The Antitumor Activity Of Tumor-infiltrating Lymphocytes In Ovarian Cancer

BackgroundOvarian cancer is one of the common female tumors worldwide. There was an estimated more than220,000new cases of ovarian cancer and140,200ovarian cancer deaths in2008.In China, ovarian cancer is one of the three malignant tumors of female genital tract. The morbidity of ovarian cancer was ranked the third in gynecological tumor, but the mortality of ovarian cancer was ranked first. Epithelial ovarian cancer is the most common histological type of ovarian cancer. It mostly occurs in older women. Patients were diagnosed when they were at the age of50～70. Epithelial ovarian cancer has serous cavity properties. When patients had malignant ascites or malignant pleural effusion, the median survival time was less than1year. It was relatively difficult to treat advanced ovarian cancer with malignant ascites or malignant pleural effusion. The life quality of patients was strongly affected by the disease.Tumor-infiltrating lymphocytes (TIL) is the infiltrated lymphocytes surrounding in the tumor tissues. It was activated by surface antigens of tumor cells. TIL had specific cytotoxicity on autologous-tumor cells. In recent years, TIL was one of the research hotspots in tumor immunology. The number and activity of TIL in tumor tissues was low. Many experiments showed that TIL could be activated by interleukin-2in vitro. As TIL was activated, it was used to treat the patients with malignant tumor in clinical practice and achieved a good therapeutic effect. Fabbri used TIL/IL-2to treat39patients, including18cases of melanoma,19cases of colon cancer,2cases of renal cell carcinoma. The results revealed that11cases of melanoma patients (64.7%) survived more than37months,8cases of colon cancer patients (44.4%) survived more than21months,1patient of renal cell carcinoma survived more than28months. In all patients, one of melanoma and one of colon cancer were completely cured. Lin Jing reported that TIL/IL-2was used to treat107patients with malignant pleural effusion (74cases of lung cancer,2cases of breast cancer,4cases of liver cancer,9cases of ovarian cancer,2cases of gastric cancer,3cases of colorectal cancer,2cases of nasopharyngeal carcinoma,8cases of cervical cancer and3cases of other cancer). A total of104patients were evaluable for efficacy.72%complete remission (73/104),22.1%partial remission (23/104), the total efficiency was92.3%.Wang Yuling reported that TIL/IL-2was used to treat12cases of advanced ovarian cancer with malignant ascites, the overall response rate was75%。The formation mechanism of malignant pleural effusion, malignant ascites was not very clear. The obstruction of lymphatic drainage due to cancer cells and the metastases of abdominal cavity might be the main reasons of malignant serous effusion. Recent studies showed that cancer cells could synthesize and secrete vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). That not only promoted cancer invasion, metastasis, angiogenesis, but also increased vascular permeability. VEGF and MMPs were thought to play an important role in the formation of malignant effusion. The good effect of TIL on malignant pleural effusion might be related to the decrease of VEGF and MMPs. TIL showed the advantages of remarkable curative effect and it had no significant adverse reactions. Because of the low immune function of cancer patients, the cytotoxicity of TIL was low when it just had been separated from malignant pleural effusion, malignant ascites or tumor tissue. As it was induced and expanded by IL-2and other factors in vitro, the cytotoxicity of TIL could be greatly enhance. Then TIL was returned into the pleural cavity, peritoneal cavity to treat the patients with malignant ascites or pleural effusion. In the clinical practice, we observed that TIL achieved good curative effect in treating the advanced ovarian cancer with malignant ascites or malignant pleural effusion. Now we are working on a study to discuss the antitumor activity and mechanisms, to find systematic review to evaluated the curative effect of TIL on molecular level. It might provide a strong evidence for the clinical practice of TILObjectiveThe cytotoxic effects of ovarian cancer TIL after induction, activation and amplifying by the IL-2for different time on ovarian cancer autologous-tumor cells, K562cells and Raji cells were explored. The phenotypic changes of ovarian cancer TIL were analyzed before and after the induction, activation and amplifying by the IL-2. The changes of the molecular markers (CA-125, CA-153, VEGFand MMP-9) in the culture supernatant of ovarian cancer autologous-tumor cells were analyzed. The apoptosis of ovarian cancer autologous-tumor cells inducing by TIL was analyzed.Methods1. The obtaining and culture of autologous-tumor cells and TILTwelve fresh surgical specimens of solid tumor tissues with ovarian cancer were obtained from obstetrics and gynecology department of Nanfang Hospital Affiliated to Southern Medical University. All the patients were diagnosed epithelial ovarian cancer using histopathological detection (both were serous cystadenocarcinoma). The solid tumor tissues were digested with type Ⅱ0.1%collagenase under4℃and placed overnight. Then0.02%hyaluronidase and0.004%DNA enzyme were added at37℃and digested the tissue for4hours.100mesh and200mesh sieve filters were used to obtain single cell suspension. Human lymphocytic isolation solution (100%Ficoll-Hypaque), low speed centrifugalization and discontinuous density gradients centrifugation were used to separate tumor cells and tumor-infiltrating lymphocytes. The autologous-tumor cells were cultured with20%fetal bovine serum (FBS)(Hyclone) RPMI-1640(Gibco) complete culture medium, the TIL was cultured with10%FBS RPMI-1640complete medium and2500U/ml IL-2. The solution was changed according to the cell growth situation for subculture, and IL-2was supplemented after the TIL was cultured for1-2days.2. The culture of the cell linesHuman leukemia cell lines K562and B cell lymphoma cell line Raji were kept by the hematology laboratory of Nanfang Hospital. And10%FBS RPMI-1640complete medium was used. The solution was changed according to the cell growth situation for subculture.3. Identification of autologous-tumor cellThe hematoxylin-eosin staining method (HE staining) was used to observe the morphology of autologous-tumor cell. The tumor markers of culture supernatant of autologous-tumor cell were detected and compared with the serum tumor markers in the patients for identification of autologous-tumor cells.4. Determination of the immune phenotype of TIL The flow cytometry was used to detect the immune phenotype (CD3+, CD4+, CD8+, CD4+/CD8+) changes of TIL on0and14th day after the induction, activation and amplifying by IL-2.5. Cytotoxic effect of TILThe Methyl Thiazolyl Tetrazolium(MTT) was used to detect the cytotoxic effects of the TIL on0,3rd,7th and14th day after the induction, activation and amplifying on autologous-tumor cells, K562cells and Raji cells. And the changes of the cytotoxic effects according to the culture time were explored.6. Detection of autologous-tumor cell apoptosis induced by TILThe TIL (effector cell) was applied to treat the autologous tumor cells (target cells) for24hours in accordance with the proportion of50:1. Equivalent culture supernatant fluid was added in the blank group for the same time as control. The autologous-tumor cell apoptosis was detected using flow cytometry and Annexin V-FITC/PI double staining methods after the treatment.7. Detection of molecular markers of cell culture supernatantThe enzyme-linked immunosorbent assay (EIISA) was used to detect the changes of molecular markers in autologous-tumor cells before and after the treatment:(CA-125and CA-153, VEGF and MMP-9).8. Statistical processingExperiment results were showed as mean±standard deviation (x±s). SPSS13.0for Windows was used for data statistical analysis. Paired Samples T Test and two relevant Samples nonparametric tests (2Related Samples Test) were applied for the data. One-Way ANOVA Test was applied for multiple comparisons. P<0.05(on both sides) was considered to be statistically significant different. Results1. The immune phenotype changes of TILThe immune phenotype of CD3+, CD4+, CD8+on the14th day during the induction, activation and amplifying by IL-2were higher than these on the0day in different degree. CD3+was increased from48.2±14.5%to69.6±10.7%.CD8+was increased from21.7±11.9%to37.0±10.5%.CD4+was increased from20.4±16.7%to25.6±16.0%. The difference had statistical significance (P<0.05).CD4+/CD8+was decreased from1.5±1.7to0.8±0.8. The difference had no statistical significance.2. The cytotoxic effects of TIL on autologous-tumor cells, K562cells and Raji cellsThe cytotoxic effects of the TIL on0day on autologous tumor cells, K562cells and Raji cells were low. With the culture time of TIL cells increased, the cytotoxic effects of three kinds of cells increased to some extent, of which the cytotoxic effect on autologous-tumor cells increased the most obviously. The cytotoxic effects of the TIL on autologous-tumor cells was increased from5.0±1.8%(0day) to57.8±10.4%(14day). The difference had statistical significance(P<0.01).The cytotoxic effects of the TIL on14day after the induction, activation and amplifying on autologous-tumor cells, K562cells and Raji cells were57.8±10.4%、32.5±17.5%、27.1±11.1%。The cytotoxic effects of the TIL on autologous-tumor cells was higher than K562cells and Raji cells. The difference had statistical significance(P<0.01).3. TIL inducing autologous-tumor cells apoptosisAutologous-tumor cells were treated for14days TIL induced and activated by IL-2for24h. The Annexin V-FITC/PI double staining methods were used to detect cell apoptosis. The apoptosis rate of the treatment group was11.4±3.5%and the apoptosis rate of the control group was1.3±0.6%. The apoptosis rate of the treatment group was higher than the control group and the difference was statistically significant (P<0.05).4. The changes of tumor markers, VEGF and MMP-9After treatment of TIL, CA-125, CA-153, VEGF and MMP-9of culture supernatant for autologous-tumor cells decreased in different degree. CA-125was decreased from1051.4±583.8U/ml to240.2±195.7U/ml. CA-153was decreased from115.1±48.9pg/ml to93.1±50.9pg/ml. VEGF was decreased from1480.0±520.0pg/ml to697.6±273.7pg/ml. MMP-9was decreased from1732.1±560.8pg/ml to865.7±435.9pg/ml。 The differences of CA-125, VEGF and MMP-9were statistically significant(P<0.05), but the difference of CA-153was no significant difference.ConclusionsThe ovarian cancer TIL could be separated, induced, activated and cultured in vitro. It showed high efficiency of cytotoxic effect on malignant tumor cells after it was induced and activated by the IL-2. And also it could induce autologous-tumor cell apoptosis. After the treatment, the molecular markers of CA-125, VEGF, MMP-9were significantly decreased. That showed the good cytotoxic effect of TIL on ovarian cancer cells.