Korean J Urol.  2010 Feb;51(2):139-144.

The Establishment of Dendritic Cell-Tumor Fusion Vaccines for Hormone Refractory Prostate Cancer Cell

• Kim TB
• Park HK
• Chang JH
• Choi IH
• Kim KH
• Yoon SJ
• Lee MS
• Jung H
• Kim CS

Abstract

PURPOSE: Dendritic cell (DC)-based tumor vaccine is an attractive modality for the treatment of hormone-refractory prostate cancer (HRPC) because it has some efficacy and few side effects in patients with poor general conditions. The aim of this study was to establish which is the most effective DC vaccine for the treatment of HRPC. We compared DC vaccine sensitized with tumor lysate and a fusion vaccine of DCs and tumor cells.
MATERIALS AND METHODS
The DU145 cancer cell line was purchased from the American Type Culture Collection. DCs were cultured from peripheral blood monocytes. Peripheral blood monocytes were cultured in RPMI 1640 medium supplemented with interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor, and 10% fetal calf serum. Tumor necrosis factor-alpha was added on day 7 to support maturation. Functional activity was measured in three groups: the DC single-culture group, the DC culture group with DC vaccine sensitized with tumor lysates, and the DC culture group prepared with tumor fusion vaccine made from irradiated tumor cells and monocyte-derived DCs by the polyethylene glycol method.
RESULTS
By FACS analysis, the rate of DC-tumor fusion vaccine was 20.3+/-3%. The IL-12 level produced by the DC-tumor fusion vaccine was significantly higher than that of DCs pulsed with tumor lysate (p<0.05). Also, the generation of interferon-gamma by tumor-specific T cells in the DC-tumor fusion vaccine group was superior to that of DCs pulsed with tumor lysate (p<0.05). In addition, the T cells of the tumor lysate-pulsed DCs and tumor fusion vaccine had 1.6 and 2.5 times the functional activity, respectively, of the DC single-culture group in killing tumor cells in the cytotoxicity assay.
CONCLUSIONS
The DC-tumor fusion vaccine seems to be more effective than DC single-culture or DC-tumor lysate vaccine in the treatment of HRPC.

Keyword

Prostatic neoplasms; Dendritic cells; Cancer vaccines

MeSH Terms

Cancer Vaccines
Cell Line
Dendritic Cells
Granulocyte-Macrophage Colony-Stimulating Factor
Homicide
Humans
Interferon-gamma
Interleukin-12
Interleukin-4
Monocytes
Polyethylene Glycols
Prostate
Prostatic Neoplasms
T-Lymphocytes
Tumor Necrosis Factor-alpha
Vaccines
Cancer Vaccines
Granulocyte-Macrophage Colony-Stimulating Factor
Interferon-gamma
Interleukin-12
Interleukin-4
Polyethylene Glycols
Tumor Necrosis Factor-alpha
Vaccines

Figure

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  FIG. 1 Photograph of dendritic cells on day 7. Aggregates of dendritic cells developed from adherent peripheral blood monocytes under the influence of GM-CSF and IL-4 (×1,200, PENTAX A10, Pentax Corporation, Japan, OLYMPUS CK2, Olympus Optical Co., Ltd. Japan). GM-CSF: granulocyte-macrophage colony-stimulating factor, IL-4: interleukin-4.

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  FIG. 2 Phenotypic characterization of immature and mature DCs by flow cytometry. (A) Phenotypes of immature DCs. (B) Phenotypes of mature DCs. Immature dendritic cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy donors by culturing with IL-4 and GM-CSF. Differentiation into mature DCs was achieved by the addition of inflammatory cytokine cocktail for 24 hours. Mature DCs have a higher CD83 and CD86 count than do immature DCs. DCs: dendritic cells, IL-4: interleukin-4, GM-CSF: granulocyte-macrophage colony-stimulating factor.

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  FIG. 3 Efficacy of PEG hybridization of dendritic cells and the DU145 tumor cell line. Before hybridization, tumor cells were stained with PKH26PE and DCs were stained with monoclonal antibody against CD86FITC. FACS analysis showed that 20.3% of DC-tumor hybrids were obtained (circled area). Tumor cells were irradiated at 150 Gy and pooled with mature DCs at a 1:2 ratio. DC: dendritic cell.

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  FIG. 4 Mixed lymphocyte reaction of tumor lysate-pulsed DCs and DC-tumor hybrid. Cells were co-cultured with 105 allogenic T cells/well in 96-well plates for 5 days. For 15-20 hours before harvesting, 1 uCi of H3-thymidine was added to each well. Cells were harvested and incorporated BrdU was measured colorimetrically (a: p<0.05). DC: dendritic cell.

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  FIG. 5 Dendritic cell function estimated by IL-12 assay. Supernatants from tumor lysate-pulsed DCs and DC-tumor hybrid were analyzed for production of IL-12 by ELISA (a: p<0.05). DC: dendritic cell, IL-12: interleukin-12.

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  FIG. 6 Generation of IFN-γ-producing tumor-specific T cells. T cells re-stimulated by co-culture with tumor lysate-pulsed DCs and DC-tumor hybrid were analyzed for production of IFN-γ by ELISA (a: p<0.05). IFN-γ: interferon-gamma, DC: dendritic cell.

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  FIG. 7 Generation of cytotoxicity of tumor lysate-pulsed DCs and DC-tumor hybrid. T cells stimulated by DC cells pulsed with DU145 tumor lysate and DC/DU145 hybrid cells were cocultured with DU145 cells in a 96-well plate for 24 hours at 37℃. The CytoTox-ONE assay measured the release of lactate dehydrogenase (LDH) from cells with a damaged membrane (a: p<0.05). DC: dendritic cell.

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