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Objective To observe the clinical efficacy of transcatheter arterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) and hepatic artery infusion of autologous cytokineinduced killer (CIK) cells in treating clinical stage I hepatocellular carcinoma (HCC).Methods A total of 80 patients with confirmed HCC,who were treated with comprehensive interventional therapy during the period from January 2009 to May 2010,were enrolled in this follow-up study.The patients were divided into the study group (n=38),receiving TACE,RFA and autologous CIK cells therapy,and the control group (n=42),receiving TACE and RFA only.The quality of life (QOL),changes in immune function indexes,progression free survival (PFS) and survival rate were calculated,and the results were compared between the two groups.Results (1) QOL score:after the treatment the QOL score of the study group was significantly higher than that of the control group (P＜0.05).(2) Immune function:the post-treatment immune function values were different from the pre-treatment ones in both groups,the differences were statistically significant (P＜0.05);and the differences between the two groups were also statistically significant (P＜0.05),with the changes of the study group being more obvious.(3) PFS and survival rate:the median PFS of the study group and the control group was 48.0 and 40.1 months respectively,while the one-,2-,3-,5-year survival rates of the study group and the control group were 100％,89.5％,71.1％,55.3％ and 95.2％,88.1％,64.3％,28.6％ respectively.Both the median PFS and survival rate in the study group were higher than those in the control group.Conclusion In treating clinical stage I HCC,TACE combined with RFA and hepatic artery infusion of autologous CIK ceils can improve QOL of patients,strengthen patient's immune function,prolong the median PFS,and increase the overall survival rate.
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Objective To investigate the antitumor effects and the possible mechanisms of dendrit-ic cells co-cultured with cytokine-induced killer cells(DC-CIK)in combination with sorafenib on two lung adenocarcinoma cell lines,A549 cells(harboring KRAS gene mutation)and PC-9 cells(harboring EGFR gene mutation). Methods DC and CIK cells were routinely generated in vitro by stimulating PBMCs isola-ted from lung cancer patients with different cytokines and then co-cultured after a week of culturing. Flow cy-tometry analysis(FCM)was used to analyze the phenotype of DC-CIK cells after 7 days of co-culturing. The 50% inhibitory concentration(IC50 )of sorafenib against tumor cells was detected by MTT assay. The tumor cells were treated with DC-CIK cells alone or in combination with sorafenib. The proliferation of tumor cells was tested by CCK-8 kit and dynamically monitored by real-time cellular analysis(RTCA). Annexin-V/ PI staining was used to examine the apoptosis rates in each group. Real-time fluorescent quantitative PCR and FCM were respectively performed to detect the expression of natural killer group 2 member D ligands (NKG2DLs)at mRNA and protein levels after the treatment with sorafenib for 24 h. Results There was no significant difference between the IC50 of sorafenib against A549 and PC-9 cells after a 24-hour exposure(P﹥0. 05). Compared with the DC-CIK biotherapy,treating the tumor cells with DC-CIK cells in combination with sorafenib significantly inhibited the cell proliferation and increased the total apoptosis rates of tumor cells(P﹤0. 05). Moreover,the inhibition rates to tumor cell proliferation were enhanced along with the in-crease of effect-to-target ratio(E/ T). Compared with the single-factor treatment groups,the normalized cell index(NCI)in the combined treatment group was significantly decreased. Blocking NKG2D could abate the inhibitory effect of DC-CIK cells on tumor cell proliferation(P﹤0. 05). The expression of NKG2DLs(inclu-ding ULBP1,UBLP2 and ULBP3)on tumor cells at mRNA and protein levels were increased to different ex-tent after treating with 5 μmol/ L of sorafenib for 24 h. Conclusion There was no significant different be-tween the inhibitory effects of sorafenib on the proliferation of lung adenocarcinoma cancer cells harboring KRAS or EGFR gene mutation. The antitumor effects of DC-CIK cells combined with sorafenib on lung ade-nocarcinoma cells might be induced by regulating the NKG2D-NKG2DLs pathway and enhancing apoptosis. Moreover,the antitumor effects of the combined treatment were better than those of single-factor treatments.
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Objective To observe the effect of cytokine-induced killer (CIK) cells on the apoptosis of human leukemia cells,and explore the role of miR-155 in this process.Methods The cytotoxicity of CIK cells against a variety of leukemic cell lines (NALM-6,Jurkat) was investigated by MTT technique,miR-155 was determined by real time quantitative PCR,and the apoptosis was detected by flow cytometry in NALM-6 and Jurkat cells induced by CIK cells.Psi CHECK2-CEBP/β 3'-UTR containing the binding site of miR-155 was constructed,and then it was transfected into NALM-6 and Jurkat cells.Luciferase activity of CEBP/β (CCAAT/enhancer binding protein beta) was determined with the assistance of dual luciferase report system.Results CIK cells possessed strong cytotoxicity against NALM-6 and Jurkat cells,which was time-dependent and dose-dependent (P ＜ 0.05).CIK cells could increase the expression of miR-155 in NALM-6 cells by (2.87±0.19) fold (t =2.787,P ＜ 0.05),and in Jurkat cells by (1.98±0.25) fold (t =3.513,P ＜ 0.05).Moreover,miR-155 mimics could promote the apoptosis of NALM-6 and Jurkat cells induced by CIK cells (t =4.239,P ＜ 0.05;t =3.565,P ＜ 0.05).However,miR-155 inhibitor might block this process (t =3.772,P ＜ 0.05;t =4.017,P ＜ 0.05).MiR-155 targeted at the site of CEBP/β3'-UTR,and CIK cells could decrease the luciferase activity of NALM-6 cells by (42.89±2.07) ％ (t =3.578,P ＜ 0.05),meanwhile,in Jurkat cells by (37.02±1.95) ％ (t =4.393,P ＜ 0.05).Conclusion CIK cells could enhance human leukemia NALM-6 and Jurkat cells apoptosis by upregulating miR-155,which may provide a new database to elucidate leukemia cell therapy using CIK cells.
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Cytokine-induced killer cells (CIK cells) are a group of CD3+ CD56+ T cell-based heterogeneous cells generated in vitro,with T-cell receptor (TCR) specificity of CD8+ T cells and anti-tumor activity of non-restrictive major histocompatibility complex (MHC).Recent studies indicated that vaccination with CIK cells induced strong anti-tumor activity in the treatment of malignant lymphoma.These researches show that CIK cell-based immunotherapy has the important values for patients with malignant lymphoma.
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Objective To study proliferation,secreted cytokines,immune phenotypes and cytotoxicity on Raji cells by cytokine induced killer (CIK) cells co-cultured with dendritic cells (DC).Methods The mononuclear cells from peripheral blood of healthy individuals were extracted,then cultured the cells under 5 ％ CO2 at 37 ℃ for 2 hours.DC were induced from suspended cells,and CIK cells were from adherent cells.After 9 days of nurturing,two types of cells were mixed.CIK cells were cultured alone as the control.The cytotoxic activity of CIK and DC-CIK cells were detected by MTT assay.The morphologies,proliferation,secreted cytokines,and immune phenotypes of the two cells in day 0,3,6,9,12,15 in culture were monitored.Results In day 12 in culture,comparing with CIK cells,DC-CIK cells significantly enhanced the proliferation rate [(42.44±2.68) fold vs (30.01±2.05) fold] (t =11.64,P ＜ 0.05) and had increased IL-2,IFN-γ,IL-12 and TNF-α secretion [(124.34±12.57) ng/L vs (56.32±6.58) ng/L,(496.60±95.32) ng/L vs (247.80± 69.45) ng/L,(84.92±6.07) ng/L vs (24.18±3.31) ng/L,(380.6±45.95) ng/L vs (196.61±24.19) ng/L] (t =15.16,P ＜ 0.05; t =6.67,P ＜ 0.05; t =27.78,P ＜ 0.05; t =11.20,P ＜ 0.05),and there were more CD3+ CD8+ cells and CD3+ CD56+ cells in the co-culture [(71.79±1.73) ％ vs (60.37±3.24) ％,(48.54±3.30) ％ vs (33.07±2.22) ％](t =9.83,P ＜ 0.05; t =12.30,P ＜ 0.05),and DC-CIK cells had a significandy increased cytotoxicity on Raji cells in vitro at the same ratio of effector cells to target cells.Conclusion CIK cells have higher proliferation rate and cytotoxicity against Raji cells when co-cultured with DC.
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Objective To investigate the feasibility of using cytokine-induced killer (CIK) cells as targeted vehicle to deliver hIL-21 gene for hePatocellular carcinoma (HCC) treatment. Methods We constructed pDC759-h!L-21and pDC759- CopGFP for cotransfection of HEK293 cells with PAd5 and PAd5F35 which can exPress hIL-21 and CopGFP Proteins for viral Packaging. We isolated CIK cells and Plotted the growth curve. Then Ad5/AAV-CcpGFP and Ad5F35/AA'V-CopGFP of different MOIs (0,1,5,10,50,100, and 1 000) were used to infect CIK cells; Ad5/AAV-h!L-21 and Ad5F35/AAV-h!L-21 of different MOIs (0,1,5,10,20, and 50) were used to infect SMMC-7721 cells and the hIL-21 Protein levels were examined by ELISA assay 48 h after infection. Ad5F35/AAV- hIL-21 of different MOIs (0, 1, 5, 10, 50, and 100) was used to infect CIK cells and hIL-21 Protein levelswere also examined 48 h after infection. SMMC-7721 cells were subcutaneously injected to nude mice for tumor forming, and then the tumor-bearing mice were treated with CIK cells carrying hIL-21 and the theraPeutic effects were observed. Results Plasmids for viral Packaging were constructed. After cotransfection of HEK293 cells, Ad5 and Ad5F35 which can exPress hIL-21 and CopGFP Proteins were successfully Packaged. Ad5 and Ad5F35 which can exPress CopGFP were used in this study. It was found that Ad5F35 was superior to Ad5 in CIK infection, with the suitable MOI being 50. Ad5/AAV-h!L-21 andAd5F35/AAV-h!L-21 had similar effect on IL-21 expression in SMMC-7721 cells. Results of animal study showed that mice in group CIK/hIL-21 had a better curative effect. Conclusion hIL-21 and CIK cells have synergistic anti-tumor effect in nude mice bearing HCC.
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Objective To investigate the feasibility of using cytokine-induced killer (CIK) cells as targeted vehicle to deliver hIL-21 gene for hePatocellular carcinoma (HCC) treatment. Methods We constructed pDC759-h!L-21and pDC759- CopGFP for cotransfection of HEK293 cells with PAd5 and PAd5F35 which can exPress hIL-21 and CopGFP Proteins for viral Packaging. We isolated CIK cells and Plotted the growth curve. Then Ad5/AAV-CcpGFP and Ad5F35/AA'V-CopGFP of different MOIs (0,1,5,10,50,100, and 1 000) were used to infect CIK cells; Ad5/AAV-h!L-21 and Ad5F35/AAV-h!L-21 of different MOIs (0,1,5,10,20, and 50) were used to infect SMMC-7721 cells and the hIL-21 Protein levels were examined by ELISA assay 48 h after infection. Ad5F35/AAV- hIL-21 of different MOIs (0, 1, 5, 10, 50, and 100) was used to infect CIK cells and hIL-21 Protein levelswere also examined 48 h after infection. SMMC-7721 cells were subcutaneously injected to nude mice for tumor forming, and then the tumor-bearing mice were treated with CIK cells carrying hIL-21 and the theraPeutic effects were observed. Results Plasmids for viral Packaging were constructed. After cotransfection of HEK293 cells, Ad5 and Ad5F35 which can exPress hIL-21 and CopGFP Proteins were successfully Packaged. Ad5 and Ad5F35 which can exPress CopGFP were used in this study. It was found that Ad5F35 was superior to Ad5 in CIK infection, with the suitable MOI being 50. Ad5/AAV-h!L-21 andAd5F35/AAV-h!L-21 had similar effect on IL-21 expression in SMMC-7721 cells. Results of animal study showed that mice in group CIK/hIL-21 had a better curative effect. Conclusion hIL-21 and CIK cells have synergistic anti-tumor effect in nude mice bearing HCC.
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Objective: To investigate the in vitro and in vivo inhibitory effects of DC (dendritic cell)-CIK (cytokine-induced killer cell) co-cultured cells combined with sorafenib against hepatocellular carcinoma cell line BEL-7402. Methods: DC and CIK cells were generated in vitro by stimulating human peripheral blood mononuclear cells with different cytokines, and then they were co-cultured. The cytotoxicity of DC-CIK co-cultured cells (DC-CIK) combined with sorafenib against BEL-7402 cells was determined by CCK8 kit. The apoptosis of BEL-7402 cells was measured by Annexin V-FITC Kit. BEL-7402-implanted tumor model was established by subcutaneous injection in nude mouse. Tumor-bearing mice were divided into normal saline control group, sorafenib group, DC-CIK group and DC-CIK+sorafenib group. The inhibitory effects were observed in different groups. Results: The cytotoxicity rate of BEL-7402 cells in DC-CIK+sorafenib group was significantly higher than those in the other two groups, with cytotoxicity rate in DC-CIK+sorafenib group being (75.24±1.91)%, which was 1.8 times that in DC-CIK group and 2.1 times that in sorafenib group (P<0.01). The apoptosis rate of BEL-7402 cells in DC-CIK+sorafenib group was significantly higher than those in the sorafenib and DC-CIK groups, with the apoptosis rate in DC-CIK+sorafenib group being (78.32±2.54)% (P<0.05). The volume of tumor in the combination group was significantly smaller than those in the other groups (P<0.05). In vivo results showed that DC-CIK+sorafenib treatment significantly inhibited the growth of BEL-7402-implanted tumors, and the inhibitory rate was (83.37 ±0.16)%, which was significantly higher than those of the other groups (P<0.01). Conclusion:DC-CIK co-cultured cells combined with sorafenib can inhibit the growth of hepatocellular carcinoma cell line BEL-7402 in vitro and in vivo. Molecular targeting therapy combined with immunotherapy may be a new way for the comprehensive treatment of hepatocellular carcinoma.
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[Objective] In order to explore the optimal minor-factor culture system of CIK/NK cells by proliferating CIK/NK cells using single-factor,bi-factor,and tri-factor combinations of IL-2,IL-7,and IL-12.[Methods] Ficoll-Hypaque method was used to separate cord blood mononuclear cells and divide them into 6 groups.Add single-factor,bi-factor,and tri-factor combinations (IL-7;IL-12;IL-7 + IL-12;IL-2 + IL-7;IL-2 + IL-12;IL-2 + IL-7 + IL-12) of IL-2 (80 ng/mL),IL-7 (40 ng/mL),and IL-12 (40 ng/mL) and culture them in total 21 days then harvest the cells.To collect cell suspensions of each group in culture outset and day 21 and to count the proportion of CD3+ CD56+ CIK cells and CD3- CD56+ NK cells with flow cytometry.[Results] The proportion of CIK cells in single-factor culture system of IL-12 was the highest in all groups (30.23 ± 1.18%).The proportion of CIK cells in bi-factor culture system of IL-2 + IL-12 can raised to 18.58 ± 0.68%.The proportion of CIK cells of the two groups above can reach the level of that using traditional multi-factor culture methods.NK cell proportion in IL-12 was 30.23 ± 1.18%,NK cell proportion in IL-2 + IL-7 can also reach to 29.52 ± 0.89%.[Conclusions] It is adoptable to proliferate CIK/NK cells using minor-factor culture system of IL-12,IL-2 + IL-12,or IL-2 + IL-7.
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Objective: To observe the efficacy and safety of chemotherapy combined with cytokine-induced killer (CIK) cells in the treatment of metastatic melanoma (MM). Methods: Fifty three chemotherapy-naive MM patients were administered fotemustine at 100 mg/m2 on d 1-5; dacarbazine were given at 400 mg/d on d 2-6; CIK were infused on d 7, d 14, d 16. Twenty-eight days were regarded as one cycle. The clinical efficay was evaluated every 2 cycles. Results: Thirty-four out of 53 patients were eligible to be evaluated. The overall response rate (ORR) was 23.5% including 1 case with complete response (CR) (2.9%) and 7 cases with partial response (PR) (20.6%). Fourteen cases had stable disease (44%). The clinical benefit response was 67.5%. Median progression free survival (PFS) was 8 months. Median overall survival (OS) was 11 months. The patients with normal lactate dehydrogenase (LDH) had longer OS; those with stable disease had longer PFS and OS. Adverse reaction included grade III/IV thrombopenia (41%), decrease in the number of WBC (23.5%), and hyperreactivity (2 cases). No treatment-related death occurred. Conclusion: Chemotherapy followed by the infusion of CIK was tolerable. The response rate was higher than common chemotherapy. It could prolong the survival time of tumor patients with elevated LDH and non progression disease. Phase III study is needed to confirm the results.
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Objective To investigate the biological activity of cytokine-induced killer(CIK)cells in vitro.Methods Lymphocytes isolated from peripheral blood in leukemic children were induced with interferon-?(IFN-?),anti-CD3 monoclonal antibody(CD3McAb)and interleukin-2(IL-2)and co-cultured with dendritic cells(DC)to generate DC-CIK cells.The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry,respectively.Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the methyl thiazolyl tetrazolium (MTT) assay.Interleukin-12(IL-12),tumor necrosis factor-?(TNF-?)levels released by DC-CIK cells were quantified by enzyme-linked immunosorbent assays.Results Induced DC-CIK cells were regular,round and transparent with variable cell volume and cellular aggregation.At the 0th-4th day,its amplification was very slow,and it increased quickly at the 5th-8th day,it reached its peak amplification at the 9th-10th day,at approximately 100-fold.The main effector cells in this population were CD3+CD8+ cells and CD3+CD56+ cells.DC-CIK cells were cytotoxic to B95 cells,K562 cells and HL-60 cells,with the highest cytotoxicity towards B95 cells.The expression levels of IL-12 and TNF-? in supernatant were very high.Conclusions DC-CIK cells induced with cytokines displayed powerful amplification and strongly killing activities in vitro.It suggested that DC-CIK cells induced with cytokines may play killing activities through Th1 pathway in vitro,as a result of high secretion of Th1 cytokines,such as IL-12 and TNF-?.
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Objective To investigate the cytotoxicity and mechanism of killing tumor of cytokine-induced killer (CIK) cells in vitro.Methods Mononuclear cells were acquired freshly from bone marrow of children with leukemia,and the cells obstained were induced into dendritic cells by adding granulocyte-macrophage colony-stimulating,IL-4,TNF-? and other cytokines.Lymphocytes cells were isolated freshly from peripheral blood of children with leukemia by Ficoll-Hypaque density centrifugation,and the cells obstained were induced by IFN-?,IL-2 and CD3McAb.The DC cells and CIK cells were co-cultured for 10-25 days,then DC-CIK cells were obtained.Phenotypes of DC-CIK were analyzed by flow cytomery.The cytotoxicity of DC-CIK against a variety of leukemic cell lines was investigated by MTT technique.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 and T -bet in the levels of mRNA and protein were mea-sured by using RT-PCR and Western Blot technique,respectively.Results In the first 0-6 days,DC-CIK induced slowly,the proliferation of DC-CIK got 100-fold at the 13th day,cells were rapidly proliferating in the first 13-21 days.The maximum proliferation of DC-CIK reached at the 22nd day.The phenotypes of CD3,CD11a,CD54,HLA-DR were expressed highly; CD3/CD56,CD25,CD28,CD69,FasL were expressed moderately on DC-CIK.The expression of CD16 was not increased.DC-CIK possessed the cytotoxicity against tumor cells of B95,Jhhan and M07e.The effect was stronger to B95,there was no significant difference when the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached about 50% and 60%,respectively,against tumor cells of B95.However,it was not obvious to Jhhan and M07e.When the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached to 27.21%,25.13%,33.05%,29.72%,respectively,against tumor cells of Jhhan and M07e.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 in the level of mRNA was up-regulated(t=3.425,4.523 Pa
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Objective To investigate the inhibitory effects of CIK cell on apoptosis of lung cancer cell A549 in vivo. Methods CIK cells were induced by culturing PBMC with regular method and cell apoptosis was detected.Results Electron microscopic observations showed that CIK cells could induce the lung cancer cells A549 to apoptosis. Flow cytometry(FCM)demonstrated that apoptosis cells of lung cancer cells A549 were increased in CIK group as compared with the control group. Conclution CIK cells can induce the apoptosis of lung cancer cells A549.
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Cytokine-induced killer(CIK) cells are heterogeneous lymphocytes generated by incubation of human peripheral blood mononuclear cells with various cytokines in vitro,characterized by both the T lymphocyte's powerful cytotoxic activity and the natural killer(NK) cell's non-major histocompatibility-restricted antitumor activity in vitro and in vivo.CIK cells have their peculiar superiority over standard lymphokine activated killer(LAK) cells,tumor-infiltration lymphocytes(TIL) and anti-CD3 antibody induced activated killer(CD3AK) cells in application to the cancer adoptive immunotherapy and have hence received wide attention from researchers.This article updates recent researches on CIK cells,their interaction with dendritic cells,bispecific antibodies and oncolytic viruses,as well as their use in gene transfer.
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Objective:To investigate the inhibitory effect of cytokine-induced killer cells (CIK) against implanted gastric cancer cells. Methods:Gastric cancer SGC-7901 cells were subcutaneously injected into the inguina of nude mice to establish gastric cancer model. The tumor bearing mice were randomly divided into CIK group and fibroblasts group,in which mice were subcutaneously injected with fluorescence dye SP-DiI labeled CIK and fibroblasts HFL-I cells,respectively. Distribution of CIK and HFL-I cells in different tissues of gastric cancer bearing mice were observed. Meanwhile,tumor volume was measured after different treatments and tumor inhibitory rate was calculated. Tumor necrosis areas in different groups were observed. Results:SP-DiI labeled CIK was mainly located in the gastric cancer tissues 10 d after injection,and was hardly detected at the injection sites,liver,spleen and lung tissues (P
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Objective To evaluate the clinical effect of hepatocellular carcinoma treatment with a combination therapy of transcather arterial super liquefied lipiodol embolization and cytokine-induced killer cell(CIK) infusion.Methods There were 3 groups in this study,group 1:38 cases of HCC patients treated with a combination therapy of transcather arterial super liquified lipiodol embolization and CIK infusion;group 2:80 cases of HCC patients treated with a combination therapy of transcather arterial super liquefied lipiodol embolization and percutaneous intratumoral ethanol injection;group 3:134 cases of HCC patient treated with transcather arterial super liquefied embolization.Finally,the outcomes of the 3 groups were compared.Results The short term effective rates of group 1,2 and 3 were 76.1%,41.3% and 14.9% respectively,simultaneously with significant difference of changes concerning AFP value among the three groups especially in group 1 the AFP decrease to normal level while those of the other two groups still remain in higher levels.Conclusions The living quality and survival rate of HCC patients could be improved by a combination therapy of transcather arterial super liquefied lipiodal embolization and CIK infusion.(J Intervent Radiol,2007,16:235-239)