Applied Research Nanomaterials In The Anti-tumor Immunotherapy

Objective:The significance of anti-tumor immunotherapy as an adjuvant cancer treatment has been well recognised. Among the practical immunotherapy technigues, dendritic cells (DC)-based therapy is considered an important approach that has attracted mainstream attention. Some of the prior clinical studies on DC-based therapy have generated promising results in treating malignancies such as glioblastoma or malignant melanoma. Nevertheless, the outcome of DC-based anticancer immunotherapy still needs further improvement. An important issue of DC-based therapy, therefore, is to develop new technologies that can further enhance the efficacy for DC stimulation by available tumor antigens. Carbon nanotubes (CNT) have been shown to have potential applications in multiple biomedical fields, especially as transporters for delivery of various bioactive molecules such as peptides, proteins, DNAs, RNAs, or drugs. In this study, we investigated whether the conjugates of CNT and tumor protein (CNT-TumorP) would influence the endocytosis of tumor antigen by human DC in vitro. We also evaluated the effects of CNT-TumorP on the capacity of DC to induce anticancer cytotoxicity in human lymphocytes.Methods:Human DC were cultured from fresh peripheral blood of healthy donors per standard protocol employing rhGM-CSF and rhIL-4. MCF7 human cancer cells were lysed and tumor lysate protein (TumorP) was extracted. The tumor protein was conjugated to the oxidized CNT in the presence of the coupling agent N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDAC). Tumor protein (TumorP) was labeled with fluorescein-5-isothiocyanate (TumorP-FITC), and then covalently conjugated to CNT (CNT-TumorP-FITC). Two groups of DC were co-incubated with TumorP-FITC and CNT-TumorP-FITC separately. The endocytosis of tumor protein by DC was evaluated by flow cytometry and confocal microscopy. Moreover, expression of CD40, CD83, CD86 and HLA-DR on immature DC after co-incubation with CNT, TumorP or CNT-TumorP was evaluated by flow cytometry. The lymphocytes'specific cytotoxiciy to MCF-7 was then evaluated after stimulated with CNT-TumorP loaded DC using a standard MTS cytotoxicity assay. Additionally, the toxicity of CNT in a wide range of concentrations was studied using MCF-7 cell line as target.Results:1) Approximately 2×106 immature DC were obtained by a routine protocol employing rhGM-CSF and rhIL-4, with mononuclear cells isolated from 20ml of peripheral human blood.2) The dispersibility and stability of CNT were well preserved after the conjugation.3) The flow cytometry analysis revealed that endocytosis of tumor protein by DC was significantly enhanced when TumorP was conjugated to CNT (p<0.05). The enhancements of mean fluorescence intensity were 84% and 95% for TumorP concentration of 1μg/ml and 0.1μg/ml, respectively.4) Confocal microscopy indicated that the CNT-PRO was taken into the DC.5) CNT showed no effect on the phenotype of DC and did not induce DC maturation.6) CNT-TumorP enhanced the function of DC to induce anticancer response in lymphocytes.7) The anticancer response, induced by CNT-TumorP, was specific to the tumor cells from which the TumorP was extracted, with no obvious cytotoxicity against other cell lines.8) CNT, through a range of concentration from 0.0005μg/ml to 0.5μg/ml, showed no influence on viability of MCF-7 cells.Conclusion:The conjugates of carbron nanotubes and tumor protein could enhance the endocytosis of tumor antigen by human DC and the capability of DC to induce anticancer response in vitro. The results suggest that CNT-based nanotechnology may have a prospective role in the development of more efficacious DC-based anticancer immunotherapy. Objective:Anti-tumor immunotherapy has the potential to significantly improve the prognosis of cancer treatment, though the efficacy of immunotherapy generally needs further improvement. One way to improve the efficacy is using immune adjuvants, but the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines. The identification of novel and effective adjuvants is thus of practical importance for anticancer immunotherapy. Conventional aluminum has been used as adjuvant for more than 70 years in human vaccines; however, its efficacy for enhancing antitumor immune response is limited. Nanotechnology has the potential to generate new solutions for multiple biomedical problems. In this work, we studied whether aluminum oxide nanoparticles (nano-alum) would enhance the efficacy of an anti-tumor immunotherapy that employs tumor cell vaccine (TCV), in a murine model bearing the H22 liver cancer, to assess nano-alum's applicability as adjuvant in common anticancer immune regimens.Methods:BALB/c mice were inoculated with 2×106 live hepatoma H22 cells subcutaneously on day 0. Tumor cell vaccine (TCV) was prepared by inactivating H22 cells with Mitomycin C. On day 7, the mice with subcutaneous tumors were randomly divided into four groups:1) the control group,2) the TCV treatment group, 3) the TCV plus conventional alum (TCV+alum) treatment group, and 4) the TCV plus nano-alum (TCV+nano-alum) treatment group. On day 7 and 14, the treatment groups received two subcutaneous doses of designed immune treatment agents for triggering anti-tumor reactions. Tumor dimensions were monitored every 4 days after the second vaccination. At the end of the study, the cytotoxicity of mouse peripheral blood lymphocytes against H22 cells was evaluated in vitro. Histological slides of the tumor tissues were prepared for evaluation tumor necrosis and lymphocyte infiltration. Results:1) The average tumor size of the TCV+Nano-alum group was significantly smaller than that of the control group (p<0.05). Except for the TCV+Alum group which had smaller tumor size in comparison with the control group on day 14, no difference of the average tumor sizes was observed between the other groups.2) The cytotoxicity of the lymphocytes against H22 cells was 9.9±6.9% for the control group, 30.1±3.09% for the TCV group,35.63±2.8% for the TCV+alum group, and 67.96±9.54% for the TCV+nano-alum group. TCV+nano-alum group generated significantly higher anticancer cytotoxicity compared to the other treatment groups (p<0.05).3) Compared to the control group, there was more lymphocyte infiltration in the treatment groups by histological studies. Moreover, the tumor tissue of the TCV+nano-alum group developed obvious necrosis.Conclusion:Aluminum oxide nanoparticles enhanced the anticancer immune response induced by tumor cell vaccine. The results suggest that nano-alum may potentially serve as an effective adjuvant in anticaner immunotherapies. Objective:It has been reported that hapten dinitrophenyl (DNP) modified tumor cell vaccines (TCV) can enhance the immune response against malignant melanoma and improve the prognosis of patients. However, whether DNP-modified TCV can also induce effective immune reactions against other types of malignancy has not been well evaluated. In this study, we investigated DNP-modified TCV in the enhancement of lymphocytes'cytotoxicity against breast and lung cancer cells in vitro. Moreover, we compared the enhancement of DNP-modified TCV mediated lymphocytes' cytotoxicity with that of another widely used tumor vaccine modification agent, New Castle Disease Virus of Ulster Strain (NDV Ulster),-modified TCV.Methods:Human breast cancer cell line MCF7, lung cancer cell line H23 cells were inactivated by mitomycin C and modified with or without DNP/NDV. As the effector cells for the killing of targeted tumor cells, peripheral blood mononuclear cells (PBMC) from healthy human donors were obtained via standard Ficoll gradient centrifugation. Then PBMC were divided into three groups:control lymphocytes (T), tumor cell vaccine-stimulated lymphocytes (T+TCV), and DNP-or NDV-modified tumor cell vaccine-stimulated lymphocytes (T+DNP-TCV or T+NDV-TCV). After 3-day incubation of the lymphocytes with different modified tumor vaccines, cell killing was evaluated using a standard MTS cytotoxicity assay.Results:1) Compared to the control group (T), both DNP-modified TCV and unmodified TCV caused significant lymphocyte inhibition of MCF-7 cells and H23 cells.2) DNP-modified TCV (T+DNP-TCV) induced significantly higher lymphocyte inhibition of MCF-7 cells than that of unmodified TCV (T+TCV).3) Both DNP and NDV Ulster generated similar enhancement of anti-tumor cell immune reaction but not non-tumor cells. Conclusion:DNP-modified TCV enhances the specific cytotoxicity of lymphocytes against human breast cancer cells. Our findings suggest that DNP-modification of TCV may have the potential in immunotherapy against multiple types of human cancer in addition to malignant melanoma.