The Study Of DC/tumor Fusion Vaccine Against Breast Cancer Bone Metastasis

Bone, is one of the most preferential metastatic target sites for cancers including breast, prostate, lung, colon, stomach, bladder, uterus, rectum, thyroid, or kidney cancers. The consequences of bone metastasis are often devastating. It can cause severe pain, pathologic fractures, life threatening hypercalcemia, spinal cord compression, and other nerve compression syndrome which severely affect the life quality and surviving time of patients. Breast cancer is one of most common malignant tumor in female and up to 70% of breast cancer will develop bone metastasis which takes 70% of distant organ metastasis. So, breast cancer bone metastasis is frequent and serious. However, once bone disease has developed, current therapies are rarely curative. Palliative radiotherapy and surgery are occasionally used to reduce the size of tumor deposits in bone. Chemotherapy is usually not successful, due to drug resistance that commonly develops during progression of the disease. So, novel strategies that specifically prevent or reduce metastasis of breast cancer to bone are urgently required. Cancer biotherapy is recognized as the fourth therapeutic approach except for the conventional treatment: surgery, chemotherapy and radiotherapy and it offers a promising concept against cancer. Dendritic cells (DCs) as the most potent professional antigen presenting cells is the focus of caner biotherapy research and play extremely important role in the anti-tumor immune responses. DC-based treatment consists of two major strategies. The first is DC loaded with defined TAAs (Tumor Associated Antigen) including pulsing DCs with synthetic or eluted TAA peptides, whole protein, or transfecting DC with nucleic acid (RNA or DNA) and this strategy can induce specific CTL (cytotoxic T lymphocyte) responses against the defined TAA. However, drawbacks come from few identified TAAs, heterogeneity of tumor antigen and high polymorphism of human MHC system which restrict the clinical use. The second strategy focuses on loading DC with whole tumor cell products, such as apoptotic bodies, tumor cell lysates, peptides eluted from MHCs, tumor total RNA and DC/tumor fusion [7]. This strategy targets multiple TAAs, including those yet unidentified and can result in polyclone CTL responses without the requirement to identify and characterize the specific antigens which dramatically improve the anti-tumor efficiency and broaden the clinical use. The DC/tumor fusion vaccine possesses distinct advantages. Fusion process is direct and easy and more importantly, through this approach, multiple TAAs, are endogenously processed and presented by MHC Class I pathway and eventually activate the CD8+ CTL which is the major effector cells in the anti-tumor immune response. In addition, MHC Class II pathway is also involved to active the CD4+ T cells which help the effector cells. Compared with fusion strategy, loading DC with apoptotic tumor bodies, tumor lysates or eluted peptides mainly active the CD4+ T cells through the MHC Class II pathways, even the DC transfected with tumor total RNA can present antigen through MHC Class I pathway, however, it depends on RNA amplification and effective DC transfection which requires complicate process and higher technique.In view of the considerable advantages of fusion vaccine, we used fusion strategy to construct the anti-tumor vaccine. In the fusion strategy, autologous tumor cells are preferred to be used because these cells contain both known and unknown TAAs that are best matched to the tumor bearing host. However, the tumor samples from which the autologous tumor cells originate are not available for most patients with breast cancer bone metastasis, because the primary tumor lesion has been resected for most patients and the preferred treatment for the bone metastasis lesion is usually conservation not surgery. Even the tumor lesion is obtained, it is impossible to get sufficient viable tumor cells due the length of culture time and potential contamination of bacteria and fungus.In order to resolve this problem and use fusion strategy in breast cancer bone metastasis, for the first time, we fuse DC with allogeneic tumor cell line MCF7 as a substitute of autologous breast tumor cells to deliver TAAs to DC and we further investigate the immunogenicity of this allogeneic fusion vaccine which may provide experiment basis for the DCs/allogeneic tumor cells fusion vaccination protocol.AimsIn order to use the fusion strategy in patients with breast cancer bone metastasis and provide experiment basis for the DCs/allogeneic tumor cells fusion vaccination protocol, DC was fused with allogeneic MCF7 and its anti-tumor immunogenicity was investigated.MethodsDCs were induced by GM-CSF and IL4 from PBMC. DC/MCF7 fusion cells were generated by electrofusion and laser confocal microscope and flow cytometry were used to analyze the fusion efficiency and identify the truly fusion cells. Then hybrid cells were used to stimulate the T cells responses. After 7 days'stimulation, Elispot was used to determine IFNγproduction assay and LDH release was used to perform CTL assay against autologous breast cancer cells and other targets.ResultsAfter 7 days'culture, DCs possess characteristic morphology and phenotype. The electrofusion efficiency was around 45% detected by the flow cytometry and double positive truly fusion cells were identified under laser confocal microscope. After stimulation of DC/MCF7 fusion cells, T cells presented large proliferation clusters, T cells can secret high level IFNγupon the stimulation of tumor cells. The CTL assay demonstrated DC/MCF7 fusion cells were able to induce CTLs responses against autologous breast cancer cells and other tumor targets and the responses was tumor antigen specific in HLA restriction manner. Immunogenicity of DC/MCF7 fusion vaccine was comparable with that of DC/autologous breast cancer fusion vaccine.ConclusionsOur research reach the conclusion that allogeneic tumor cell line MCF7 can be used as substitute of autologous breast tumor cells to deliver TAAs to DC when preparing the fusion vaccine and the immunogenicity of DC/MCF7 fusion vaccine was comparable with that of DC/autologous breast cancer fusion vaccine. This finding provided evidence and foundation for the use of DC allogeneic tumor fusion vaccine in the breast cancer bone metastasis and explore new vaccination protocol for the use of fusion vaccine.