Cisplatin pretreatment enhances anti-tumor activity of cytokine-induced killer cells

AIM： To investigate whether cisplatin （DDP） enhances the anti-tumor activity of cytokine- induced killer （CIK） cells in a murine colon adenocarcinoma model. METHODS： Tumor size and weight served as indicators of therapeutic response. Immunohistochemistry was performed to observe intratumoral lymphocyte infiltration and tumor microvessel density. Changes in the percentage of regulatory T （Treg） cells within the spleens of tumor-bearing mice preconditioned with DDP were monitored using flow cytometry. RESULTS： A marked T cell-dependent, synergistic anti- tumor effect of the combined therapy was observed （1968 ± 491 mm3 ys 3872 ＋ 216 mm3; P = 0,003）, Preconditioning chemotherapy with DDP augmented the infiltration of CD3＋ T lymphocytes into the tumor mass and reduced the percentage of both intratumoral and splenic Treg cells. CONCLUSION： Preconditioning with DDP markedly enhances the efficacy of adoptively transferred CIK cells, providing a potential clinical modality for the treatment of patients with colorectal cancer.

In vitro incubation of cytokine-induced killer cells from patients with and without hepatitis B virus and a cell subset analysis

Objective The aim of the study was to explore the difference between immune cell subsets during the incubation of cytokine-induced kill cells （CIKs） from patients with and without hepatitis B virus （HBV）. Methods Peripheral blood samples were extracted from 50 tumor patients, and were divided into two groups according to the presence or absence of HBV. The proliferation rate and activity of CIK cells were examined based on counts on days 1, 5, 7, 9, 11, 13, and 15 of culture. Additionally, the CD3＋, CD4＋, CD8＋, CD3＋CD8＋, C＋）3＋CD4＋, and CD3＋CD56＋ T cell populations were analyzed by flow cytometry on days 5, 7, 10, 13, and 15 of culture. Results Proliferation over a 15-day period was higher in the HBV-positive group than in the negative group （280-fold vs. 180-fold increase, respectively）, but there was no significant difference between the two groups at each time point. The frequencies of CD3＋, CD8＋ T, CD3＋CD8＋, and CD3＋CD56＋T cells increased over time, while those of CD4＋ and CD3＋CD4＋ T cells decreased over time, and these changes were greater in the positive group than in the negative group. The differences in CD8＋ T cells and CD3＋CD4＋ T cells between the two groups were significant （P 〈 0.05）. Conclusion The proliferative capacity of CIK cells was higher for patients in the HBV-positive group than those in the HBV-negative group, and immune cell subsets were more favorable in the HBV-positive group than the neaative arouD.

Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-ulsed dendritic cells and cytokine-induced killer cells

AIM： To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells （DCs） and cytokine-induced killer cells. METHODS： Freshly collected hepatocellular carcinoma （HCC） tumor tissues were incubated with a mixture of neuraminidase and recombinant αl,3-galactosyltrans- ferase （αI,3GT） to synthesize α-Gal epitopes on car- bohydrate chains of the glycoproteins of tumor mem- branes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage 111 primary HCC were randomly chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS： The evaluation demonstrated that the pro- cedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly pro- longed the survival of treated patients as compared with the controls （17.1 ± 2.01 mo vs 10.1 ±4.5 mo, P = 0.00121）. After treatment, all patients in the study group had positive delayed hypersensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-y-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO- and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the se- rum. CONCLUSION： This new tumor-specific immunotherapy is safe, effective and has a great potential for the treat- ment of tumors.

Intraperitoneal perfusion of cytokine-induced killer cells with local hyperthermia for advanced hepatocellular carcinoma

AIM:To study the effect and tolerance of intraperitoneal perfusion of cytokine-induced killer(CIK) cells in combination with local radio frequency(RF) hyperthermia in patients with advanced primary hepatocellular carcinoma(HCC).METHODS:Patients with advanced primary HCC were included in this study.CIK cells were perfused intraperitoneal twice a week,using 3.2 × 10 9 to 3.6 × 10 9 cells each session.Local RF hyperthermia was performed 2 h after intraperitoneal perfusion.Following an interval of one month,the next course of treatment was administered.Patients received treatment until disease progression.Tumor size,immune indices(CD3 +,CD4 +,CD3 + CD8 +,CD3 + CD56 +),alpha-fetoprotein(AFP) level,abdominal circumference and adverse events were recorded.Time to progression and overall survival(OS) were calculated.RESULTS:From June 2010 to July 2011,31 patients diagnosed with advanced primary HCC received intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia in our study.Patients received an average of 4.2 ± 0.6 treatment courses(range,1-8 courses).Patients were followed up for 8.3 ± 0.7 mo(range,2-12 mo).Following combination treatment,CD4 +,CD3 + CD8 + and CD3 + CD56 + cells increased from 35.78% ± 3.51%,24.61% ± 4.19% and 5.94% ± 0.87% to 45.83% ± 2.48%(P = 0.016),39.67% ± 3.38%(P = 0.008) and 10.72% ± 0.67%(P = 0.001),respectively.AFP decreased from 167.67 ± 22.44 to 99.89 ± 22.05 ng/mL(P = 0.001) and abdominal circumference decreased from 97.50 ± 3.45 cm to 87.17 ± 4.40 cm(P = 0.002).The disease control rate was 67.7%.The most common adverse events were low fever and slight abdominal erubescence,which resolved without treatment.The median time to progression was 6.1 mo.The 3-,6-and 9-mo and 1-year survival rates were 93.5%,77.4%,41.9% and 17.4%,respectively.The median OS was 8.5 mo.CONCLUSION:Intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia is safe,can efficiently improve immunological status,and may prolong survival in HCC patients.

Immunotherapy with dendritic cells and cytokine-induced killer cells for hepatocellular carcinoma: A meta-analysis

BACKGROUND Hepatocellular carcinoma(HCC) has been revealed as the second most common cause of cancer-related deaths worldwide. The introduction of cell-based immunotherapy, including dendritic cells(DCs) and cytokine-induced killer cells(CIKs), has brought HCC patients an effective benefit. However, the efficacy and necessity of cellular immunotherapy after different interventional therapy remains to be further explored.AIM To investigate the efficacy of cellular immunotherapy, involving DCs and CIKs,combined with different conventional treatments of HCC.METHODS We performed a literature search on PubMed and Web of Science up to February15, 2019. Long-term efficacy(overall survival and recurrence) and short-term adverse effects were investigated to assess the effectiveness of immunotherapy with DCs and/or CIKs. Review Manager 5.3 was used to perform the analysis.RESULTS A total of 22 studies involving 3756 patients selected by eligibility inclusion criteria were forwarded for meta-analysis. Combined with the conventional clinical treatment, immunotherapy with DCs and/or CIKs was demonstrated to significantly improve overall survival at 6 mo [risk ratio(RR) = 1.07;95%confidence interval(CI): 1.01-1.13, P = 0.02], 1 year(RR = 1.12;95%CI: 1.07-1.17, P< 0.00001), 3 years(RR = 1.23;95%CI: 1.15-1.31, P < 0.00001) and 5 years(RR =1.26;95%CI: 1.15-1.37, P < 0.00001). Recurrence rate was significantly reduced by cellular immunotherapy at 6 mo(RR = 0.50;95%CI: 0.36-0.69, P < 0.0001) and 1 year(RR = 0.82;95%CI: 0.75-0.89, P < 0.00001). Adverse effect assessment addressed that immunotherapy with DCs and/or CIKs was accepted as a safe,feasible treatment.CONCLUSION Combination immunotherapy with DCs, CIKs and DC/CIK with various routine treatments for HCC was evidently suggested to improve patients’ prognosis by increasing overall survival and reducing cancer recurrence.

Adjuvant treatment for triple-negative breast cancer: a retrospective study of immunotherapy with autologous cytokine-induced killer cells in 294 patients

Objective: To examine the efficacy and safety of a sequential combination of chemotherapy and autologous cytokine-induced killer(CIK) cell treatment in triple-negative breast cancer(TNBC) patients.Methods: A total of 294 post-surgery TNBC patients participated in the research from January 1, 2009 to January 1, 2015. After adjuvant chemotherapy, autologous CIK cells were introduced in 147 cases(CIK group), while adjuvant chemotherapy alone was used to treat the remaining 147 cases(control group). The major endpoints of the investigation were the disease-free survival(DFS) and overall survival(OS). Additionally, the side effects of the treatment were evaluated.Results: In the CIK group, the DFS and OS intervals of the patients were significantly longer than those of the control group(DFS:P = 0.047;OS: P = 0.007). The multivariate analysis demonstrated that the TNM(tumor-node-metastasis) stage and adjuvant CIK treatment were independent prognostic factors for both DFS [hazard ratio(HR)= 0.520, 95% confidence interval(CI):0.271-0.998, P = 0.049;HR = 1.449, 95% CI:1.118-1.877, P = 0.005, respectively] and OS(HR=0.414, 95% CI:0.190-0.903, P = 0.027;HR= 1.581, 95% CI:1.204-2.077, P = 0.001, respectively) in patients with TNBC. Additionally, longer DFS and OS intervals were associated with increased number of CIK treatment cycles(DFS: P = 0.020;OS: P = 0.040). The majority of the patients who benefitted from CIK cell therapy were relatively early-stage TNBC patients.Conclusion: Chemotherapy in combination with adjuvant CIK could be used to lower the relapse and metastasis rate, thus effectively extending the survival time of TNBC patients, especially those at early stages.

Effect of anti-asthma Chinese medicine Chuankezhi on the anti-tumor activity of cytokine-induced killer cells

Chuankezhi(CKZ),a new Chinese medicine,plays an important role in immunoregulation.Cytokineinduced killer(CIK)cells have been commonly used for immunotherapy in recent years.In this study,we aimed to investigate the immunoregulatory effect of CKZ on CIK cells.Peripheral blood monocytes were isolated from healthy donors,and CIK cells were generated by culturing monocytes with interferon-gamma(IFN-γ)and interleukin 2.Different concentrations of CKZ were added on day 2.After incubation for 14days in culture,the antitumor effects of CIK cells were measured by cytotoxicity assay.Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype,intracellular cytokine production,and apoptosis.The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated.CKZ increased the percentage of CD3+CD56+CIK cells but did not significantly change the percentage of CD4+,CD8+,or CD4+CD25+CIK cells.CKZ-conditioned CIK cells showed a greater ability to kill tumor cells,as well as a higher frequency of IFN-γand TNF-αproduction,compared with the CIK cells in the control group.CKZ also suppressed the apoptosis of CIK cells in vitro.Furthermore,CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK,CKZ,or normal saline control groups.Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.

Preliminary Study of Local Immunotherapy with Autologous Cytokine-Induced Killer Cells for Glioma Patients

OBJECTIVE Cytokine-induced killer （CIK） cells are T-cells that display effective anti-tumor activity. In this stud, we investigated the anti-tumor activity of CIK cells in vitro, and conducted a preliminary investigation using autologous CIK cells to treat glioma patients through local administration. METHODS The CIK cells were derived from peripheral blood monocytes （PBMCs） of the glioma patients. The anti-tumor activity of the CIK cells against human T98-G glioma cell was tested in vitro. In addition, the autologous CIK cells were locally administrated into the tumor cavity in the malignant glioma patients through an Ommaya reservoir which was pre-inserted during tumor resection. The 4 x 108 CIK cells in a 5 ml suspension were injected once a week 2 times per cycle. Five hundreds KU of IL-2 was injected every other day. RESULTS （i） With incubation, the CIK cells showed dual staining of CD3^＋CD56^＋ with a positive rate of 3.45% on day 10 and 55.2% on day 30. In vitro anti-tumor activity （against T98-G cells） of the CIK cells reached the highest level after 18 days of incubation with different effector/target （E：T） ratios. （ii） Six patients received autologous CIK cell treatment （10 cycles）. Two patients showed no recurrence and are still alive （24 and 10 months）, while 4 cases had a recurrence 3 of which have died. The mean survival time from the first CIK cell treatment to the end of follow-up was 12.5 months. The main side-effects of the local CIK cell treatment was brain edema, which was controlled by mannitol in most of the cases. However for one patient injection of CIK cells and IL-2 had to be discontinued. CONCLUSION In vitro CIK cells are effective anti-glioma T-cells. Local therapy with CIK cells has potential anti-glioma efficacy and tolerable side-effects.

Autologous cytokine-induced killer cells in equal to liver protectant in a patient with metastatic rectal cancer

The cytokine-induced killer (CIK) therapy was an effective treatment for many cancers. We report a patient with postoperative rectal cancer received autologous CIK therapy combined with raltitrexed chemotherapy. After the adjuvant therapy, the serum transaminase was persistently elevated, and lung metastases was observed. Due to hepatic injury, only cytokine-induced killer therapy was administered, and it rectified transaminase. The following regimens of CIK therapy and low-dose raltitrexed could diminish the metastatic lesion, improve the quality of life and prolong the survival time. It reveals that the CIK cells may repair the hepatic injury.

Harnessing cytokine-induced killer cells to accelerate diabetic wound healing:an approach to regulating post-traumatic inflammation

Impaired immunohomeostasis in diabetic wounds prolongs infiammation and cytokine dysfunction,thus,delaying or pre-venting wound-surface healing.Extensive clinical studies have been conducted on cytokine-induced killer(CIK)cells recently,as they can be easily proliferated using a straightforward,inex-pensive protocol.Therefore,the function of CIK cells in regulat-ing inflammatory environments has been drawing attention for clinical management.Throughout the current investigation,we discovered the regenerative capacity of these cells in the chal-lenging environment of wounds that heal poorly due to diabe-tes.We demonstrated that the intravenous injection of CIK cells can re-establish a proregenerative inflammatory microenviron-ment.promote larizationand.ultimatel accelerateskin healing in diabetic mice.The results indicated that CIK cell treatment affects macrophage polarization and restores the function regenerative cells under hyperglycemic conditions.This novel cellular therapy offers a promising intervention for clinical applic tions through specific inflammatory regulation functions.

A randomized controlled trial of postoperative tumor lysate-pulsed dendritic cells and cytokine-induced killer cells immunotherapy in patients with localized and locally advanced renal cell carcinoma

Background It remains a challenge to inhibit the local recurrence or distant metastasis of localized or locally advanced renal cell carcinoma （RCC） after surgical resection. We investigated the feasibility, safety and efficacy of immunotherapy using autologous tumor lysate （TL）-pulsed dendritic cells （DCs） and cytokine-induced killer （CIK） cells in patients with localized or locally advanced RCC.

Antitumour activities of cytokine-induced killer cells and dendritic cells in vitro and in vivo

Solid tumour cells show a resistance to immunological effector cells in vitro. The resistance may be one reason why these tumours withstand immunotherapeutic approaches in humans. Dendritic cells （DC） play an important role in the immune response to tumour associated antigens in humans. DC in the periphery capture and process antigens, express lymphocyte costimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune response.

Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin

Objective： The aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer （CIK） cells and then demonstrate the appropriate mechanism. Methods： Tumor-derived exosomes （TEXs）, which are derived from RBE cells （human cholangiocarcinoma line）, were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting （FACS） was performed to determine the phenotypes of TEX-CIK and N-CIK （normal CIK） cells. The concen- trations of tumor necrosis factor-a （TNF-a） and perforin in the culture medium supematant were examined by using an enzyme-linked immunosorbent assay （ELISA） kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line. Results： The concentrations of TNF-a and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml （P〈0.01） and 3.39 ng/ml （P〈0.05）. The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK （47,35% （P〈0.01））. The population of CD3＋, CD8＋, NK （CD56＋）, and CD3＋CD56＋ cells decreased in the TEX-CIK group （63.2±6.8）%, （2.5±1.0）%, （0.53±0.49）%, （0.45±0.42）%） compared with the N-CIK group （（90.3±7.3）%, （65.7±3.3）%, （4.2±1.2）%, （15.2±2.7）%）, P〈0.01. Conclusions： Our results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3＋, CD8＋, NK （CD56＋）, and CD3＋CD56＋ cells and the secretion of TNF-a and perforin. TEX may play an important role in cholangiocarcinoma immune escape.

Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation

A female patient diagnosed with acute myelocytic leukemia M5a （AML-M5a） relapsed 986 days after her allogeneic peripheral blood stem cell transplantation （alIo-PBSCT） from an unrelated male donor with matched human leukocyte antigen （HLA）. Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer （CIK） cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion （DLI） and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

Cytokine-induced killer (ClK) cells:from basic research to clinical translation

The accumulation of basic researches and clinical studies related to cytokine-induced killer(CIK) cells has confirmed their safety and feasibility in treating malignant diseases.This review summarizes the available published literature related to the biological characteristics and clinical applications of CIK cells in recent years.A number of clinical trials with CIK cells have been implemented during the progressive phases of cancer,presenting potential widespread applications of CIK cells for the future.Furthermore,this review briefly compares clinical applications of CIK cells with those of other adoptive immunotherapeutic cells.However,at present,there are no uniform criteria or large-scale preparations of CIK cells.The overall clinical response is difficult to evaluate because of the use of autologous CIK cells.Based on these observations,several suggestions regarding uniform criteria and universal sources for CIK cell preparations and the use of CIK cells either combined with chemotherapy or alone as a primary strategy are briefly proposed in this review.Large-scale,controlled,grouped,and multi-center clinical trials on CIK cell-based immunotherapy should be conducted under strict supervision.These interventions might help to improve future clinical applications and increase the clinical curative effects of CIK cells for a broad range of malignancies in the future.

Ex vivo Expansion of Cytokine-Induced Natural Killer Cells from Tumor Patients for Autologous Therapy

To optimize the expansion conditions for cytokine-induced natural killer cells (CINK), including major CD3 - CD56 + NK cells, and by using the selected method to expand the CINK cells from patients with tumors for the application in the autologous immunotherapy, peripheral blood mononuclear cells (PBMC) from healthy donors were cultivated in stem cell growth medium supplemented with 5% human AB serum, and monoclonal anti-CD3 antibody, IL-2 and phytohaemagglutin (PHA) at different concentrations were added to select the optimal cultural conditions for the CINK cells. The optimized method was used to expand the CINK cells from 12 patients with various malignant tumors, the proportions of CD3 + and CD56 + cells in the expanded cells were determined by flow cytometry, and the cytotoxicity of the CINK cells from patients was detected by MTT assay in comparison with those of cytokine-induced killer (CIK) cells or anti-CDC3 antibody-activated killer (CD3AK) cells. It was found that the expansion of CINK cells could be obtained when these cells were activated with 500 ng/ml of monoclonal anti-CD3 antibody and 100?U/ml of IL-2. The highest degree of cell proliferation (51.3 folds) could be attained in the presence of PHA after 14?d cultivation of cells, and the expanded cell populations included CD3 - CD56 + NK cells (49.8%±7.2%) and CD3 + CD56 + T cells (14.7%±3.9%) could lyse 83.7% of K562 cells and 55.8% of Raji cells at a 10∶1 of effector/target ratio. Also the CINK cells of clinical scale could be expanded from tumor patients by using the optimized method with a similar expansion efficiency and cytotoxicity. These cells showed a higher cytotoxic activities against target cell lines (K562 and Raji cell lines) than those of CIK and CD3AK cells. This improved method has several advantages over other methods, such as the easy manipulation, low costs and the production of functionally active NK cells on a clinical scale from cancer patients without the need for special equipments. This might facilitate the realization of further protocol for evaluating the clinical effects of NK cells in autologous immunotherapies of tumor patients.

Immune evasion and therapeutic opportunities based on natural killer cells

Natural killer(NK)cells can elicit an immune response against malignantly transformed cells without recognizing antigens,and they also exhibit cytotoxic effects and immune surveillance functions in tumor immunotherapy.Although several studies have shown the promising antitumor effects of NK cells in immunotherapy,their function is often limited in the tumor microenvironment because tumor cells can easily escape NK cell-induced death.Thus,for efficient tumor immunotherapy,the mechanism by which tumor cells escape NK cell-induced cytotoxicity must be fully understood.Various novel molecules and checkpoint receptors that mediate the disruption of NK cells in the tumor microenvironment have been discovered.In this review,we analyze and detail the major activating and inhibitory receptors on the surface of NK cells to delineate the mechanism by which tumor cells suppress NKG2D ligand expression and increase tumor receptor and inhibitory receptor expression[NKG2A,programmed cell death1(PD-1),and T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif(TIGIT)]on the NK cell surface,and thus inhibit NK cell activity.We also reviewed the current status of treatments based on these surface molecules.By comparing the therapeutic effects related to the treatment status and bypass mechanisms,we attempt to identify optimal single or combined treatments to suggest new treatment strategies for tumor immunotherapy.

Investigation of the Cytotoxicity of CAR-NK-92 Cells Targeting CEA Against Gastric Cancer Cells

Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 cells with MKN-45 gastric cancer cells,the killing effect of CAR-NK-92 cells was detected by a lactate dehydrogenase release assay.The secretion levels of gamma interferon and granulocyte-macrophage colony-stimulating factor were measured using an ELISA assay.Results:The lactate dehydrogenase release assay showed that CAR-NK-92 cells had a significant killing effect on MKN-45 cells compared to CON-NK-92 cells,and the difference was statistically significant(P<0.001).ELISA results indicated that the levels of gamma interferon and granulocyte-macrophage colony-stimulating factor secreted by CAR-NK-92 cells and MKN-45 target cells were significantly increased after co-culture(P<0.001).Conclusion:CAR-NK-92 cells targeting CEA exhibit a significant killing effect on CEA-positive gastric cancer cells.

Targeted Therapy of CEA-CAR-NK Cells Against Colorectal Cancer Cells

Objective:Investigate the cytotoxic effect of CAR-NK cells targeting CEA on colorectal cancer cells with positive CEA expression.Methods:The mRNA and protein levels of CEA in different CRC cell lines were detected by qRT-PCR and Western blot analysis.Lentiviral transduction was used to construct CAR-NK cells and empty vector CON-NK cells targeting CEA.Fluorescence microscopy and WB were used to determine whether the cells successfully constructed and expressed CAR structures.The effector NK cells were co-cultured with target cells,and the levels of LDH,IFN-γ,and GM-CSF were detected.The killing rate of effector cells was calculated,and the release of cytokines during the killing of target cells by different effector cells was compared.Results:The expression level of CEA in colorectal cancer patients was significantly higher than that in normal samples and other tumor samples,and the prognosis survival time of patients with high CEA expression was lower than that of CRC patients with low or no CEA expression(P<0.05).The CEA expression of the HT29 cell line was significantly higher than that of the SW1116 cell line at both the mRNA and protein levels.CEA-CAR-NK92 cells and CON-NK92 cells expressed green fluorescence under a microscope,and WB results showed that CEA-CAR-NK92 cells successfully expressed the CAR structure.Compared with CON-NK92 cells and NK92 cells,CEA-CAR-NK92 cells effectively killed HT29 cells(P<0.05).CEA-CAR-NK92 cells secreted a large amount of IFN-γand GM-CSF during the killing of HT29 cells,while the cytokine secretion of CON-NK92 cells and NK92 cells was not significant(P<0.05).Conclusion:CAR-NK92 cells targeting CEA can effectively kill CEA-positive colorectal cancer cells.