Dendritic Cell (DC) Vaccine in Mouse Lung Cancer Minimal Residual Model; Comparison of Monocyte-derived DC vs. Hematopoietic Stem Cell Derived-DC

The anti-tumor effect of monocyte-derived DC (MoDC) vaccine was studied in lung cancer model with feasible but weak Ag-specific immune response and incomplete blocking of tumor growth. To overcome this limitation, the hematopoietic stem cell-derived DC (SDC) was cultured and the anti-tumor effect of MoDC & SDC was compared in mouse lung cancer minimal residual model (MRD). Therapeutic DCs were cultured from either CD34+ hematopoietic stem cells with GM-CSF, SCF and IL-4 for 14 days (SDC) or monocytes with GM-CSF and IL-4 for 7 days (MoDC). DCs were injected twice by one week interval into the peritoneum of mice that are inoculated with Lewis Lung Carcinoma cells (LLC) one day before the DC injection. Anti-tumor responses and the immune modulation were observed 3 weeks after the final DC injection. CD11c expression, IL-12 and TGF-beta secretion were higher in SDC but CCR7 expression, IFN-gamma and IL-10 secretion were higher in MoDC. The proportion of CD11c+CD8a+ cells was similar in both DC cultures. Although both DC reduced the tumor burden, histological anti-tumor effect and the frequencies of IFN-gamma secreting CD8+ T cells were higher in SDC treated group than in MoDC. Conclusively, although both MoDC and SDC can induce the anti-tumor immunity, SDC may be better module as anti-tumor vaccine than MoDC in mouse lung cancer.

Generation and Qualification of Functionally Active Leukemia-derived DCs from Malignant Blasts in Acute Leukemia / 대한혈액학회지

BACKGROUND: Dendritic cells (DCs) are increasingly being utilized for anti-cancer immunotherapy. Acute myeloid leukemia (AML) blasts are able to generate leukemia-derived DC. Advances in culture techniques and AML-DC characterization justify possible clinical applications. We investigated the ability of AML, acute lymphoblastic leukemia (ALL) and biphenotypic acute leukemia (BAL) blasts to differentiate into DCs in vitro and the qualified function of the leukemia-derived DCs. METHODS: Leukemia cells from 11 patients with AML, 3 patients with ALL and 2 patients with BAL were cultured with GM-CSF, IL-4 and with or without SCF. Cultured leukemia cells were evaluated by phenotype, mixed lymphocyte reaction (MLR), cytokine production and cytotoxic T cell (CTL) inducing activity. RESULTS: DCs were generated with GM-CSF and IL-4 from the leukemic blasts in 72% of the AML patient cells. MHC class I/II, CD11c and ICAM-1 were highly expressed in the AML-derived DCs. MLR and enzyme linked immunospot (ELISPOT) assays demonstrated that AML-DCs were able to induce T cell proliferation and activation into IFN-gamma secreting effector cells. The ALL blasts from two out of three patients differentiated into DCs with MHC class I/II+, CD11c+ only in the presence of GM-CSF, SCF and IL-4 for 14 days. CONCLUSION: These results suggest that functionallyactive DCs can be differentiated from AML blasts using GM-GSF and IL-4 and ALL, BAL blasts were differentiated into DCs only under stem cell-DC culture conditions.

Mechanism of Differential Ag-specific Immune Induction by Different Tumor Cell Lysate Pulsed DC

BACKGROUND: Tumor cell lysate has been considered as a preferential antigen source for the therapeutic dendritic cell pulsing. Our experiences with in vivo study with animal tumor model indicate the tumor cell lysate dependent differential effect of DC therapy. Our previous data show that MC38 lysate pulsed-DC induced stronger ag-specific immunity than CT26 lysate pulsed-DC in vitro. In this study we tried to reveal the mechanism for differential induction of ag-specific immunity of different colon cancer cell lysate pulsed-DCs. METHODS: MC38 and CT26 cell lines were prepared as lysate by freezing-thawing procedure. Tumor cell antigenicity was confirmed by detecting the surface expression of MHC I/II & B7.1/2 molecules. IL-10, IL-12 and TGF-beta in the tumor cell lysate were detected by ELISA and the presence of heat shock proteins were analysed by western blotting. RESULTS: The secretion of IL-10, a immune-inhibitory cytokine was about 470% higher in CT26 lysate than in MC38. Hsp 70 was detected only in the MC38 lysate but not in the CT26. On the other hand, Hsp 60 and 90 expression were not different in two colon cancer cell lysates. CONCLUSION: In two different colon caner cell lysate, immune inhibitory IL-10 (higher in CT26) and Hsp70 (MC38 superiority) were differentially expressed. These data indicate that higher ag- specific immunity induction by MC38 lysate pulsed-DC may due to the expression of hsp70 and lower secretion of IL-10, a immune-inhibitory cytokine than CT26 lysate. The significance of other cytokine and the surface marker expression will be discussed.

Anti-cancer Effect of Hematopoietic Stem Cell-derived Allogeneic-DC Vaccine in Melanoma Metastasis Model

BACKGROUND: Dendritic cell (DC)-based cancer immunotherapy is studied for several years. However, it is mainly derived from autologous PBMC or leukapheresis from patient, which has limitations about yield and ability of DC production according to individual status. In order to solve these problems, inquiries about allogeneic DCs are performed but there are no preclinical trial answers for effect or toxicity of allogeneic DC to use for clinical trial. In this study, we compared the anti-tumor effect of allogeneic and autologous DCs from mouse bone marrow stem cells in mouse metastatic melanoma model. METHODS: B16F10 melanoma cells (5 x 10(4)/mouse) were injected intravenously into the C57BL/6 mouse. Therapeutic DCs were differentiated from autologous (C57BL/6: CDC) or allogeneic (B6C3F1: BDC) bone marrow stem cells with GM-CSF, SCF and IL-4 for 13days and pulsed with B16F10 tumor cell lysate (Blys) for 18hrs. DC intra-peritoneal injections began on the 8th day after the tumor cell injection by twice with one week interval. RESULTS: Anti-tumor response was observed by DC treatment without any toxicity especially in allogeneic DC treated mice (tumor burden score: 2.667+/-0.184, 2.500+/-0.463, 2.000+/-0.286, 1.500+/-0.286, 1.667+/-0.297 for saline, CDC/unpulsed-DC: U-DC, CDC/Blys-DC, BDC/U-DC and BDC/Blys-DC, respectively). IFN-gamma secretion was significantly increased in allogeneic DC group stimulated with B16F10 cell lysate (2,643.3+/-5,89.7, 8,561.5+/-2,204.9. 6,901.2+/-141.1 pg/1 x 10(6) cells for saline, BDC/U-DC and BDC/Blys-DC, respectively) with increased NK cell activity. CONCLUSION: Conclusively, promising data was obtained that allogeneic DC can be used for DC-based cancer immunotherapy.

Immunocell Therapy for Lung Cancer: Dendritic Cell Based Adjuvant Therapy in Mouse Lung Cancer Model

BACKGROUND: The anti-tumor therapeutic effect of autologous tumor cell lysate pulsed-dendritic cells (DCs) was studied for non-immunogenic and immune suppressive lung cancer model. To test the possibility as an adjuvant therapy, minimal residual disease model was considered in mouse in vivo experiments. METHODS: Syngeneic 3LL lung cancer cells were inoculated intravenously into the C57BL/6 mouse. Autologous tumor cell (3LL) or allogeneic leukemia cell (WEHI-3) lysate pulsed-DCs were injected twice in two weeks. Intraperitoneal DC injection was started one day (MRD model) after tumor cell inoculation. Two weeks after the final DC injection, tumor formation in the lung and the tumor-specific systemic immunity were observed. Tumor-specific lymphocyte proliferation and the IFN-r secretion were analyzed for the immune monitoring. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 for 7 days and pulsed with tumor cell lysate for 18 hrs. RESULTS: Compared to the saline treated group, tumor formation was suppressed in 3LL tumor cell lysate pulsed-DC treated group, while 3LL-specific immune stimulation was minimum. WEHI-3-specific immune stimulation occurred in WEHI-3 lysate-pulsed DC treated group, which had no correlation with tumor regression. CONCLUSION: The data suggest the possible anti-tumor effect of cultured DCs as an adjuvant therapy for minimal residual disease state of lung cancer. The significance of immune modulation in DC therapy including the possible involvement of NK cell as well as antigen-specific cytotoxic T cell activity induction was discussed.

Effect of Dendritic Cell Based Cancer Vaccine Using Allogeneic Tumor Cell Lysate in Melanoma Pulmonary Metastasis Model

BACKGROUND: To perform the successful dendritic cell-based cancer immunotherapy one of the main issues to be solved is the source of antigen for DC pulsing. Limitations occur by using auto-tumor lysate due to the difficulties obtaining enough tumor tissue(s) quantitatively as well as qualitatively. In this study the possibility of allogeneic tumor cell lysate as a DC pulsing antigen has been tested in mouse melanoma pulmonary metastasis model. METHODS: B16F10 melanoma cells (1x10(5)/mouse) were inoculated intravenously into the C57BL/6 mouse. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 (1,000 U/ml each) for 7 days and pulsed with lysate of either autologous B16F10 (B-DC), allogeneic K1735 (C3H/He origin; K-DC) or CloneM3 (DBA2 origin; C-DC) melanoma cells for 18 hrs. Pulsed-DCs (1x10(6)/mouse)[CGP1] were injected i.p. twice with one week interval starting from the day 1 after tumor cell inoculation. RESULTS: Without observable toxicity, allogeneic tumor cell lysate pulsed-DC induced the significantly better anti-tumor response (tumor scale: 2.7+/-0.3, 0.7+/-0.3 and 0.3+/-0.2 for saline, B-DC and C-DC treated group, respectively). Along with increased tumor specific lymphocyte proliferations, induction of IFN-gamma secretion against both auto- and allo-tumor cell lysates was observed from the DC treated mice. (w/B16F10-lysate: 44.97+/-10.31, 1787.94+/-131.18, 1257.15+/-48.27, w/CloneM3 lysate: 0, 1591.13+/-1.83, 1460.47+/-86.05 pg/ml for saline, B-DC and C-DC treated group, respectively) Natural killer cell activity was also increased in the mice treated with tumor cell lysate pulsed-DC (8.9+/-[CGP2]0.1, 11.6+/-0.8 and 12.6+/-0.7% specific NK activity for saline, B-DC and C-DC treated group, respectively). CONCLUSION: Conclusively, promising data were obtained that allogeneic-tumor cell lysate can be used as a tumor antigen for DC-based cancer immunotherapy.

Dendritic Cell Based Cancer Immunotherapy: in vivo Study with Mouse Renal Cell Carcinoma Model

BACKGROUND: As a potent antigen presenting cell and a powerful inducer of antigen specific immunity, dendritic cells (DCs) are being considered as a promising anti-tumor therapeutic module. The expected therapeutic effect of DCs in renal cell carcinoma was tested in the mouse model. Established late-stage tumor therapeutic (E-T) and minimal residual disease (MRD) model was considered in the in vivo experiments. METHODS: Syngeneic renal cell carcinoma cells (RENCA) were inoculated either subcutaneously (E-T) or intravenously (MRD) into the Balb/c mouse. Tumor cell lysate pulsed-DCs were injected twice in two weeks. Intraperitoneal DC injection was started 3 week (E-T model) or one day (MRD model) after tumor cell inoculation. Two weeks after the final DC injection, the tumor growth and the systemic immunity were observed. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 for 7 days and pulsed with RENCA cell lysate for 18 hrs. RESULTS: Compared to the saline treated group, tumor growth (E-T model) or formation (MRD model) was suppressed in pulsed-DC treated group. RENCA specific lymphocyte proliferation was observed in the RENCA tumor-bearing mice treated with pulsed-DCs. Primary cytotoxic T cell activity against RENCA cells was increased in pulsed-DC treated group. CONCLUSION: The data suggest the possible anti-tumor effect of cultured DCs in established or minimal residual disease/metastasis state of renal cell carcinoma. Systemic tumor specific immunity including cytotoxic T cell activity was modulated also in pulsed-DC treated group.

Production of IFN-gamma by TNF-alpha in Macrophages from Tumor Micro Environment; Significance in Angiogenic Switch Control

BACKGROUND: The role of macrophages in tumor angiogenesis is known to be the production of angiogenic cytokines and growth factors including TNF-alpha. Recently, macrophage also can produce the INF-gamma that is being studied to be involved in angiogenic inhibition. Thus, the importance of macrophages in tumor angiogenesis is might being an angiogenic switch. Thus, the hypothesis tested here is that TNF-alpha can modulate the INF-gamma production in the macrophages from tumor environment as a part of tumor angiogenic switch. METHODS: Macrophages in tumor environment were obtained from the peritoneal cavity of C57BL/6 mice injected with B16F10 melanoma cell line for 6 or 11 days. Mac1(+) -macrophages were purified using magnetic bead (MACs(TM); Milteny Biotech, Germany) and cultured with various concentrations of TNF-alpha for various time points at 37degreeC. The supernatants were analyzed for IFN-gamma or VEGF by ELISA kit (Endogen, Woburn, MA). RESULTS: Residential macrophages from the peritoneal cavity did not respond to LPS or TNF-alpha to produce INF-gamma. However, the cells from tumor environment produced IFN-gamma as well as VEGF and upregulated by the addition of LPS or TNF-alpha. RT-PCR analysis revealed the external TNF-alpha-induced IFN-gamma gene expression in the macrophages from tumor environment. CONCLUSION: The overall data suggest that the macrophages in tumor environment might have an important role not only in angiogenic signal but also in anti-angiogenic signal by producing related cytokines. And TNF-alpha might be a key cytokine in tumor angiogenic switch.