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AIM: To explore the effects of dendritic cells (DC) and cytokine-induced killer cells (CIK) carrying melanoma-associated antigen gene A3 (MAGE-A3) on endometrial cancer tumor stem cells and malignant progression. METHODS: Human peripheral blood was collected to separate mono-nuclear cells, and DC and CIK cells were induced by cytokines, respectively. DCs were incubated with MAGE-A3 and then co-cultured with CIK, and the phenotypes of DC-CIK and MAGE-A3-DC-CIK were detected by flow cytometry; The CD133
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Objective:To investigate the effect of immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy on apoptosis-related genes and immune function in patients with middle- and advanced-stage non-small cell lung cancer.Methods:A total of 100 patients with middle- and advanced-stage non-small cell lung cancer who received treatment in Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, China from February 2018 to May 2019 were included in this study. They were randomly divided into control and observation groups ( n = 50/group). The two groups were given chemotherapy with pemetrexed and cisplatin. The observation group was given immunotherapy with dendritic cells and cytokine-induced killer cells based on chemotherapy with pemetrexed and cisplatin. Changes in apoptosis-related genes [primary autosomal recessive microcephaly gene (MCPH1), ataxia-telangiectasia mutated (ATM), ataxia telangiectasia mutated and Rad3 related (ATR), transcription factor 21 (TCF21)] and immune function were monitored. Clinical efficacy of immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy with pemetrexed and cisplatin in the treatment middle-and advanced-stage non-small cell lung cancer was assessed. Results:After treatment, expression of MCPH1, ATM, ATR and TCF21 in the observation group was 301.11 ± 41.12, 239.98 ± 30.15, 270.01 ± 36.01, 270.01 ± 34.02, respectively, which was significantly higher than that in the control group [101.32 ± 15.32, 103.00 ± 13.97, 101.12 ± 14.90, 100.20 ± 14.99, t = 32.194, 29.149, 30.644, 32.299, all P < 0.001]. The proportion of the number of Th1-positive cells in the number of CD +4 T cells in the observation group was significantly higher than that in the control group [(29.00 ± 3.41)% vs. (22.61 ± 3.22)%, t = 9.634, P < 0.001]. The proportion of the number of Th17-,Th2 and CD +4CD +25Treg-positive cells in the number of CD +4 T cells in the observation group were (0.89 ± 0.10)%, (12.01 ± 1.36)%, (11.02 ± 1.92)%, respectively, which were significantly lower than those in the control group [(1.70 ± 0.20)%, (17.61 ± 2.20)%, (18.70 ± 2.40%)%, t = 25.614, 15.310, 17.670, all P < 0.001]. Total effective rate in the observation group was significantly higher than that in the control group [52.0% (26/50) vs. 30.0% (15/50), χ2 = 5.002, P < 0.05]. Conclusion:Immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy with pemetrexed and cisplatin can induce apoptosis and regulate immune function. The combined therapy exhibits better clinical efficacy in the treatment of non-small cell lung than chemotherapy alone.
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@#Objective To investigate the influence of programmed cell death protein-1 (PD-1) monoclonal antibody on the anti-lung cancer effect of cytokine-induced killer cells (CIK) which were programmed in vitro. Methods Peripheral blood mononuclear cells from 20 patients (8 males and 12 females with an average age of 56.45±5.89 years ranging from 42 to 65 years) diagnosed with advanced lung cancer from January to May 2019 at the Department of Oncology of Dalian Central Hospital were collected and induced to amplify into CIK cells in vitro. PD-1 monoclonal antibody combined with CIK cell culture group, individual cell culture group and PD-1 monoclonal antibody group were set up to detect the cell killing activity of CIK cells against lung cancer under different effective target ratio conditions, and the ratio of perforin and granzyme positive expression in PD-1 monoclonal antibody combined CIK cell culture group and individual CIK cell culture group was detected by flow cytometry. ELISA method was used to detect the interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) cytokine secretion levels in the two groups. Results The killing effect of CIK cells on A549 lung cancer cells increased with the increase of effective target ratio by CCK8, and PD-1 monoclonal antibody increased the killing effect of CIK cells on A549 lung cancer cells under different effective target ratio, E∶T=5∶1 (28.5%±1.9% vs. 20.3%±1.8%), 10∶1 (40.6%±2.4% vs. 31.7%±2.1%), 20∶1 (57.4%±3.5% vs. 44.7%±3.8%), 40∶1 (74.1%±8.3% vs. 60.8%±5.3%). The killing effect of PD-1 monoclonal antibody combined with CIK cells and CIK cells alone on A549 lung cancer cells was statistically different (P<0.05). The killing effect of cells in both groups on lung cancer A549 cells was stronger than that of the PD-1 monoclonal antibody group (P<0.01). The results of flow cytometry showed that PD-1 monoclonal antibody increased the positive ratio of perforin and granzyme release in CIK cells, and the positive ratios of perforin release (46.7%±3.5%% vs. 35.1%±2.2%) and granzyme release (34.6%±3.8% vs. 25.7%±3.3%) in PD-1 monoclonal antibody combination with CIK cells group and CIK cells group were statistically different (P<0.05). Similarly, the secretion levels of IL-2, TNF-α, and IFN-γ cytokines were also increased in the PD-1 monoclonal antibody combined with CIK cells group compared with the CIK group (5 409.0±168.8 pg/mL vs. 4 300.0±132.3 pg/mL, 252.7±16.7 pg/mL vs. 172.5±8.6 pg/mL, 327.2±23.5 pg/mL vs. 209.7±16.0 pg/mL, P<0.05). Conclusion PD-1 monoclonal antibody can promote the release of tumoricidal substances in CIK cells and improve the killing effect of CIK cells on lung cancer A549 cells. It is speculated that the infusion of PD-1 monoclonal antibody before CIK cell adoption in lung cancer patients may be more beneficial to the treatment of disease. PD-1 monoclonal antibody combined with CIK cell therapy is promising as a new type of lung cancer immunotherapy.
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At present, coronavirus disease 2019 (COVID-19) caused by 2019 novel coronavirus (2019-nCoV) infection has spread rapidly in China and more than 70 countries around the world and thus become a public health event of international concern. In addition to fever and respiratory symptoms, varying degrees of liver injury is also observed after 2019-nCoV infection. This article reviews the clinical features, pathology, pathogenic mechanism, and therapeutic strategies of liver injury associated with COVID-19, hoping to provide a reference for clinical decision-making on the prevention and treatment of COVID-19.
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@# Objective: To evaluate the long-term clinical efficacy and follow-up of dendritic cell (DC) vaccines in combination with cytokine-induced killer cell (CIK) treatment in metastatic renal cell carcinoma. Methods: From January 2011 to December 2013, 29 patients with metastatic renal cell carcinoma (pathologically confirmed as renal clear cell carcinoma) were treated by DC vaccines-CIK at the Department of Hematopoietic Stem Cell Transplantation, the Fifth Medical Center of Chinese PLA General Hospital. The 29 patients included 24 male and 5 female, with a median age of 57(32-81) years old. Mature DC vaccine was obtained by gene transfection technology and CIK cells were obtained by i n v i t r o culture; and DC vaccine-CIK was infused back to patients through lymphatic drainage area and vein by each course. Twelve patients received first line treatment, 6 patients received second line treatment after the disease progression by targeted drug therapy or cytokine therapy, and 11 patients received third-linetreatment or above. The long-term clinical efficacy and overall survival rate were evaluated. Results: The median follow-up time was 5 (1-7) years. Treatment cycle was over 2 (2-23) cycles. One case (3.4%) achieved complete remission, 9 cases (31%) achieved partial responses, 13 cases (44.8%) demonstrated stable disease over 3 months and 6 patients (20.7%) developed progressive disease. The objective response rate was 34.4%,and the disease control rate was 79.2%. Stable disease for more than one year realized in 19 cases (65.5%). The 1-, 3- and 5-year survival rates were 93.1% (27/29), 65.5% (20/29) and 51.7% (15 / 29), respectively. Neither the median progression-free survival (PFS) nor the median survival time was achieved. No adverse reactions above grade 3 were observed during treatment. Conclusion: DC vaccines-CIK therapy for the treatment of metastatic renal cell carcinoma is affirmative; it achieved good disease control and long-term survival with controllable safety, and prolonged the survival time for advanced renal cell carcinoma patients.
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@# Objective: To investigate the function of CIK (cytokine induced killer) cells cultured using ATG-F (anti-human T lymphocyte rabbit immunoglobulin-Fresenius) and IFN- γ, IL-2 system and its feasibility in clinical practice. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and were used to culture CIK cells by different activating antibodies; the total cell count was calculated on Day 7 and 14. The CIK cell composition, cell surface activation and proportion of inhibitory receptor molecular in ATG-F group, CD3 group and TG (Thymoglobulin) group were analyzed by Flow cytometry, and the cytotoxicity of CIK cells against K562 cells were also determined by flow cytometry at day 14 in ATG-F high-dose group, CD3 group and TG group. Results: CIK cells were successfully cultured by ATG-F, IFN-γ, IL-2 system. The proliferation rate of ATGF high-dose group was significantly higher than that in TG group (27.25±1.25 vs 16.60±1.72, P <0.01), but the proportion of CD3+ CD56+ cells showed no statistical difference compare with the CD3 group ( P >0.05). The percentage of CD3-CD56+ NK cells in ATG-F high-dose group was significantly higher than that in TG group and CD3 group [(11.19±2.60）% vs（5.66±1.00）%,（1.42± 0.51）% ， P <0.01], while the proportion of CD4+T cells was significantly lower than that in CD3, TG group [(4.35±1.47）% vs （26.88±5.01）%,（14.52±6.22）%, P <0.01]; the proportion of CD56+CD94+, CD56+CD158a+, CD56+CD158b cells was significantly higher than those in CD3 group (all P <0.01). The ATG-F high does group showed significantly higher cytotoxicity against K562 cells than that of CD3 group at the target/effect ratio of 1∶10. Conclusion: CIK cells cultured by ATG-F culture system has higher NK cell proportion than other ordinary culture system, and its activated receptor has more stronger cytotoxicity against K562 cells.
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PURPOSE: The treatment of triple-negative breast cancer (TNBC) remains challenging, due to the absence of estrogen, progesterone, and human epidermal growth factor receptors. This study was designed to evaluate the efficiency and safety of cytokine-induced killer (CIK) cell immunotherapy, following regular chemotherapy, for patients with TNBC. METHODS: A total of 340 patients with postmastectomy TNBC, from January 1, 2010 to June 30, 2014, were included in this retrospective study. Seventy-seven patients received CIK cell immunotherapy, following regular chemotherapy (arm 1), and 263 patients received regular chemotherapy alone (arm 2). The primary aim was overall survival (OS) and disease-free survival (DFS), and the treatment responses and adverse events were also evaluated. RESULTS: The 5-year DFS and OS rates in arm 1 were 77.9% and 94.3%, compared with 69.8% and 85.6% in arm 2, respectively (p=0.159 and p=0.035, respectively). This clearly shows that there was no statistical difference in the 5-year DFS between the two groups. Multivariate analyses of arm 1 indicated that a Karnofsky performance score (KPS) ≥90 and stage I/IIA disease were significantly associated with a prolonged DFS period (hazard ratio [HR], 0.25; 95% confidence interval [CI], 0.09–0.74; p=0.012; and HR 0.21; 95% CI, 0.06–0.82; p=0.024, respectively), but a KPS ≥90 and stage I/IIA disease were not independent prognostic factors for OS. Toxicity was mild in patients who received the CIK therapy. CONCLUSION: The data suggested that CIK cell immunotherapy improved the efficiency of regular chemotherapy in patients with TNBC, and the side effects of CIK cell immunotherapy were mild.
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Humans , Arm , Cytokine-Induced Killer Cells , Disease-Free Survival , Drug Therapy , Estrogens , Immunotherapy , Multivariate Analysis , Progesterone , Prognosis , ErbB Receptors , Retrospective Studies , Triple Negative Breast NeoplasmsABSTRACT
The treatment outcomes of relapsed or refractory neuroblastoma have been unsatisfactory till date. We reported two cases of adoptive immunotherapy using cytokine-induced killer (CIK) cells against relapsed or refractory neuroblastoma. CIK cell production was attempted in three patients, out of which two patients exhibited adequate levels of CIK cell production. Two patients completed full term of CIK cell infusions (weekly for 6 weeks and then biweekly for 8 wk) without serious adverse events. The progression-free survivals for the two patients were 1.9 and 4.1 months. Their overall survivals were 16.7 and 28.7 months. Although the efficacy was unclear, CIK cell infusion combined with other treatment strategies may have prolonged overall survival in refractory neuroblastoma patients. Further studies are needed to determine the exact role of CIK cell-based immunotherapy in relapsed or refractory neuroblastoma patients.
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Humans , Cytokine-Induced Killer Cells , Disease-Free Survival , Immunotherapy , Immunotherapy, Adoptive , NeuroblastomaABSTRACT
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 evaluate the clinical efficacy and safety of dendritic cell (DC) combined with cytokine induced killer cell (CIK) in treatment of advanced non-small-cell lung carcinoma (NSCLC).Methods Peripheral blood mononuclear cells (PBMCs) were collected from 39 patients with advanced NSCLC,who were admitted to Affiliated Hospital of Academy of Military Medical Sciences,and cultured in vitro to produce DCs and CIK cells which,after phenotypic characterization by flow cytometry,were then returned to the patients.DCs were given subcutaneously on day 7,9,11 and 13 and CIK cells were given intravenously on day 11 and 13.The clinical efficacy and safety were analyzed before and after DCs-CIK cells treatment.Results Following up displayed that,in 39 patients with advanced NSCLC and eligible for evaluation,the objective response rate (ORR) was 30.8％ including 2 cases of completed response (CR) and 10 cases of partial response (PR),the disease control rate (DCR) was 69.2％.No significant difference existed pre-and post-treatment on the proportion of T cell subsets including CD3+CD4+CD8ˉ,CD3+CD4ˉ CD8+,CD3+CD19ˉ,CD3ˉCD 19+,CD3 CD16+CD56+,CD3+CD16+D56+,CD3+HLA-DRˉ,CD3+HLA-DR+ and CD3+CD28+CD8+ (P＞0.05),while obvious changes were found in Th1,Th2 and CD3+CD4+CD25+ T cells (Treg cells) (P＜0.05).No serious adverse events were observed.Conclusion Combined DCs-CIK cells immunotherapy provides a safe and effective treatment for patients with advanced NSCLC,improves the quality of life and relieves the probability of metastasis and recurrence.
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Objective:To obtain a high specificity and high affinity anti-human PD-L1 monoclonal antibody which can be used for clinical diagnosis and block PD-L1 and PD-1 binding.Methods:BALB/c mice were immunized with recombinant PD-L1 protein.The positive cell clones stably secreting anti-human PD-L1 monoclonal antibody were obtained by classical hybridoma cell fusion technique.The specificity,affinity,subtype and other characteristics of the antibody were identified by ELISA.Immunofluorescence and indirect immunofluorescence were used to detect the tumor cells.Antibody blocking activity was confirmed by tumor killing test.Results:Two cell strains stably secreting monoclonal antibodies against human PD-LI were screened out.Abl and Ab2 had high titer and affinity.The antibody titers were 1:2.56×106 and 1:3×105,and the affinity was 1.5×109 L/mol and 2.5×10s L/mol respectively.There was no cross reaction between these two antibodies and PD-L2.Immunoblotting,indirect immunofluorescence confirmed that the antibody can be used to the diagnosis.Experiment showed that PD-L1 antibodies can increases tumor-killing activity of CIK cells.Conclusion:Two hybridoma cell lines capable of stably secreting highly specific and high affinity anti-human PD-L1 monoclonal antibody are obtained.They can specifically bind to PD-L1 molecules on tumor cells and can be used to the diagnosis of tumor phenotype and prognosis.Antibody blocking function can be applied to combined CIK cell immunotherapy.
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Objective:To investigate the anti-tumor effects of cytokine-induced kill cells(CIK)methods combined with chemotherapeuticdrug Gemcitabine against Cholangiocarcinoma cancer cell lines QBC-939.Methods:Peripheral blood mononuclear cells(PBMC) of healthy people were stimulated by different cytokines,and were induced into CIK cells.CIK cells were cultured for 14 days as effector cells.The phenotype of CIK cells were analyzed by flow cytometer.QBC939 cells were cultured with the CIK cells at different effector-target ratio or various concentrations of Gemcitabine for 24 and 48 hours.The antitumor effects were measured by CCK8 methods.The expression of Bax was detected by using Western blot.Results:The CD3+CD4+,CD3+CD8+,CD3+HLA-DR+,CD3+CD56+,CD4+CD25+ double positive cel1 was up to 10.89％,60.27％,71.82％,9.03％ and 4.01％ after 14 days' cultivation.The killing effect of CIK and Gemcitabine increased with the increase of effector-target ratio and drug concentration or the extension of time.The killing effect of combination of CIK and Gemcitabine was obviously higher than that of each single factor.The protein levels detected hints of CIK cells and gemcitabine can up-regulated the expression of Bax,and the joint action of both is more significant.Conclusions:CIK cells have strong anti-tumor effect against QBC939 cells by inducing apoptosis of QBC939 cells,and it can enhance the anti-tumor effects of Gemcitabine against QBC939 cells when CIK and Gemcitabine are combined together.
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Objective To evaluate the clinical efficacy of dendritic cells-cytokine induced killer cells combined with surgical treatment for primary liver cancer.Methods Totally 78 patients with primary liver cancer were randomly divided into experiment group (n =30) and control group (n =48).The patients in experiment group received hcpatectomy combined with dendritic cells-cytokine induced killer cell treatment while those in control group were given hepatectomy treatment.The median time to recurrence,progression-free survival,survival and quality of life were evaluated.Observed side effects of cell therapy in experiment group.Results Experiment group received a total of dendritic cells-cytokine induced killer cell treatment 78,an average of 2.6 times per person.Fever occured in 8 patients who received dendritic cells-cytokine induced killer cell treatment.After one cycle of immune therapy,the KPS score of 20 cases was improved,8 cases were stable and and 2 case was worsen in the experiment group.The KPS score of 10 cases were improved,32 cases were stable and and 6 cases were worsen in the control group,and the difference is statistically significant (P ＜ 0.05).The progression-free survival rates for 1,2 and 3 years in the experiment group were 73.3％,40.0％,23.3％ and 68.7％,27.0％,14.5％ in the control groups,respectively.The progression-free survival rates in the experiment group were improved compared to the control group and the difference is statistically significant (P ＜ 0.05).The median time of recurrence in the experiment group were (16.9 ± 2.6) months and (13.5 ± 2.9) months in the control group,respectively (P ＜ 0.05).The 1-2-3-years survival rates in the experiment group were 85.0％,50.0％,35.0％ and 85.0％,40.0％,23.3％ in the control group respectively.There was no statistically significant difference between these two groups (P ＞ 0.05).Conclusions Dendritic cellscytokine induced killer cells combined with surgical treatment on primary liver cancer is safe and effective,it can improve quality of life,and delay the recurrence time after surgery.But not improve long-term survival.
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Objective To evaluate cytotoxic effects of cytokine-induced killer cells (CIK cells) transfected with the interleukin-2 (IL-2) gene on malignant melanoma cells.Methods Mouse spleen cells were extracted,lymphocyte cells were separated,and CIK cells were prepared from these lymphocyte cells.PEGF-N1 plasmids containing IL-2 gene (PEGF-NI-IL-2) were transfected into CIK cells.Fluorescence microscopy was used to observe transfection products,and reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to determine the IL-2 mRNA expression.Then,effector cells such as CIK cells and IL-2-transfected CIK cells were separately co-cultured with target cells (B16 melanoma cells) at effector-target ratios of 10∶ 1,20∶1 and 40∶1,then 4-hour lactate dehydrogenase release assay was performed to evaluate cytotoxic effects of the two kinds of CIK cells on B 16 cells.After effector cells were cocultured with target cells at the effector-target ratio of 40∶1 for 48 hours,enzyme-linked immunosorbent assay (ELISA) was conducted to detect levels of IL-2,interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant of the two kinds of CIK cells.Finally,mouse models of melanoma were established,and a total of 28 melanoma-bearing mice were divided into 4 groups to be peritumorally injected with 0.2 ml sodium chloride physiological solution (control group),100 IU IL-2 solution (IL-2 group),CIK cell suspension at a cell density of 1 × 106 cells per milliliter (CIK group) and IL-2-transfected CIK cell suspension at a cell density of 1 × 106 cells per milliliter (IL-2-transfected CIK group) respectively.Tumor morphology,tumor inhibition rate and cell apoptosis rate were used to evaluate tumor growth in the above groups.If data were normally distributed,t-test was used for comparing means between two groups,and analysis of variance and least significant difference (LSD)-t test were used for comparing means among multiple groups.Results Fluorescence microscopy and RT-PCR both showed that IL-2 was successfully transfected into CIK cells.The cytotoxic effect of IL-2-transfected CIK cells on B16 cells was strongest at the effector-target ratio of 40:1.Levels of IL-2,IFN-γ and TNF-α were also significantly higher in the supernatant of IL-2-transfected CIK cells [(1107.26 ± 6.49) pg/ml,(50.01 ± 3.35) pg/ml,(39.86 ± 3.25) pg/ml] than those in that of CIK cells [(51.09 ± 3.85) pg/ml,(32.71 ± 2.43) pg/ml,(30.11 ± 3.08) pg/ml,t =442.60,14.93,6.89,all P ＜ 0.01].Animal experiments showed that the tumor volume obviously increased in the control group (P ＜ 0.05),but significantly decreased in the IL-2 group,CIK group and IL-2-transfected CIK group (all P ＜ 0.001) after intervention compared with those before intervention.Furthermore,the tumor volume in the IL-2-transfected CIK group was significantly less than that in the other three groups (all P ＜ 0.01),but no significant difference was observed between the IL-2 group and CIK group (P ＞ 0.05).In addition,the apoptosis rate was significantly higher in the IL-2 group,CIK group,and IL-2-transfeeted CIK group than that in the control group (all P ＜ 0.01).The apoptosis rate and tumor inhibition rate were significantly higher in the IL-2-transfected CIK group than those in the IL-2 group and CIK group (all P ＜ 0.01),but insignificantly different between the IL-2 group and CIK group (P ＞ 0.05).Conclusion IL-2-transfected CIK cells had stronger killing effects on malignant melanoma.
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Objective@#To explore the inhibitory effect of icotinib combined with cytokine induced killer (CIK) on various human lung adenocarcinoma cell lines in vitro.@*Methods@#The inhibitory effect of icotinib alone or icotinib combined with CIK on HCC827 and A549 cells was detected by cell counting kit-8(CCK-8). The apoptosis was detected by flow cytometry via Annexin V/PI staining. The effect of icotinib on CIK phenotype was detected by flow cytometry.@*Results@#The inhibitory rates of HCC827 cells treated with 1.5, 3, 6, 12 μmol/L icotinib were (5.64±0.05)%, (8.62±0.45)%, (14.57±0.65)% and (18.52±0.91)%, respectively. The inhibitory rates of A549 cells were (1.64±0.48)%, (2.09±0.28)%, (3.69±0.45)%, (4.41±0.58)%, respectively. At the same concentration, the inhibitory rate of HCC827 cells with icotinib treatment was significantly higher than that of A549 cells (P<0.05). When the effector/target ratio was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells co-cultured with CIK were (15.17±2.33)%, (42.59±7.18)%, (62.59±8.95)%, respectively, and the inhibitory rates of A549 were(16.99±2.81)%, (46.31±1.89)%, (58.24±4.23)%, respectively. The inhibitory rate of HCC827 cells co-cultured with CIK was not significantly different from that of A549 cells at the same effector/target ratio (P10∶1=0.299, P20∶1=0.318, P40∶1=0.366). When the effector/target ratio of CIK combined with 6 μmol/L icotinib was 10∶1, 20∶1 or 40∶1, the inhibitory rates of HCC827 cells were (37.07±3.50)%, (76.03±6.55)%, (80.34±10.69)%, respectively, and the inhibitory rates of A549 cells were(25.72±1.41)%, (52.76±3.82)%, (62.26±1.94)%, respectively. The inhibitory rates of 6 μmol/L icotinib combined with CIK were significantly higher than those of icotinib group and CIK group alone at the same effector/target ratio (P<0.05), except for the effector/target ratio at 40︰1 on A549 cells (P=0.089). Moreover, all of the combination index (CI) of combined group were <1 (P<0.05). The apoptotic rates of HCC827 and A549 cells induced by icotinib combined with CIK were significantly higher than those of icotinib group and blank control group (P<0.05), especially the proportion of late apoptotic or necrotic cells.Increasing effector/target ratio of CIK contributed to stronger inhibition(P<0.05). The expressional rate of CIK phenotype with or without icotinib treatment was not significantly different from each other(P>0.05).@*Conclusions@#EGFR mutant lung adenocarcinoma cells are more sensitive to icotinib, while the EGFR mutation status has no effect on the killing effect of CIK cells. icotinib combined with CIK has a synergistic effect on the inhibition of tumor growth, and icotinib has no any impact on the phenotype of CIK cells.
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Objective To describe the phenotype of NK cells in Bama miniature pigs, and establish an efficient activation and culture method for porcine cytokine-induced killer ( CIK) cells in vitro. Methods The porcine peripheral blood mononuclear cells ( PBMCs) were isolated by Percoll gradient centrifugation, and the phenotype of NK cells was test-ed by detecting the CD2 + /CD8 + /CD3 - cell compartment. To establish an efficient activation and culture method for por-cine CIK cells, we optimized the culture conditions to improve the CIK activation efficiency. Results Using the optimized induction culture conditions, the ratio of CIK ( CD2 + /CD8 + /CD3 -) cells was up to 43. 63% at the fifth day, approxi-mately 5. 59 times increased compared with the initially separated PBMCs. Cell proliferation experiments showed that three obvious fluorescence peaks were observed on the fifth day. The results indicated that the induced CIK cells underwent three times cell division, in theory, about increased 8-fold compared with the initial separation of PBMCs. Furthermore, the qRT-PCR result of the surface markers of porcine NK cells also showed a similar variation tendency as the flow cytometry results. Conclusions Our findings demonstrate the successful establishment of an efficient activation and culture method for porcine CIK cells in vitro.
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Objective:To observe the efficacy of cytokine-induced killer (CIK) cells on patients with advanced lung cancer. Methods:A total of 90 patients with advanced lung cancer were identified from January 2011 to December 2013. CIK therapy was given to 41 pa-tients in the observation group, whereas the other 49 patients in the control group received the best support treatments without che-motherapy or radiotherapy within one month of inclusion. Following up was conducted for the patients in the two groups, and KPS scores, median survival, and adverse reactions compared. Results:The KPS score in the observation group was higher than that of the control group after treatment (P=0.034). The median survival period of the observation group was eight months, which was one month longer than that of the control group (P=0.044). Major adverse reactions included fever, joint pain, and insomnia, which were recorded 51.22%, 36.58%, and 29.27%of occurrence, respectively. Conclusion:CIK cell therapy improved the quality of life and pro-longed the survival of advanced lung cancer patients with tolerable adverse reactions.
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Objective To evaluate effects of autologous cytokine-induced killer cell (CIK) transfusion on the survival and hepatitics B virus (HBV) reactivation after radiofrequency ablation (RFA) combined with transcatheter arterial chemoembolization (TACE).Methods A retrospective analysis was conducted on 185 patients with hepatocellular carcinoma treated from Mar 2007 to Oct 2013.Patients were divided into study group (RFA,TACE,CIK) of 98 cases and control group (RFA,TACE) of 87 cases.According to tumor size,numbers and vascular invasion,patients were stratified into 4 subgroups:the high and the low risk group respectively with tumor ≤ 5 cm and ＞ 5 cm.Results The 1-,3-,5-year survival rate between study and control group were not significantly different:75.5％ (74/98),57.1％ (56/98),20.4％ (20/98) vs.71.2％ (62/87),54.0％ (47/87),21.8％ (19/87) (P ＞ 0.05).Only the study group's 1-,3-,5-year survival rate of high risk patients with tumor ≤ 5 cm were higher than the control group:75.0％ (21/28),53.6％ (15/28),35.7 ％ (10/28) vs.61.9％ (13/21),42.9％ (9/21),23.5％ (5/21) (P ＜ 0.05).The incidence of HBV reactivation was lower in dunantiviral patients who received CIK therapy than those who had 6.0％ (3/50) vs.23.5％ (12/61) (P ＜ 0.05).Conclusion Postoperative adjuvant CIK therapy with tumor≤5cm after RFA combined with TACE was beneficial to the survival of high risk patients and decreased the risk of HBV reactivation.
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Objective To study the killing effect of cytokine-induced killer cells (CIK cells) on human lung adenocarcinoma cell line (A549) and the lung adenocarcinoma' s radiation resistant cell line (A549RR).Methods Peripheral blood mononuclear cells (PBMC) of healthy volunteers were stimulated by different cytokines,and were induced into killer activity CIK cells.The phenotype of CIK cells were analyzed by flow cytometer.A549 and A549RR cell lines were cultured separately with the CIK cells.The absorbance value (A) of the cells was measured by CCK8,and the killing rates of all cells which were cultured for 24 and 48 hours with the CIK were calculated.Results The rate of CD3+ CD56+ cell was 45.8 ％ after culture for 14 d.The killing rates of CIK cells to lung adenocarcinoma A549 cells and its radiation resistant cells A549RR were increased with the rise of the ratio of effective cells to target (5∶1-40∶1) and the increasing of culturing time (all P ＜ 0.001).The killing effect of CIK to A549 and A549RR cells had no obvious difference in the same culturing time and the same ratio of effective cells to target(all P ＞ 0.05).Conclusion CIK cells have strong anti-tumor effect against lung adenocarcinoma and its radiation resistant cells with high clinical application value.
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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.