Evaluation of anti-tumor effect of a novel manganese adjuvant collaborated with tumor antigens / 中国生物制品学杂志

@#ObjectiveTo investigate the anti-tumor effect of agonist MnCl_2of a novel cyclic guanosine monophosphate-adenosine monophosphate synthase(c GAS)/stimulator of interferon genes(STING)pathway collaborated with tumor cell lysate(Lysate)and the neo-antigen 10K-Adpgk of mouse colon cancer MC38 cell line.MethodsBone marrow-derived dendritic cells(BMDCs)were extracted from mouse bone marrow and divided into three groups:PBS,1 μmol/L MnCl_2and 10 μmol/L MnCl_2,which were analyzed for the maturation by flow cytometry,determined for the concentration of IL-6 in supernatant by ELISA,and detected for the transcription levels of IL-6,IFN-α,IFN β and CXCL9 genes by q PCR.Mouse tumor model was established by using MC38 cell line.When the tumor volume reached 100 mm3,the mice were randomly divided into two groups for administration,PBS,Lysate,MnCl_2,10K-Adpgk,Lysate + MnCl_2group and Lysate +10K-Adpgk + MnCl_2combined treatment group,which were administered subcutaneously through the tail for 3 times,with each interval of 1 week,and measured for the tumor volume every 2 days.One week after the last dose,serum samples were collected and determined for the concentrations of IFNγ and TNFα by ELISA.The tumor and spleen were isolated.The proportions of tumor infiltrating T cells and T cells in peripheral blood mononuclear cells(PBMCs)and the ratio of T cells to memory T cells in spleen were detected by flow cytometry,and the proportion of antigen specific T cells in spleen was detected by ELISPOT.Results10 μmol/L MnCl_2stimulated the maturation of BMDCs and activated the subsequent immune process.The tumor volumes of mice in the combined treatment group were considerably smaller than those in PBS group,the contents of IFNγ and TNF-α in serum were higher than those in other groups,and the proportions of tumor infiltrating T cells,T cells in PBMCs and ratio of T cells to memory T cells in spleen were also significantly higher than those in PBS group.Combined therapy caused strong antigen-specific T cell immune response.ConclusionThe addition of the novel adjuvant MnCl_2significantly enhanced the treatment effect of tumor cell lysate and neo-antigen,which provided an experimental basis for the development of the combination tumor treatment method based on MnCl_2and tumor antigens.

Immune Mechanism of Tumor Cell Lysate Combined with IL-2 Preventing Melanoma and Inhibiting Tumor Growth / 肿瘤防治研究

Objective To investigate the preventive and inhibitory effects of tumor cell lysate(TCL) combined with IL-2 on melanoma and the potential immune mechanism. Methods The B16F10 melanoma TCL cell were prepared using an ultrasonic disruptor. Twenty-four C57BL/6 mice were randomly divided into four groups which were immunized with PBS, IL-2, TCL and TCL+IL-2 for three weeks, and contra lateral tumors were implanted in the fourth week. We observed onset time of tumor and tumor size, collected peripheral blood continuously and monitored the expression of CD4+T and CD8+T cell subsets dynamically by flow cytometry. Spleen and tumor tissues of mice were also tested for CD4+T and CD8+T cell subsets by flow cytometry and immunohistochemistry, respectively. Results The preventive immunization of the TCL+IL-2 group significantly delayed the onset time of tumor (P=0.034); moreover, the tumor volume (P=0.023) and tumor weight (P=0.0015) were also significantly smaller than those in the control group. The expression of CD8+T cell subsets in the TCL+IL-2 group and the TCL-only group were significantly higher than that in the control group (P=0.0016, P=0.012). However, the CD4+T cell subsets of the TCL+IL-2 group decreased after tumor implantation, compared with the control group (P=0.0089). The expression of CD4+T and CD8+T cell subsets in spleen and tumor tissues were as same as those in peripheral blood. Conclusion The tumor vaccine of TCL combined with IL-2 could prevent the occurrence of melanoma in mouse and effectively inhibit tumor growth by activating CD8+T cells.

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.

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.

Specific T-cells induced by human dendritic cells pulsed with tumor cell lysate against pancreatic carcinoma cells in vitro / 中国普通外科杂志

Objective To observe the effect of a specific T-cell induced by lysate-pulsed human dendrtic cells against pancreatic cancer(PC) cells . Methods Dendritic cells were amplified and purified from peripheral blood of PC patient and were pulsed with tumor cells.Then they were co-cultured with autologus T-cell in vitro and divided into lysate-pulsed DCs group, unplused DCs group, lysates group,and control group. The supernatants of every co-culture group were collected and the concentration of IL-12 and IFN-? were measured by ELISA. The capacity of DC induced proliferation of T-cells was tested.The specific cytolytic activity of CTL was assessed in a 4-hr 51 Cr-release assay. Results Difference in the concentration of IL-12 and IFN-? was statistically significant in lysate-pulsed DCs group(1161?239 pg/ml and 1044?312 pg/ml)than these in unplused DCs group and lysates group (P