The Correlation Studies Of The Anti-tumor Effect Of Chemotherapy In Combination With CIK Cells And The Clinical Observation Against Ovarian Cancer

The pathogenesis of ovarian cancer is concealed. Most patients are already inadvanced stage when they are in treatment. This is the leading cause of death in patientswith gynecological tumor. The incidence and mortality of colon cancer are first in thedigestive system tumors also. Therefore, it is urgent to find a more effective treatment forthem. With the continuous development of tumor immunology and molecular biology,tumor immunotherapy has become the fourth treatment mode. Cytokine-induced killercells (CIK) is a new kind of adaptive immune cells characterized by its dramaticproliferation ability and cytotoxicity, non-major histocompatibility antigens (MHC)restriction and low side effects, which draw an increasing attention in recent years. CIKhas been widely used in clinical treatment of a variety of solid tumors, but there has fewreport about this therapy of ovarian cancer.Firstly, we investigated the anti-tumor effects of chemotherapeutic drug cis-Dichlorodiamine in platinum (CDDP or DDP) and CIK or in combination against ovarian cancercell lines SKOV-3in vitro. And then, to investigate the anti-tumor effects of fluorouracil(5-FU) and cytokine-induced killer cells (CIK) or in combination against colon cancer inmouse syngeneic model. Finally, we retrospectivly analyzed the clinical data of6patientswith ovarian cancer who have been treated with CIK therapy. To explore the safety andactivity of CIK cells treatment of patients with ovarian cancer and to search for biologicaltreatment strategies for ovarian cancer.Part I The Anti-tumor Effects of DDP Combined with CIK AgainstOvarian Cancer Cell Lines SKOV-3in vitroObjective: T0investigate the anti-tumor effects and mechanism of chemotherapeuticdrug cis-Dichloro diamine in platinum (CDDP or DDP) combined with cytokine-induced kill cells (CIK) against ovarian cancer cell lines SKOV-3in vitro.Methods: CIK cells were induced from peripheral blood mononuclear cells (PBMC)of healthy human stimulated by different cytokines. The CIK cells were counted and thephenotype of CIK cells were analyzed by flow cytometry (FCM) at the cultivation of0,7,14,21,28and35days; The CIK cells were harvested at day14as effector cells and thetarget cells was logarithmic phase SKOV-3lines. SKOV-3cells was cultivated with theCIK cells culturing supernatant. The normal cultivated SKOV-3cells as control group.24and48hours later, the SKOV-3cells apoptosis was analyzed by a flow cytometer. Killingexperiment were divided into A1~4CIK groups (The effector-target ratios were5:1,10:1,20:1and40:1respectively); B1~7DDP groups (The concentrations of DDP were0.625,1.25,2.5,5,10,20and40μg/ml) and C1~2CIK combined with DDP groups (Theconcentration of DDP was2.5μg/ml and the effector-target ratios were5:1and10:1respectively). At the same time, take the effector cells and target cells as control. Theinhibition rate of each group of SKOV-3cells were measured by MTT.Results: CIK cells numbers in vitro cultivation of0,7,14,21,28and35days were(1.8±0.2)×107,(14.67±1.53)×107,(230.0±20.0)×107,(225.0±22.91)×107and (220.0±17.32)×107in sequence. The number increased to about127.78fold at culturing21days,which reached to proliferation peak; The percentage of CD3+CD56+double positive cellswas (0.47±0.17)%、(11.30±1.96)%、(35.50±2.30)%、(38.23±1.92)%、(31.40±3.91)%and (28.61±3.45)%in sequence. which also reached peak after14days; The apoptosisrates of SKOV-3cell lines were (12.30±1.47)%and (27.13±2.03)%respectively after24hours and48hours cocultured with CIK cells’ supernatant; The inhibition rates of A1~4CIK groups were (16.52±2.52)%、(24.18±4.05)%、(35.98±5.19)%and (53.98±5.02)%respectively at24hours, And the inhibition rates reached to (24.20±5.59)%、(41.23±3.64)%、(57.27±6.68)%and (74.74±6.87)%respectively at48hours. The difference ofcell inhibition rates among groups were statistically significant at the same time (P <0.05).And the difference of cell inhibition rates of the same effect-target ratio between24h and48h were statistically significant too (P <0.05). The killing activity increased with theculture time prolonged and the effector-target ratio enhanced; The inhibition rates of B1~7DDP groups were (15.09±4.03)%、(25.95±3.36)%、(37.11±3.05)%、(50.07±5.21)%、(62.93±5.85)%、(77.93±3.28)%and (92.55±1.39)%respectively at24hours. And the inhibition rates reached to (23.42±5.63)%、(32.39±1.47)%、(46.04±2.76)%、(60.46±3.26)%、(72.77±2.38)%、(86.42±1.46)%and (96.24±0.59)%respectively at48hours.The differences of inhibition rates of each group between24h and48h were statisticallysignificant except for DDP0.625μg/ml group. The antitumour activity increased with thecultured time prolong and the DDP concentration enhanced; The inhibition rates of C1~2CIK combined with DDP groups were (50.50±3.69)%and (68.19±2.07)%at24hours.And they were (69.87±2.67)%and (80.11±6.87)%at48hours. There were statisticallysignificant in the differences of inhibition rates among the groups treated with CIK or DDPalone and the group treated in combination. But factorial analysis results show that the lowdose group (effector-target ratios5:1)24h and48h inhibition rates did not see the synergyeffect (P values were0.171and0.895), but in the high dose group (the effector-target ratiowas10:1) the synergistic inhibition effect can be observed (P <0.05).Conclusion: PBMC could be induced into CIK cells by a variety of cytokines in vitrostimulation and get a large number of amplification. CD3+CD56+CIK, the main effectorcells reached to the high expression of plateau in14~21days. We can obtain the idealeffect CIK cells during the meantime; CIK cells have strong anti-cancer activity againstSKOV-3cells in vitro by secreting cytokines and inducing apoptosis of SKOV-3cells. Itcan enhance the anti-tumour effects of DDP against SKOV-3cells when combined withCIK cells.Part II The Experimental Study on the Anti-tumor Effect ofFluorouracil Combined with CIK Cells Against Colon Cancer in MouseSyngeneic ModelObjective: To investigate the anti-tumor effects of fluorouracil (5-FU) combinationwith CIK cells against colon cancer in mouse syngeneic model.Methods: BALB/C mouse colon cancer models were established. Murine CIK cellswere induced by culturing in vitro. The phenotype of main effect CIK cells were analyzedby FCM at the cultivation of0and7days. Ten tumor-burdened rats were divided randomlyinto two groups. One group were treated with5-FU (12.5mg/kg) intraperitoneal injection.The other group were treated with NS (0.2ml each) intraperitoneal injection. FCM wasused to detect the expression of the lymphoid-specific cytotoxic T lymphocyte-associated antigen-4(CTLA-4), and nonlymphocytic CD80, CD86in the the tumor tissue derivedfrom the treated mice after10days. Another40tumor-burdened rats randomly divided intofour groups. Group A (normal control group): NS (0.2ml intraperitoneal injection, day0)and NS (0.2ml intravenous injection, day3). Group B (5-FU group):5-FU (12.5mg/kgintraperitoneal injection, day0) and NS (0.2ml intravenous injection, day3). Group C(CIK group): NS (0.2ml intraperitoneal injection, day0) and CIK (1×107intravenousinjection, day3). Group D (5-FU combined with CIK group):5-FU (12.5mg/kgintraperitoneal injection, day0) and CIK (1×107intravenous injection, day3). The tumorgrowth was observed and the tumor growth curve was drawn. The survival data of thetreatment was analyzed using Kaplan-Meier method and the log-rank test. Five mice ofeach group were executed10days later. The expression level of IFN-γ and TNF-in tumortissue were detected by RT-PCR.Result: The major effector CIK cells of mice amplied to (18.4±6.8)%at the seventhday in vitro. The expression of CTLA-4in CD45+cells and CD80/CD86in CD45-cellswere increased with the treatment of5-FU compared with the control group. Comparedwith treatment of5-FU or CIK cells alone, the combination group can significantlydecreased the growth rate of tumor in mice, and the difference has statistical significance(P <0.05). The expression of IFN-γ and TNF-in tumor tissue were also increased in thecombination group. There was statistically significant difference in expression of IFN-γbetween combination treatment groups and single treatment group (P <0.05).Conclusion: The treatment of5-FU and CIK cells in combination was better thanalone in inhibiting the growth of transplanted colon carcinoma and also it can enhanceimmune function in mice with colon cancer.Part III Clinical Observation of Chemotherapy in Combination withCIK Cells for Ovarian CancerObjective: Preliminary discussion the safety and activity of CIK cells treatment ofpatients with ovarian cancer. To explore the biological treatment strategies for patients withovarian cancer.Methods: Peripheral blood mononuclear cells (PBMC) of patients with ovariancancer who have been treated with conventional surgery and chemotherapy were stimulated by different cytokines and were induced into CIK cells. The phenotype of CIKcells were analyzed by FCM. The CIK cells cultivated14~21days in vitro weretransfused back to patients through the venous.The changes of peripheral blood T cellsubsets in patients were detected by FCM. Six cases of ovarian cancer with the treatmentof CIK cells were retrospectively observed. Observe the difference of Karnofskyperformance score (KPS) therapy. performance score (KPS), tumor markers anddistribution of the peripheral blood T cell subsets of patients before and after CIK therapy,and also the related adverse reaction were observed after CIK therapy.Results: The KPS of5cases were obviously increased and one case remainunchanged after CIK cells treatment. All patients with CIK therapy does not appearobvious adverse effects. There is one patient whose tumor marker CA199elevated aftersurgery and chemotherapy. However, the level dropped to normal gradually after threeperiods of CIK cells therapy. The progression free survival (PFS) of whom has reached to3years and she is in good condition now. Tumor markers and radiographic follow-up showno sign of tumor recurrence. One patient with stage II, whose histology type is clear celland serosity carcinoma, her PFS is close to4years. And her tumor markers andradiographic follow-up tumor visit shows no sign of tumor recurrence. There is anotherEOC patient with stage II who had accepted eight periods of CIK cells treatment, Her PFShas reached to5years and she is in good condition at present with no sign of tumorrecurrence either.Conclusion: CIK therapy for patients with ovarian cancer can improve the quality ofsurvival, It is a promising treatment for ovarian cancer.To sum up, through the basic research of the anti-tumor mechanism of CIK cells andretrospective analysis of ovarian cancer cases who have been treated with CIK cells, wecan conclude that CIK cells could get a large number of amplification induced by a varietyof cytokines stimulation in vitro. The14~21days’cultiviation could get the ideal effectorcells; CIK cells have strong anti-tumor activity against SKOV-3cells in vitro by secretingcytokines and inducing apoptosis of SKOV-3cells. It can enhance the anti-tumor effects ofDDP against SKOV-3cells when combined with CIK cells.5-FU and CIK cells incombination was better than alone in inhibiting the growth of the transplanted tumor andenhancing immune function in mice with colon cancer. CIK therapy for patients withovarian cancer can improve the quality of life, It is a promising treatment for patients with ovarian cancer.