After 7 days, the mice were sacrificed using abdominal arterial blood collection method to obtain the lymphocytes from the spleen. Immunohistochemistry Paraffin-embedded sections were prepared from tumor tissue samples that had been previously fixed in 4% buffered formalin. EpCAM peptides resulted in the efficient generation of mature DCs (mDCs), thus enhancing T cell stimulation and generating potent cytotoxic T lymphocytes (CTLs). The activation of CSC peptide-specific immune responses by the DC vaccine in combination with standard chemotherapy may provide better clinical outcomes in advanced carcinomas. Introduction Tumor cells express antigens that can be recognized by the immune system of their host. Cancer patients can be inoculated by these tumor-associated antigens (TAAs) to induce systemic immune responses that may result in the destruction of various cancers. This procedure is defined as active immunotherapy, or vaccination . Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that exist in the immune system [2, 3]. DC vaccines aim to stimulate cancer-specific effector T cells to eradicate tumor cells and to stimulate immunological memory to control cancer recurrence . Human DCs are commonly generated from monocytes that are isolated from peripheral blood mononuclear cells (PBMCs) and differentiated to produce immature DCs (iDCs). The iDCs then undergo maturation and an antigen-loading step to produce mature DCs (mDCs) . DCs have been pulsed/activated Rat monoclonal to CD4.The 4AM15 monoclonal reacts with the mouse CD4 molecule, a 55 kDa cell surface receptor. It is a member of the lg superfamily,primarily expressed on most thymocytes, a subset of T cells, and weakly on macrophages and dendritic cells. It acts as a coreceptor with the TCR during T cell activation and thymic differentiation by binding MHC classII and associating with the protein tyrosine kinase, lck with tumor lysates, recombinant proteins, or peptides, and peptide pulsing has been most widely investigated [6C10]. Studies have shown that peptide-pulsed DCs can present antigens to na?ve T lymphocytes, and in turn activate and induce T lymphocytes to become antigen-specific cytotoxic T lymphocytes (CTLs) that target tumor cells . Both the proliferative and cytolytic functions of tumor-specific CTLs require antigen recognition by the T cell receptor (TCR) in the context of major histocompatibility complex class one (MHC class I) molecules presented on APCs or target cells . Hepatocellular carcinoma (HCC) is a malignant disease that is often associated with a very poor prognosis . While considerable efforts have been made to improve HCC treatmentwhich mainly depends on surgical resection, liver transplantation KU-55933 and chemotherapythe HCC mortality rate remains high, largely due to cancer recurrence after surgery or intra-hepatic metastasis that develop through invasion of the portal vein or spread to other parts of the liver . Breast cancer ranks first among the causes of mortality among females aged between 20 and 59 years . In recent years, the encouraging trend towards earlier detection and the increased use of systemic adjuvant treatments have improved breast cancer survival rates; however, nearly half of all breast cancer patients treated for localized disease develop metastasis . Cancer stem-like cells (CSCs) typically represent a small fraction of tumor cells that can self-renew and differentiate into many more mature cancer cells . The failure of conventional cancer KU-55933 therapy may be due to the presence of residual CSCs that can survive in a dormant state for many years after remission and result in tumor relapse . In the present study, we investigated the effect of CSC peptides as antigen sources for DC vaccination against human breast cancer and HCC. Our results revealed that pulsing DCs with CD44 or EpCAM peptides enhanced T cell stimulation thus resulting in the induction of cell cytotoxicity. Furthermore, pulsing KU-55933 DCs with EpCAM peptides significantly suppressed tumor growth. The results of the present study suggest that the capacity of this vaccine to target CSCs could be exploited as a novel therapeutic strategy to inhibit tumor relapse. Materials and methods Cell culture conditions The human breast adenocarcinoma cell line MCF-7 and the human hepatoma cell line HepG2 were purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 100 U/ml penicillin/streptomycin (Gibco, Carlsbad, CA, USA) in a humidified atmosphere with 5% CO2 at 37C. Flow cytometry and cell sorting Cells were trypsinized and suspended in phosphate-buffered saline (PBS) containing 2% FBS at a density of 1108 cells/ml. For flow cytometry, the MCF-7 cells were incubated with anti-CD24-FITC and anti-CD44-APC monoclonal antibodies (mAbs) (BD Biosciences, Bedford, MA, USA), and the HepG2 cells were incubated with the anti-EpCAM-PerCP-Cy5.5 mAb (BD Biosciences, San Jose, CA, USA) on ice for 60 min. FITC mouse anti-IgG2a, APC mouse anti-IgG2b and PerCP/Cy5.5 anti-mouse IgG1 (BD.