Abstract Objective To investigate the load autologous antigen DC induced CTLs against autologous breast cancer cell killing effect. Load autologous DCs in vitro tumor cell antigen-induced CTLs, ELISA was used to detect the expression of IFN  γ and IL  12 level, measured by LDH CTL autologous cancer cells in vitro. Results load higher than the self-antigens DC group IFN  γ and IL  12 concentration not sensitized the DC group antigen group and mononuclear cells in the control group (P <0.05), and antigen-loaded DC group stimulated CTLs killing role stronger than non-sensitized DC group, the antigen group monocytes group (P <0.01) and control MCF-7 group and the HT-29 group (P <0.01). Conclusions Tumor lysate pulsed DC sustainable stimulate specific CTLs autologous tumor cells in vitro anti-breast cancer patients, indicating that the tumor antigen pulsed DC vaccine for the treatment of residues and (or) may be chemotherapy-resistant breast cancer suitable, which is likely to become a standard remedies.

Key words breast cancer; dendritic cells; cytotoxic T cells

Cytotoxic Effects of CTLs Activated by DCs to Kill Autologous Breast Cancer Cells

    ZHANG Xi  mei1, ZHOU Fu  xiang1, 2, WU Jian  ping3, XU Yu1, XIE Cong  hua1, 2, CHEN Huan  chao3, ZHOU Yun  feng1, 2

    1.Department of Radio  Chemotherapy, Zhongnan Hospital of Wuhan University, Whuhan 430071, China, 2.Hubei Key Laboratory of Tumor Biological Behaviors; 3.Hubei Cancer Hospital

    Corresponding Author: ZHOU Fu  xiang, E  mail: happy zhoufx@sina.com; zhou Yun  fen, E  mail: yfzhouwhu@163.comAbstract: Objective To investigate T cell  mediated antitumor effects induced by DCs pulsed with tumor lysates in patients with breast cancer in vitro.Methods CTLs were activated by DCs pulsed with autologous breast cancer cell lysates.The concentration of IFN  γ and IL  12 were measured by ELISA.The specific cytolytic activity of CTLs was assessed by LDH assay.Results The results showed that IFN  γ and IL  12 were higher in the autologous breast cancer cell lysates pulsed DCs group than those in the unpulsed DCs group, PBMC group and Breast cancer lysate group (P <0.05). The CTLs stimulated by the autologous breast cancer cell lysates pulsed DCs had much higher cytotoxicity to autologous breast cancer cells, as compared with MCF  7 and HT  29 cells (P <0.01). Also higher than other three groups just like the level of IFN  γ and IL  12 (P <0.01). Conclusion The results indicated that tumor lysates  pulsed DCs could stimulate specific CTLs and could kill autologous tumor cells in patients with advanced breast cancer in vitro.Autologous tumor antigen  pulsed DC  based vaccinations may be appropriate for the treatment of residual and / or chemotherapy  resistant breast cancer, and may be as standard salvage treatment modalities.

    Key words: Breast cancer; Dendritic cell; CTLs

 Breast cancer is the most common tumor in women worldwide and the second bit of cancer death, 2003, only the United States alone 211,300 new cases [1]. Surgery, radiation therapy, and chemotherapy can improve the survival rate of patients, but the treatment of tumor cells to resist early diffusion metastasis, recurrence and mortality. Biological treatment is rapidly developing in recent years, another important treatment. The present study suggests that dendritic cells (DC) cell activation of cytotoxic T lymphocytes (CTLs)-mediated immune responses play an important role in the anti-tumor immune reaction [2]. In this study, the freeze-thaw method to obtain tumor cell antigen-pulsed autologous DC observed killing effect induced CTLs against autologous breast cancer cells, and to explore the potential therapeutic value of this pathway for breast cancer patients.

    1 Materials and methods

    1.1 Materials

    Control tumor cell lines our laboratory cultured breast cancer cell line MCF-7 and colon cancer cell line HT-29. rh GM-CSF, rh IL  4 rh of TNF  a purchased from Biological Engineering Co., Ltd. of Xiamen special treasure. Human IL  12, IFN  γELISA kit was purchased from Bio-Engineering Co., Ltd. of Shenzhen Jingmei. Ficoll  Hypaque lymphocyte separation medium was purchased from Shanghai Hanson. Acid dehydrogenase test kit was purchased from Nanjing Jiancheng Bioengineering Institute.

    1.2 Methods

    1.2.1 tumor antigens obtained

    8 cases of breast cancer patients are women, pathological type catheter invasive aged 35 to 68 years old, who had not received chemotherapy and radiotherapy before surgery. Whichever is the specimen after surgery, the digestion and processing into a single cell suspension, some cells continued to train, some of the cells to freeze-thaw prepared antigen.

    1.2.2 dendritic cells in culture

    The mononuclear cells (PBMC) was separated from the peripheral blood of patients with breast cancer, DC precursor cells cultured adherent seeded in 96-well cell culture plate, divided into three groups, a, b, c, wherein a, b group Add rhGM  CSF 800u/ml, rhIL  4 1000u/ml culture and TNF  α 10ng/ml, c group without any cytokines. To the freeze-thaw method to obtain tumor antigen 50μl/ml only three days in a group. 9th day of the culture, digestion, the three groups of cells were collected and counted.

    1.2.3 cytotoxic T lymphocytes (CTL) in vitro induced

    Ficoll  Hypaque density gradient centrifugation in peripheral blood of breast cancer patients of PBMC with the homemade nylon wool column separation for T lymphocytes, with complete medium to adjust cell concentration of 2 × 105/ml. Placed in 96-well culture plate, each hole 1ml. Divided into four groups: A, B, C, D first three groups were added to the collection of a, b, c cells (effector cells and stimulate cell ratio of 40:1), and the fourth group (D) by adding freeze-thaw antigen 50μL, groups of eight wells. Co-incubated for 4 days, and Determination of the killing activity of the part of the multiple holes. The remaining complex hole cells were cultured IFN  γ, IL  12 detection to six days.

    1.2.4 CTL s killing effect on breast cancer cells

    MCF  7, HT  29 cells and in patients with breast cancer cells were seeded in 96-well plates were divided into four groups, were added to the A, B, C, D group CTL (effector to target ratio of 40:1), each group three wells (8 patients repeat all experimental specimens were taken), also set up the same concentration of effector cells and target cells corresponding to the experimental group as the control in the incubator. 24h after centrifugation, the supernatant 100μL in 4ml Eppendorf tube, the cytotoxic activity is detected by lactate dehydrogenase release assay in accordance with the kit of the method shown.

    1.2.5 IFN  γ, IL  12 detection

    CTLs cells supernatant to take the above induced in vitro to six days every hole 100μL, Jingmei ELISA kit shown the determination of the content of IFN-γ and IL  12.

    1.2.6 Statistical Methods

    The statistical analysis of experimental data with a ± s, using SPSS13.0 statistical software. Groups were compared using the t-test, P <0.05 was considered statistically significant.

    2 Results

    2.1 cells by Factor of IFN-γ and IL  12 detection

    Breast tumor cell lysates DC lymphocytes cultured with cytokines IFN  γ and IL  12 content than the unsensitized DC group, simple breast cancer cell lysates group and the control group of monocytes ( P <0.05). Table 1 cytokine IFN  γ and IL  12 test results (pg / ml)

2.2 CTLs killing activity detected

    Cytotoxic activity of CTLs to target cells induced self-the body tumor antigen pulsed DC group than unsensitized the DC group monocytes group, CTL killing activity of pure antigen group (P <0.01), and the killing of autologous tumor cells The activity was also significantly better than the cytotoxic activity of autologous tumor cells (MCF  7, HT  29) (P <0.01), as shown in Table 2. Autologous tumor killing activity of CTLs induced by DC pulsed with specificity. Table 2 different groups CTLs cytotoxic activity of different target cells

3 Discussion

    The body’s anti-tumor immune response to the local cellular immune dendritic cells is an important anti-tumor immune immune cells, and its role in the antitumor increasing attention. Dendritic cells mainly in the peritumoral lymphocytic intensive areas, often co-exist with lymphocytes, but also scattered in cancer tissue and its processes in close contact with cancer cells. The distribution characteristics suggesting that dendritic cells its projection uptake of tumor-associated antigens and to identify tumor-specific antigens, and then pass this antigen to the T lymphocytes, so that infiltration of lymphocytes in the tumor next to the organization, thereby starting the body’s anti-tumor immune response. While freshly isolated breast tissue can provide the identification and distribution of the antigen peptide is poorly understood, recent studies prove beyond doubt that already exists a variety of tumor antigens can be recognized by CTL can be used as a target molecule to induce vitro autologous tumor cell killing [3]. So for cancer patients effective anti-tumor immune strategy might be to use the non-degradation of the tumor-derived antigen (such as the tumor cells, proteins or peptides isolated from the tumor cells) sensitized autologous DC. Is consistent with this view, the fusion DCS and breast cancer cells [4] or HER-2/neu [5] derived peptide sensitized autologous DCs in breast cancer patients are capable of activating the CTL response. Another DCs cell vaccine achieved some positive results of the clinical trials in the treatment of malignant melanoma [6], prostate cancer [7,8], malignant glioma and other tumors.

    And was positively correlated with the increase in CTL secrete IFN  γ to load tumor lysates DCs can be seen from our test results increased ability to secrete IL  12. This is mainly due to the IL  12 can affect the direction of the polarization of T cells, T cells increase the expression of IFN  γ, killing activity increased by secreting IFN  γ. These results suggest that IL  12, IFN  γ secretion reaction vitro induced body tumor antigen pulsed DC CTLs, may be a useful method. The present study, the CTLs induced by the antigen loading DCs to autologous good killing activity of breast cancer cells in vitro, and the unsensitized group DC CTLs and the CTLs induced by immature DC (mononuclear cell group) were significantly different, indicating that mature DC capable of presenting tumor antigen to stimulate the cytotoxic effect of CTLs. Poor killing activity rather than autologous tumor cells (MCF  7, HT  29) (P <0.01), and the specificity of this with a load of autologous tumor cell antigen CTLs cytotoxicity induced by DC. This specific killing activity showed that the stimulation of tumor lysate-pulsed DC vaccine CTLs immunotherapy can be used as a new treatment for patients with breast cancer, but the transition to clinical applications need to in vivo experiments confirmed.