CIK cells,cpmpared with control group,cloning efficiency decreased 75.8%,64%respectively.After blocking with anti-NKG2D neutralizing antibody,cloning efficiency restored 45%,32.5%respectively.The clone formation number at different groups was statistically significant difference?CNE2:F=724.422,P<0.0001 SUNE1:=125.354,P<0.001?.Tumorsphere formation assay showed that when NPC cells treated with CIK cells,cpmpared with control group,tumorsphere efficiency decreased 80.5%,78.7%respectively.After blocking with anti-NKG2D neutralizing antibody,tumorsphere efficiency restored 56.5%,61.2%respectively.The tumorsphere formation number at different groups was statistically significant difference?CNE2:F=250.832,P<0.001:SUNE1:F=103.096,P<0.001?.TUNEL and ELISA assay showed that CIK cells also can induce apoptosis and secret IL-2,IL-4,IL-6,IL-10,TNF-?,IFN-? to kill NPC cells.For Nanog-GFP?+?CSCs,We examined that Blocking of NKG2D receptor with antibody partially restored clone formation ability.When Nanog-GFP?+?CSCs treated with CIK cells,cpmpared with control group,cloning efficiency decreased 72.5%,67.5%respectively.After blocking with anti-NKG2D neutralizing antibody,cloning efficiency restored 46.5%,46.1%respectively.The clone formation number at different groups was statistically signi:ficant difference?CNE2:F=198.743,P<0.001;SUNE1:F=275.482,P<0.001?.Tumorsphere formation assay showed that when Nanog-GFP?+?CSCs cells treated with CIK cells,cpmpared with control group,tumorsphere efficiency decreased 81.2%,53.3%respectively.After blocking with anti-NKG2D neutralizing antibody,tumorsphere efficiency restored 60.2%,23.8%respectively.The tumorsphere formation number at different groups was statistically significant difference?CNE2:F=286.118,P<0.0001;SUNE1:F=99.125,P<0.001?.In addition,TUNEL assay also showed that CIK cells can induce apoptosis to kill Nanog-GFP?+?CSCs.Taken together,our results demonstrated that CIK cells kill NPC cells and CSCs via the NKG2D receptor recognition and the induction of apoptosis and secretion of immune cytokines.6.In vivo killing activity of CIK cells against CNE2 cells transduced with LV-Nanog-GFP-Luc in NOD/SCID miceTo more closely simulate the real clinical situation,we evaluated the activity of CIK cells in vivo against tumor xenografts in NOD/SCID mice.NOD/SCID mice?n=20?were subcutaneously implanted with 1x106 CNE2 cells transduced with LV-Nanog-GFP-Luc.7 days after tumor implantation,1×107 CIK cells or 3×107 CIK cells were infused by tail vein injection every two or three days?n=13?.When tumor growth in untreated mice?n=7?was more than 1.5 cm in at least one dimension,all animals were euthanized.Tumors were excised and analyzed for evaluating proliferative index Ki-67,BrdU and sternness marker GFP.The presence of lymphocytic infiltration and the extension of necrotic tissue areas were also observed.At the end of the experiment,a significant delay in the tumor growth was observed in treated mice compared to untreated controls.The tumor volume at different groups was statistically significant difference?F=9.024,P<0.001?,and the tumor weight at different groups was statistically significant difference?F=9.250,P<0.001?.Moreover,immunohistochemistry of Ki-67,BrdU,GFP detected decreased expression in tumors from animals treated with CIK cells,proliferative index Ki-67,BrdU and stemness marker GFP at different groups was statistically significant difference?F=59.084,P<0.001;F=86.216,P<0.001;F=37.444,P<0.001?.Additionally,tumors from animals treated with CIK cells had significantly larger necrotic areas compared to untreated controls,and we could confirm the presence of CIK cells?CD5,CD56?infiltrating the tumor.In order to evaluate whether CIK cells kill Nanog-GFP-Luc?+?CSCs in vivo,before and during treatment,Nanog-GFP-Luc?+?CSCs were monitored using IVIS;the photon intensity,a measurement of viable tumor volume,was also determined by the system.The average photon intensity increased slowly following treatment with CIK cells,whereas,a significant increase in the photon intensity was observed in the control group.The photon intensity at different groups was statistically significant difference?F=51.357,P<0.001?.Immunohistochemistry of sternness marker GFP and detected decreased expression in tumors treated with CIK cells?P<0.05?.Part ? Identify the stemness of Nanog-GFP?+?CSCs1.Nanog-GFP?+?accounted for CNE2 cells about?5.4±1.0?%,for SUNE1 cells about?1.4±0.5?%.2.Western blot assay showed:Compared to Nanog-GFP?-?cells,Nanog-GFP?+?cells highly expressed sternness protein of Nanog,Klf4,Oct4,Sox2.Which showed Nanog-GFP?+?cells have stem cells properties.3.Tumorsphere formation assay showed:The tumorsphere formation capacity of Nanog-GFP?+?cells was significantly higher than Nanog-GFP?0?cells?CNE2:t=7.064,P=0.002;SUNE1:-10.199,P=0.001?.It showed that Nanog-GFP?+?cells had a stronger self-renewal,proliferation.4.Colony formation assay showed:The colony formation capacity of Nanog-GFP?+?cells was significantly higher than Nanog-GFP?-?cells?CNE2:t=6.298,P=0.003;SUNE1:t=13.298,P<0.001?.It showed that Nanog-GFP?+?cells had a stronger self-renewal,proliferation.5.Transwell and Boyden assay showed:The migration capacity of Nanog-GFP?+?cells was significantly higher than Nanog-GFP?-?cells?CNE2:t=9.33,P=0.001;SUNE1:t=10.261,P=0.007?.The invation capacity of Nanog-GFP?+?cells was significantly higher than Nanog-GFP?-?cells?CNE2:t=20.108,P<0.001;SUNE1:t=21.387,P<0.001?.6.Western blot assay showed:Compared to Nanog-GFP?-?cells,Nanog-GFP?+?cells highly expressed Vimentin,N-cadherin protein,lowly expressed E-cadherin,a-catenin.Which showed Nanog-GFP?+?cells could induce EMT.Conclusions1.We successfully established CIK cells culture system.2.CIK cells can inhibit NPC cells proliferation in vitro and in vivo.3.CIK cells can inhibit cancer stem cells proliferation in vitro and in vivo.4.CIK cells kill NPC cells and CSCs via the NKG2D receptor recognition and the induction of apoptosis and secretion of immune cytokines.5.Nanog-GFP?+?cells have the characteristics of cancer stem cells.Using Nanog promoter to drivesthe expression of GFP and Luc gene to label cancer stem cells is feasible. |
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