Low-dose Chemotherapy Combined With Gene-transducted Dendritic Cell Vaccine For Hepatocellular Carcinoma

Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors which seriously affect the body health of human being. HCC’s incidence ranked the fifth in common cancers while its mortality ranked the third. In our country HCC has the incidence rate of20.4/10ten thousand and the second mortality rate in the total cancer mortality. Though many methods can be performed in HCC in clinic practice, surgical resection is preferred and most effective treatment for HCC. Because of HCC insidious onset, only less than20%of patients have surgical resection of the tumor. However, even with radical resection,60%to70%of patients would have reeurrences five years after surgery. Metastasis and recurrence is the major factor of further improvement on survival rate of HCC patients. Systemic chemotherapy is commonly used in the advanced cancer, but the traditional chemotherapy and radiotherapy for HCC is poor efficacy. Therefore, there is an urgent need to find new and effective way to reduce liver cancer recurrence and metastasis of HCC treatment, to extend the survival of patients. In recent years, with efficiency and low toxicity of the chemotherapy drugs are discovered and combinational chemotherapeutics with multiple mechanisms develops, the advanced HCC chemotherapy has achieved a new breakthrough. EACH and other studies have shown that local control and survival benefit for advanced HCC patients with FOLFOX4chemotherapy. Oxaliplatin (OXA) and fluorouracil (5-FU) are widely used chemotherapy drugs in the clinical. Chemotherapy as an important tumor treatment, chemotherapy drugs have acute and accumulation on normal tissue toxicity limits the dose and the use of time. Low-dose chemotherapy has been used clinically, and achieved a certain effect. Different mechanisms of chemotherapy and immunotherapy, makes sequential immunochemotherapy to become a new therapeutic concept.With the rapid development of tumor immunology and molecular biology, tumor biotherapy has become the fourth model of cancer treatment, and indicated a good prospect. Tumor immunology denmonstrated that the occure and development of the cancer was closely related with the functional defection of T lymphocyte, the further studies found the reason of which was the down-regulation of the mature dendritic cells(DC)by the body. DC is the most effective antigen presenting cell(APC) at activating naive T Cells, which can activate antigen specific CTL with the antigen successful loading. Transduced DC with antigen peptide has advantages such as antigen peptide is specific and which can immunize times, but which also has one main problem is the half-life of this kind of antigen peptide usually very short, so only after several times of immunization can we get CTL with good activity. Moreover, the transduction efficiency is low. Transduced DC with adeno-associated virus(AAV) carting antigen gene can make the DC produce a constant and large amount of antige peptide, which can increase the expression of DC surface markers of CD1a, HLA-DR, CD40, CD80, CD83and CD86, etc., induce antigen-specific CTL to enhance its cytolytic activity. Now, DC-based specific active immunotherapy is the most promising one. Its anti-tumor mechanism is through the activation of the immune system, especially cytotoxic T lymphocytes specific killing of tumor cells without damaging normal tissue and cells. Clinical trials show that dendritic cell vaccines have achieved encouraging efficacy in prostate cancer, melanoma, kidney cancer, malignant lymphoma. April29,2010, the U.S. FDA approved the prostate cancer therapeutic vaccine (Provenge) listed. DC-based tumor vaccine is considered to be the most potent means of tumor immunotherapy, however, the DC vaccine in the majority of malignant tumors achieved only limited clinical efficacy. The study found that the surface of tumor cells can express a variety of inhibitory cytokines related to the immunogenicity and tumor immune escape, which have an important impact on anti-tumor immune response. Tumors may directly down-regulate the expression of tumor antigens either spontaneously, or in response to a targeted immune-based intervention. Alternatively, tumors may down-regulate various components of the antigen-processing machinery (MHC class I). These mechanisms together provide a means for the outgrowth of antigen loss variant tumors intrinsically resistant to antigen-specific immunotherapy. Importantly, this phenotype has been correlated with poor clinical outcome. Moreover, the goal of DC immunotherapy is still to remove the tumor micrometastasis in the control of tumor recurrence and metastasis. High tumor burden or low tumor immunogenicity under the conditions of the limited efficacy.Therefore, traditional therapy combined with emerging immunotherapy will become the trend of cancer therapy. Different mechanisms of chemotherapy and immunotherapy makes sequential immunochemotherapy become a new therapy concept. Certain cytotoxic drugs can modulate these mechanisms of immune escape, thereby facilitating the activity of activated CD8+CTL. How to develop standardized and individualized of chemoimmunotherapy program is the main direction for future research. This study investigate the killing effect of chemotherapy combination with gene-transducted dendritic cell vaccine on HCC cells in vitro, and explore the mechanism of synergy effects.We hope that these results may provide new strategies and methods for the comprehensive therapy of HCC.Methods:1. The effects of cytotoxic drugs (OXA and5-FU) on the expression of immunological related molecules in the human HCC cell.Cell inhibition rate and viability of HepG2following treatment with OXA and5-FU were determined by MTT and trypan blue assay. Select low-dose chemotherapy drugs (5μg/ml OXA and20μg/ml5-FU) and pretreatment of HepG2cells for24hours. Cells and culture supernatant were collected, then expression of HepG2cell surface marker HLA-A2, bcl-2and IL-10were analysised by flow cytometry, Western blot and ELISA respectively.2. Generation of DC infected by AAV/AFP and CTL, immunophenotype analysis2.1Generation of DC infected by AAV/AFP and CTLAfter the PBMC (8×107) were cultured for five hours with AIM-V medium, the non-adherent cells were removed. The adherent (monocytes) were infected immediately with AAV/AFP virus or the control. After eight hours the medium/virus solution was removed and the cells were finally fed with the medium containing recombinant human GM-CSF (Prospec,800IU/ml). At day2and4, to induce the maturation of monocytes (Mo) into DC, recombinant human IL-4and TNF-α (Prospec) at1,000IU/ml and100IU/ml were added to the medium, respectively.The medium and cytokines were replaced every2days. Finally, at day6the DC were mixed with PBL (ratios from20:1, T:DC).The mixtures were cultured in AIM-V containing recombinant human IL-2(100IU/ml) and IL-7(40IU/ml). The medium and cytokines were replaced every2days. After8days post-priming the cells were harvested and analyzed further.2.2Analysis of AAV/AFP efficient infection and Cell surface marker of DCAt day6of Mo/DC culture, intracellular staining assay was employed to analyze the expression and efficient infection of AAV/AFP virus by FACS analysis according to the routine method. For the analysis of DC a panel of FITC-or PE-labeled monoclonal antibodies recognizing the following antigens was used: CD14, CD40(Chemicon International), HLA-DR, CD80, CD8, CD86and isotype-matched antibodies (BD Pharmingen). Stained cells were assayed for these CD markers by FACS analysis as described previously.2.3Analysis of level of IL-10and IL-12in the DCAt day3of Mo/DC culture (before the addition of TNF-a) and day6(after the addition of TNF-a) the supernatants were separated from the culture. IL-10and IL-12p70secretion was measured by ELISA in duplicate according to the manufacturer’s instructions.2.4CD marker analysis of activated T cell populationsFor the analysis of activated T cells, at day14of the mixed cell culture the stimulated T cell populations were analyzed for their surface markers with immunofluorescence staining by flow cytometry. A panel of FITC-, APC-or PE-labeled monoclonal antibodies recognizing the following antigens was used: CD4, CD8, CD25, CD28, CD69and FoxP3(BD Pharmingen).3. The killing effect of chemotherapy combination with gene-transducted dendritic cell vaccine on HCC cells in vitro3.1Analysis of IFN-y in the activated T cell populationsHepG2cells were treated with cytotoxic drugs for24hours, as described above. At day14post-priming T cells were harvested. Then the CTL cells and the pre-treated HepG2cells were mixed (20:1) and incubated for24h. Cell culture supernatants were collected and human IFN-γ was measured in the cells culture supernatants by ELISA.3.2Analysis of AFP-specific and MHC Class I-restricted CTL killing activityThe HepG2cells were pre-treated with or without cytotoxic drugs OXA (5μg/ml) plus5-FU (20μg/ml) for24hours. The CTL cells and target cells were mixed (20:1) and incubated for6h at37℃with5%CO2. Cytotoxic assays were assessed with LDH release assay kit (Promega) according to the manufacturer’s instructions. All experiments were repeated at least3times. For the HLA blocking experiments, anti-HLA Class I monoclonal antibody was used to block cytotoxicity. The HepG2cells were pre-incubated with mouse anti-human HLA class I antibody (w6/32) for1h before the cytotoxic assay. K562cells were used as targets to observe natural killer (NK) cell activity and a series of cells (without AFP expression) were used as negative controls.4. Statistical analysesAll analyses were performed with SPSS19.0software. Data are presented as means±SD. Comparison of means between two samples was performed using Student’s t test. Statistical comparisons of more than two groups were performed using One-way ANOVA, and then multiple comparisons were performed by leas-signifieant differenee(LSD) and Dunnett’s test. In all cases, Two-sided p values <0.05were considered to be statistically significant.Results:1. Cell survival rate assessed by MTT assaySurvival rate of HepG2was assessed with MTT assay. The OXA and5-FU are widely used chemotherapeutic drugs in the clinic. Therefore, we chose these two agents to investigate whether they sensitize HCC cells to CTL lysis. The doses of the drugs were titrated by MTT assay, and subtoxic doses were used in this study. Exposure of HepG2to OXA (5μg/ml) or/and5-FU (20μg/ml) for24-48h had no significant cytotoxic effects, as determined by trypan blue assay. The viability of cells in untreated and5-FU-and OXA-treated groups was all over85%. But at these dose, OXA and5-FU showed significant inhibition on the growth of HepG2.2. The effects of chemotherapeutic drugs on cell surface molecule expression in HCC cell line HepG2.HepG2cells were treated with low-dose of OXA and5-FU for24hours. Effects of the two drugs on cell surface molecule expression were monitored. We observed that OXA or/and5-FU can increase HLA-A2expression, while reduce the expression of Bcl-2and IL-10on HepG2(P<0.05). Compared to separate of OXA or5-FU, the effects of two-drug combination were more significant.3. Immunophenotype analysisAAV/AFP load DC successfully. The transduction efficiency of DC by AAV/AFP is91.3%. AAV/AFP-loading of DCs resulted in:(1)The AFP expression in DCs,(2) high CD40、CD80、CD83、CD86and low CD14expression in DCs. Thus, the up-regulation of these multiple costimulatory molecules could help to explain the rapid CTL expansion observed.(3) high level of IL-12and low level of IL-10in DC,(4) high CD8+/CD4+T cell ratios, high CD8+、CD8+CD69+、CD8+CD28+T cell levels, and lowCD8+CD28-T cell and CD4+CD25+FoxP3+Treg levels (P<0.05). These data were consistent with DC stimulating a more robust Th1immune response.4. Treatment of HepG2cells with OXA and5-FU sensitizes HepG2to lysis of antigen-specific and MHC Class I-restricted CTLChemotherapeutic drugs have been reported to sensitize tumor cells to T cell killing through up-regulation of class I expression on tumor cells. To investigate whether anticancer drugs sensitize HepG2cells, which are relatively resistant to chemotherapy and CTL killing. We observed that HepG2cells treated with OXA and5-FU can stimulate CTL to secret high level of IFN-y. Compared to separate of OXA or5-FU, the effects of two-drug combination were more significant (P<0.001).Analysis of CTL for cytotoxicity assays and MHC class-I limitation by LDH-release assay. The AFP positive targets HepG2cells were killed by the CTL stimulated by AAV/AFP-loaded DC, while the other AFP-negative cell lines were not (P<0.05). These data are fully consistent with the resulting CTL being highly AFP-specific. The CTL killing activity was blocked by anti-MHC class I antibody, strongly suggesting MHC class I-restriction (P<0.001).Chemotherapy combined with the DC/CTL showed much stronger killing activity to HepG2cells than the two separate applications to the kill HepG2cells (P<0.001) with appropriate chemotherapeutic drugs in a certain dose. These data suggest DC can synergy and enhance the role of low-dose chemotherapy.Conclusions:1. In summary, we demonstrate that:The chemotherapeutic drugs OXA and5-FU could dramatically upregulate surface expressions of HLA-A2, and downregualte the bcl-2expression and the immunosuppressive cytokines IL-10secretion in human HCC cells, suggesting that low-dose cytotoxic drugs as a chemo-modulation, can be used to modify the tumor microenvironment, and change the cytologic features of tumor cells, thereby making it more receptive to an effective immune response.2. These data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens and that the AFP antigen is a potentially appropriate target for cell-mediated immunotherapeutie treatment for the HCC. 3. Low doses of chemotherapy combined with gene-transducted dendritic cell vaccine showed much stronger killing activity to HepG2cells, suggesting that the appropriate species, appropriate doses of chemotherapeutic drugs combined with dendritic cell vaccine have mutual synergy anti-tumor effect. Our study may provide new strategies and methods for the comprehensive therapy of HCC.