| Immunotherapy is a potentially promising approach for the treatment ofhuman tumors, especially for aggressive and metastatic cancers. Dendritic cells(DCs), initiators of antigen-specific immune responses, can efficiently uptake,process, and present antigens. DCs play a key role in the initiation andregulation of immune responses by significantly activating naive T cells. MatureDCs highly express major histocompatibility complex I and II (MHCI I andMHC II) molecules and present tumor antigens to T cells to activate CD4+Tcells and CD8+T cells. Therefore, successfully activating DCs in the host toefficiently present tumor antigens is of critical importance for the treatment ofcancers. The DC-based cancer vaccine is an extremely promising anti-tumorstrategy. However, clinical applications of DC-based cancer vaccines areusually limited due to the high cost of culture DCs in vitro, complex techniques,degradation of DC-based cancer vaccines in vivo, and the weak antigenicity.Thus, improving the anti-tumor activity of DC-based cancer vaccines iscritically important for the treatment of cancers. In this study, mannose (M)/CpG oligodeoxynucleotide (CpG-ODN)-conjugated liposome (Lipo)loaded with tumor-associated antigens were used in the immunotherapy oftumor-bearing mice. Our results demonstrated that M/CpG-ODN-conjugatedLipo loaded with tumor-associated antigens specifically induced the activationand maturation of DCs in vivo and the activated DCs stimulated effector cells tokill tumor cells in mice. The detailed investigations and results were shownbelow.1. The effects and mechanisms of M/CpG-ODN-conjugated liposome loadedwith hepatoma H22cell lysates (M/CpG-ODN-H22-Lipo) in the treatment ofhepatocellular carcinoma (HCC).Background: Immunotherapy has been an important approach for the treatmentof HCC by improving the cure rate and prolonging the survival of HCC patients.DC-based cancer vaccine has become one of the research hotspot inimmunotherapy. Developing a novel approaches to efficiently improve theactivity of DC-based cancer vaccines in vivo is of great significance.Objective: Preparation of the M/CpG-ODN-H22-Lipo and investigation of theanti-tumor effects of M/CpG-ODN-H22-Lipo in mouse HCC model and theunderlying mechanisms.Methods: Mannose glycolipids (mannoselipid) generated by dehydrationcondensation reaction between palmitic acid and mannosamine were used toproduce mannose-conjugated liposome (M-Lipo). CpG-ODN was conjugated toM-Lipo by Post-insertion method to prepare the M/CpG-ODN-Lipo. Then, thehepatoma H22cell lysates were encapsulated into M/CpG-ODN-Lipo to obtainDC-based cancer vaccine (M/CpG-ODN-H22-Lipo). Fourier transform infraredspectroscopy (FTIR) and X-ray diffraction (XRD) were used to evaluate theproduction of mannoselipid. Dynamic light scattering instrument (DLS) andtransmission electron microscope (TEM) were applied to characterize the particle size, morphology, and zeta potential of M/CpG-ODN-H22-Lipo. Thephagocytosis experiment was conducted to confirm the specifically targeting toDCs of M-Lipo. The stability of M/CpG-ODN-H22-Lipo in serum and the effectof activation DCs by M/CpG-ODN-H22-Lipo were evaluated using flowcytometry. HCC model was established in BALB/c mice and subcutaneousinjection of M/CpG-ODN-H22-Lipo was applied to treat HCC in mice. In vivooptical imaging was used to track the distribution and metabolism ofnanoparticles in tumor-bearing mice. To evaluate the anti-tumor effects ofM/CpG-ODN-H22-Lipo in HCC mice, flow cytometry was used to measure thenumber of myeloid-derived suppressor cells (MDSCs) and T-regulatory cells(Tregs). Enzyme-linked immunospot assay (ELISPOT) and enzyme-linkedimmunosorbent assay (ELISA) were used to measure the levels of interferon-gamma (IFN-γ) and IgG, respectively. In addition, local tumor vascular density,proliferation, and apoptosis of tumor cells were evaluated using immunologicalhistological chemistry (IHC). The mice were monitored daily for survival andtumor volume was calculated according to the formula. Finally, to furtherinvestigate the anti-tumor effects of M/CpG-ODN-H22-Lipo, HCC-bearingmice were treated and monitored daily for survival after depletion the CD8+Tcells and nature killer (NK) cells respectively.Results: The diameter of M/CpG-ODN-H22-Lipo was around130nm withround surface and negative charge, and the encapsulation efficiency ofM/CpG-ODN-H22-Lipo was52.9%. This DC-based cancer vaccine keptstability in PBS containing50%serum and activated DCs efficiently. M/CpG-ODN-H22-Lipo significantly inhibited the growth of tumor and prolonged thesurvival of mice in HCC-bearing mice, decreased the MDSCs in tumor tissuesand the bone marrow, and the Tregs in the spleen. The significant increase in thenumber of IFN-γ-positive cells in spleen, and the effect of anti-angiogenesis, inhibition of tumor cell proliferation, and induction of apoptosis in HCC-bearingmice were obsvered. In addition, it was found that the IgG level in HCC miceincreased with the application of M/CpG-ODN-H22-Lipo. CD8+T cells and NKcells were the major effector cells for anti-tumor effects of M/CpG-ODN-H22-Lipo in vivo.Conclusion: M/CpG-ODN-H22-Lipo activated DCs efficiently in vivo anddisplayed highly effective anti-tumor immune response in HCC-bearing mice.2. The effects and mechanisms of M/CpG-ODN-conjugated liposome loadedwith tyrosinase-related protein2(TRP2) peptide (M/CpG-ODN-TRP2-Lipo) inthe treatment of melanoma.Background: Melanoma is an extremely malignant tumor with high rate ofmetastasis. A large portion of clinical research on the effects of DC-basedcancer vaccines have been conducted for the treatment of melanoma due to therelatively clear antigens and the good sensitivity to DC-based cancer vaccine ofthis tumor. Inducing tumor-specific CD8+cytotoxic T lymphocytes (CTLs) isthe key step in the immunotherapy of cancers using DC-based cancer vaccine.Objective: Preparation of M/CpG-ODN-TRP2-Lipo for the treatment in amouse melanoma model and investigation of the underlying mechanisms of theanti-tumor effects of M/CpG-ODN-TRP2-Lipo.Methods: M/CpG-ODN-TRP2-Lipo was prepared as mentioned above insection1. DLS and TEM were used to characterize the particle size, morphology,and zeta potential of M/CpG-ODN-TRP2-Lipo. Melanoma model wasestablished in C57BL/6mice. Subcutaneous injection of M/CpG-ODN-TRP2-Lipo was applied to treat the melanoma in mice. Flow cytometry was used tomeasure the number of activated T cells, MDSCs, Tregs, and tumor-specificCD8+CTLs in mice. ELISPOT was used to measure the secretion of IFN-γ. Thevascular density in tumor tissues, proliferation and apoptosis of tumor cells were evaluated using IHC. In addition, in situ tetramer staining (ISTS) was used toevaluate the tumor-specific CD8+CTLs in tumor tissues. The mice weremonitored daily for survival and tumor volume was calculated according to theformula. The anti-tumor effects and underlying mechanisms of M/CpG-ODN-TRP2-Lipo were further investigated by comparing the survival betweenwild-type and myeloid differentiation primary response gene88(MyD88)knockout mice.Results: The diameter of M/CpG-ODN-TRP2-Lipo was around100nm withround surface and negative charge, and the encapsulation efficiency was60.8%.This DC-based cancer vaccine inhibited the tumor growth and prolonged thesurvival of mice efficiently with melanoma. M/CpG-ODN-TRP2-Lipo reducedthe number of MDSCs in tumor tissues, bone marrow, and spleen, and the Tregsin spleen, increased the frequency of tumor-specific CD8+CTLs, activated Tcells to secret IFN-γ. It was observed that M/CpG-ODN-TRP2-Lipo efficientlyreduced the vascular density in tumor tissues, inhibited proliferation and inducedapoptosis of tumor cells. Based on ISTS experiment, tumor-specific CD8+CTLsaccumulated in tumor tissues after the treatment of M/CpG-ODN-TRP2-Lipo.The anti-tumor effect of M/CpG-ODN-TRP2-Lipo was mediated by signaltransduction pathway of MyD88.Conclusion: M/CpG-ODN-TRP2-Lipo induced specific and efficient anti-tumorimmune response in melanoma-bearing mice. |
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