The Killing Activity And Mechanism Of CIK Cells Against Hepatocellular Carcinoma Stem Cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies in China. And it is the first and second leading cause of cancer death in country and city, respectively. In China, the leading causes of HCC is hepatitis B infection, hepatiis C infection and so on.Currently, surgical resection is considered the most conventional treatment for HCC worldwide. However, surgical resection is usually limited by many factors, such as liver dysfunction, the condition of patient, and multifocality. It is reported that, only 15% of patients have the opportunity to get the surgery resection when be diagnosised. Therefore, various alternative treatments, including transarterial chemoembolization (TACE) and radiofrequency ablation therapy (RFA), have been performed for HCC, and encouraging outcomes have been reported in some early-stage patients. Unfortunately, despite the remarkable improvements in therapeutic procedures, the 5-year survival rate of HCC has remained poor because intrahepatic recurrence is common, reported as 70% after surgery. It seems that as hypothesized for other tumors, HCC includes a small cell subpopulation referred to as cancer stem cells or tumor-initiating cells(TICs) endowed with the sternness features that sustain drug resistance and disease relapse. In Reya’s opinion, tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulated self-renewal in stem cells, and cancer cells may include "cancer stem cells"-rare cells with indefinite potential for self-renewal that drive tumorigenesis. Thus, this challenging clinical scenario demands new therapeutic approaches, ideally the ones that are able to target tumor-initiating cells.Nanog is a homeodomain-containing transcription factor that is crucial for maintaining the pluripotency of the inner cell mass, and is specifically expressed in human ES cells, primordial germ cells and fetal testis. Many studies showed that Nanog was expressed in many human tumors, including breast carcinomas, Glioma, bladder cancer and so on. 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 a promoter of CSC-like characteristics in many tumors. shan et.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 over-expression of many CSC-associated molecules, including Oct4, Sox2, CD 133, ABCG2 and CD44. Therefore, we constructed LV-PNanog-GFP-T2A-Luc vector to label CSCs in HCC. Through this method, CSCs can be visualized so as to test the therapeutic efficacy of targeting CSCs in vivo.Much promise lies in adoptive immunotherapy for the treatment of metastatic HCC. Cytokine-induced killer (CIK) cells are heterogeneous ex vivo-expanded T lymphocytes, with a mixed T-NK phenotype, able to exert a wide MHC-unrestricted antitumor activity against both solid and hematologic malignancies. CIK cells can be massively expanded from peripheral blood mononuclear cells(PBMC) cultured with the timed addition of IFN-y,Ab-anti-CD3, and interleukin(IL)-2 through simple standardized culture conditions. Initial clinical trial resulting in various tumor setting are encouraging, including HCC, more and more evidence indicating that the combination of CIK with RFA for HCC patients could improve PFS, and be helpful in the prevention of the recurrence. Whereas, whether or not the tumor-killing ability of CIK cells affects the subpopulation of HCC TICs is completely un explored. The aim of this study was to explore whether CIK cells can kill CSCs of HCC and its mechanism.MethodsPart.l CIK cells preparation and the Killing activity of CIK cells against HCC cells in vitro1. CIK cells culture20 ml of heparinized peripheral blood was obtained from healthy donnor. PBMCs were separated by Ficoll-Hypaque density centrifugation, resuspended in fresh medium containing IFN-y,recombinant human IL-2, PHA and anti-CD3 monoclonal antibody.2. Phenotype analysisCells were obtained from CIK cultures for phenotype analysis with appropriate monoclonal antibodies, including CD3-FITC, CD4-FITC, CD8-PE, CD56-APC and NKG2D-APC. The cells were incubated with various conjugated monoclonal antibodies for 20 min at 4℃, washed with PBS, and resuspended in 400ul PBS. Flow cytometric analysis were performed.3. CCK8 assay and colony formation assay were employed to assess the killing activity of CIK cells against HCC cells (SMMC7721 and Huh7).4. Transwell and Boyden assay were employed to examine the migration and invasion ability of HCC cells after treated with CIK cells5. Tumor sphere formation assay was employed To examine the tumor sphere formation ability of HCC cells after treated with CIK cells6. Statistical analysisStatistical analysis was performed using a SPSS 20.0 software package. Data were presented as mean+SD (X±S). Two-tailed Student’S t test was used for comparisons of 2 independent groups. One-way ANOVA was used for comparisons of more than two groups. If homogeneity of equal variance assumed, multiple comparison were performed using LSD method, if homogeneity of equal variance not assumed, multiple comparison were performed using Dunnett T3 method. P<0.05 was considered as statistically significant.Part.2 Killing activity of CIK cells againes cancer stem cells in vitro and vivo1. Construction of the Plasmid of pLV-PNanog-GFP-T2A-Luc2.The purification and extraction of plasmid.3. 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. Then we use the produced lentivirus to infect HCC cells To construct stable HCC cell lines carrying PNanog-GFP-T2A-Luc.4. Flow cytometric analysis were performed to detect the proportion of Nanog-GFP positive cells. IVIS imaging system was employed to examine the correlation between the number of tumor cells and bioluminescent light signals.5. The GFP positive cells and GFP negative cells were sorted by Flow cytometer.6. RNA isolation and quantitative real-time PCR was employed to detect the expression of sternness genes.7. The stemness of the GFP positive cells was detected by the tumor sphere formation assay, colony formation assay, and Transwell, Boyden assay.8. CCK8 assasy was performed to detect the killing activity of CIK cells against GFP positive cells and GFP negative cells.9. The interaction between CIK cells and tumor cells/tumor sphere was observed by Time-Lapse analysis.10. In vivo activity of CIK cells against SMMC7721 cells transduced with LV-Nanog-GFP-Luc in NOD/SCID mice.11. The mice were treated with CIK cells in different proportion according to different groups, Tumor growth was determined by caliper measurement or in vivo bioluminescence (BLI)and fluorescence imaging.12. The presence of CIK cells in the tumor tissue was identified by IHC of CD5 and CD56.13. BrdU, Ki67,GFP expression were examined by IHC.14. Statistical analysis was performed using a SPSS 20.0 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. One-way ANOVA was used for comparisons of more than two groups. If homogeneity of equal variance assumed, multiple comparison were performed using LSD method, if homogeneity of equal variance not assumed, multiple comparison were performed using Dunnett T3 method. The comparison of tumor growth curve was analysed using multi-factor analysis of variance (two-way ANOVA). P<0.05 was considered as statistically significant.Part.3 The mechanism of CIK cells against cancer stem cells.1. NKG2D blocking assayTo investigate the mechanisms underlying the tumoricidal effect of CIK cells, CIK cells were firstly pre-incubated for 30 minutes with 20 μg/mL of inhibitory anti-NKG2D neutralizing antibodybefore co-culture of HCC cells with CIK cells for 24 hours.2. TUNEL stainingAfter HCC cells were treated with CIK cells for 24 hours, HCC cells were washed three of four times with PBS to fully remove suspended CIK cells, dried at room temperature, fixed with 4% formalin for 15 minutes, and then underwent TUNEL staining by using the TUNEL Apoptosis Detection Kitaccording to the manufacturer’s instruction.3. Cytokine detection by ELISAAfter HCC cells were treated with CIK cells for 24 hours, cell culture supernatants were collected by centrifuging at 2000xg for 10 min at 4℃ and stored at-80℃ before use. The cytokine levels in the collected supernatants weredetectedby ELISA kit (for IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-y) according to the manufacturer’s instructions.4. Statistical analysisStatistical analysis was performed using a SPSS 20.0 software package. Data were presented as mean+SD (X±S). Two-tailed Student’S t test was used for comparisons of 2 independent groups. One-way ANOVA was used for comparisons of more than two groups. If homogeneity of equal variance assumed, multiple comparison were performed using LSD method, if homogeneity of equal variance not assumed, multiple comparison were performed using Dunnett T3 method. P<0.05 was considered as statistically significant.ResultsPart.1 CIK cells preparation and the Killing activity of CIK cells against HCC cells in vitro1. 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 number of immune cells increased by more than 50 folds for 14 days. Most CIK cells were CD3+/CD56+ cells. When we examined the phenotypes of the cultured cell population with fluorescence-activated cell sorting analyses, the proportion of CD3+/CD56+ cells increased from 4.7±2.5% at day 7 to 55.2±13.2% at day 21; and the proportion of CD8+ cells increased from 69.1±5.1% at day7 to 92.1±2.9% at day 21; the proportion of CD4+ cells decreased from 25.6±4.7% at day 7 to 2.7±1.0% at day 21. The median membrane expression of the NKG2D receptor, main responsible for tumor recognition, on expanded CIK cells was 73.1±3.0% at 7 days, increased to 92.1±2.9% at 21 days.2. Killing activity of CIK cells against HCC cell linesThe cytotoxic effect was obviously increased when the HCC cells were treated with higher effector cells/target cells ratio (E/T). The specific tumor killing rate increased from 58.3±1.5%(SMMC7721) and 67.3±2.5%(Huh7) at 1:5 ratio to 86±2.0%(SMMC7721) and 94±2.0%(Huh7) at 1:50 ratio, respetivety. Colony formation assay showed that the colony formation ability of HCC cells decreased significantly when treated with increasing effector/target ratio CIK cells (F=59.644,P<0.001; F=65.519, P<0.001)3. The tumor sphere formation ability and migration/invasion ability of HCC cells decreased after treated with CIK cells.Transwell and Boyden assay showed that the migration and invasion ability of HCC cells decreased after treated with CIK cells. Compared with control group, the migration ability deceased by 83.5%(SMMC7721) and 58.1% (Huh7) respectively, and the invasion ability decreased by 58.7%(SMMC7721) and 82%(Huh7) with statistically significant difference (t=6.945, P=0.001; t=6.743, P<0.001; t=13.663, P<0.001; t=8.265, P=0.001). Tumor sphere formation assay showed that the number of sphere significantly decreased by 36.8%,87.2%(SMMC7721) and 40.9%,89.2% (Huh7) in the ratio of 1:10 and 1:30 respetively after treated with CIK cells (F=81.214, P<0.001; F=70.813, P<0.001)Part.2 Killing activity of CIK cells against cancer stem cells in vitro and vivo1. Stable cell lines containing PNanog-GFP-T2A-Luc construction and the identification of sternnessTo identify putative CSCs, we transduced HCC cells with a lentiviral vector encoding the GFP controled by the human Nanog promoter. The average GFP expression was 5.3% in SMMC7721 cell line and 3.9% in Huh7 cell line. And the activity of firefly luciferase (Luc) was detected by the Xenogen IVIS Lumina Ⅱ Imaging System. Nanog positive cells were sorted based on the expression of GFP. Compared to Nanog-GFP (-) cells, Nanog-GFP (+) cells highly expressed stemness genes including Nanog, Oct4, Sox2. Futhermore, we found that the tumor sphere fomation ability of Nanog-GFP (+) cells was significantly higher than Nanog-GFP (-) cells (t=13.714, P<0.001). Transwell and Boyden assay showed that the GFP positive cells had increased migrated cells (t=14.442, P=0.004; t=6.189, P=0.003). The colony formation capacity of Nanog-GFP (+) cells was significantly higher than Nanog-GFP (-) cells (t=10.575; P=0.00). In summary, we found that Nanog-GFP (+) cells have the characteristics of cancer stem cells. Using Nanog promoter to drive the expression of GFP and Luc gene to label cancer stem cells is feasible.2. The killing activity of CIK cells against GFP positive cells and tumor sphereWe performed CCK8 assay to assess the killing ability of CIK cells against Nanog-GFP (+) and Nanog-GFP (-) cells. The Result showed that CIK cells could both kill Nanog-GFP (+) and Nanog-GFP(-) HCC cells. The cytotoxic effect was obviously increased with the increase in effector cells/target cells ratio (E/T). The inhibition rate between the GFP+ cells and GFP-cell had no significant difference (F=0.201, P=0.66; F=0.465, P=0.505). In order to visualize the killing activity of CIK cells against HCC cells and cancer stem cells, we applied Time-lapse to observation the interaction of CIK cells with HCC cells, GFP+HCC cells and tumor sphere. The results showed CIK cells can directly kill HCC cells, Nanog-GFP (+) CSCs and tumor sphere. In summary, we found that CIK cells were capable to eliminate the HCC cells,tumor sphere and GFP+HCC cells which represent a subtraction of cancer stem cells.3. In vivo killing activity of CIK cells against SMMC7721 cells transduced with LV-Nanog-GFP-Luc in NOD/SCID miceWe evaluated the activity of CIK cells in vivo against tumor xenografts in NOD/SCID mice. NOD/SCID mice (n=18) were subcutaneously implanted with 1x106 SMMC7721 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. Finally, We found a significant delay in the tumor growth in the CIK(1:30) group compared to untreated controls and the CIK(1:10) group. The tumor volume at different groups was statistically significant difference(P<0.05), and the tumor weight at different groups was statistically significant difference (F=4.615, P=0.027). In order to evaluate whether CIK cells has the ability to kill Nanog-GFP-Luc (+) CSCs in vivoduring the treatment, Nanog-GFP-Luc (+) CSCs were monitored using I VIS. 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=17.206, P<0.001). Moreover, immunohistochemistry of BrdU, Ki-67 showed decreased expression in the CIK treated groups (F=1065.06, P<0.001; F=387.165, P<0.001). and the expression of sternness marker GFPat different groups was statistically significant difference (F=39.857, P=0.004). And we could confirm the presence of CIK cells infiltrating in the tumor tissues by IHC of CD5, CD56.Part.3 The mechanism of CIK cells against cancer stem cells.1. CIK cells kill HCC cells and CSCs via the NKG2D receptor recognitionCCK8 assay showed that, the killing activity of CIK cells against GFP positive HCC cells decreased significantly after blocking of the NKG2D with anti-NKG2D neutralizing antibody (F=24.953,P<0.001; F=46.369, P<0.001). Futhermore, we found that the colony formation ability and tumor sphere formation ability of the HCC cells restored after blocking of the NKG2D (F=22.282, P=0.002; F=31.903, P=0.001); (F=16.884, P=0.003; F=17.889, P=0.003)2. CIK cells could induce the apoptosis of HCC cellsTUNEL assay showed that CIK cells can induce apoptosis of HCC cells, including the GFP positive cells.3. Detection of cytokines released by CIK cells by ELISAAfter HCC cells were treated with CIK cells for 24 hours, cell culture supernatants were collected and the cytokine levels were detected by ELISA. We found a high concentration of IFN-y were released by CIK cells. The concentration was 295.3±22.7Pg/ml and 88.4±4.2Pg/ml respectively.Conclusions1. We successfully established CIK cells culture system. CD3/CD56 positive cells could be expanded rapidly during week 2-3. And the expression of NKG2D increased during the culture.2. CIK cells can inhibit NPC cells proliferation in vitro and in vivo.3. The tumor sphere formation ability of HCC cells decreased after treated with CIK cells, indicating that CIK cells can kill cancer stem cells in vitro.4. 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.5. CIK cells could eliminating the GFP positive cells in vitro.6. CIK cells could inhibited the tumor growth and eliminated the cancer stem cells in vivo.7. CIK cells kill HCC cells and CSCs via the NKG2D receptor recognition and the induction of apoptosis.