Application Of Dendritic Cells To The Immunotherapy Of Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is one of the major malignancies worldwide. Most of the HCC patients are inoperable at the time of diagnosis.Despite several palliative therapeutic options there is a high rate of recurrence or of intra-hepatic metastases.Therefore,novel treatment strategies such as immuno-gene therapy are necessary to be developed to lower the frequency of tumor recurrence.Dendritic cells(DCs)represent the class of antigen presenting cells(APCs)that initiate most immune responses.And their unique efficiency in capturing,transporting, and presenting antigen,as well as attracting and activating specific T cells,make mature DCs the most potent APCs known.Critical to the antigen-presenting function of DCs is the fact that they can present tumor-associated antigens and stimulate cytotoxicity T cells to induce specific immune responses in the context of both MHC classⅠandⅡ; thus,they can stimulate both cytotoxicity and helper T cells.Much attention has been directed to the problem of how and what antigens should be pulsed to DCs.At present, DCs pulsed with tumor-associated antigens in various forms,including whole cell lysate,peptides,proteins,RNA or DNA,have been studied for anti-tumor effects in experimental tumor models.In these models,immunization with tumor antigens presented by DCs has been shown much promise in effectively priming the cellular immune response.Some clinical applications using DC-based tumor vaccines have been reported.Although anti-tumor cellular immune responses could be induced by DCs vaccination in most patients,clinical objective responses were limited in tumor models.Thus,a new strategy for DC-based tumor vaccines is expected to improve the clinical effectiveness of treatment.In this regard,we exerted GPC-3 modification of DCs for presenting tumor antigen to T cells so as to produce specific cytotoxicity against HCC with minimal side effects.Glypican-3(GPC-3)potentially behaves as an oncofetal liver protein. Overexpression of GPC-3 is detected in fetal liver,whereas it is absent in normal adult liver and benign hepatocellular nodules,including cases of focal nodular hyperplasia and adenoma.And some researches have reported that GPC-3 was overexpressed in most HCC.By comparing AFP and GPC-3 immunostaining in different types of HCC, Wang et al.confirmed that GPC-3 is more sensitive,especially in HCC developed from cirrhosis.In general,oncofetal proteins do not seem to play a critical role in tumor progression,but may be used as tumor markers or as targets for immunotherapy. Hiroyuki Komori et al.reported that they had identified the HLA-A2 restricted cytotoxicity epitopes possibly useful for GPC-3 specific immunotherapy of HCC and raised the possibility that some GPC-3 peptides may be applicable to cancer therapy.In this study,human peripheral blood monocytes derived DCs were obtained,and HepG2 lysate pulsed DCs had more power in promoting effector cells proliferation and IFN-γproduction than GPC-3 transfected DCs only at the ratio of 1:10.However,DCs with GPC-3 modified showed intensive ability to induce highly specific cytotoxicity against HepG2 cells.Thus,genetic modification of human monocyte-derived DCs with cDNA sequences encoding GPC-3 might be a promising strategy for effective HCC immunotherapy.MethodsGeneration of DCs from a healthy donor.DCs were generated from peripheral blood mononuclear cells(PBMCs),as described previously,with few modifications. Briefly,PBMCs were isolated from 50ml peripheral blood by using a Ficoll-Pague gradient(Haoyang,China).The cells were resuspended in complete medium(CM) using RPMI1640(Gibco,USA)supplemented with 10%FCS,100U/ml penicillin, 100mg/ml streptomycin,and allowed to adhere into a six-well plate.After 2h at 37℃, non-adherent cells were transferred to another plate as responder cells and adherent cells were cultured in CM supplemented with 100ng/ml recombinant human granulocyte-macrophage colony-stimulating factor(rhGM-CSF)and 50ng/ml interleukin-4(rhIL-4)(both from PeproTech,USA).Fresh medium containing cytokines was substituted every other day.Cell lines culture.HCC cell line HepG2 and colon cancer cell line SW620 were selected in this study for their identical phenotype of HLA-A2+.The liver cell line THLE-3 was used as a normal control.The cells were all maintained in DMEM supplemented with 10%FCS,100U/ml penicillin and 100mg/ml streptomycin.Previous studies have demonstrated that GPC-3 was expressed specific for HepG2 cells,while SW620 cells and THLE-3 cells were GPC-3 negative,which was demonstrated preliminarily.Preparations of HepG2 lysate.HepG2 cells were allowed to grow until confluent and washed three times in phosphate-buffered saline(PBS).Cells were collected and underwent 4 times of freeze(liquid nitrogen)and thaw(37℃)cycles,after which particles were removed by centrifugation.The supernatant was passed through a 0.2μm filter,and protein concentrations were determinated by BCA assay(Pierce,USA).Generation of mature DCs with GPC-3 transfected or HepG2 lysate pulsed.The pEF-hGPC-3 plasmid containing full-length GPC-3 was generous gift from Dr.Jorge Filmus(Toronto University,Canada).5d after GM-CSF plus IL-4 incubation, transfections were performed according to the manufacturer's instructions.Briefly,cells were reuspended in FCS-free CM supplemented with 4μg plasmids plus 10μl Lipofectamine2000(Invitrogen,USA)and 100ng/ml TNF-α.Simultaneously,cells were reuspended in CM supplemented with 200μg HepG2 lysate and 100ng/ml TNF-α. 48h later,the non-adherent cells were harvested and regarded as mature DCs.Flow cytometric analysis.The cell surface expression of DCs markers was assessed by flow cytometric analysis.Antibodies used to evaluate the phenotype of DCs were anti-CD80-FITC,anti-CD86-FITC,and anti-CD83-FITC(Biolegend,USA).DCs were stained for 40 min at 4℃and analyzed by the FACScan using CellQuest software. About ten thousand cells were examined for each determination.Immunocytochemical staining.Cytospin preparations of GPC-3 transfected or HepG2 lysate pulsed DCs were fixed in 4%paraformaldehyde and incubated overnight with mouse monoclonal antibody detecting GPC-3(Santa Cruz,USA),following 5% rabbit serum treatment at 37℃for 1h.After which they were incubated with rabbit anti-mouse IgG and SP complex for 30min(both from Maixin,China),followed by DAB development.HCC cells were used as positive controls for GPC-3,and negative controls were prepared by non immune rabbit serum at the same dilution as for the primary antibody.Cells with brown particles appearing in membrane or cytoplasm were as regarded as positive cells. Western blot.Cells were lysed in appropriate amounts of lysing buffer containing 150mM NaCl,50mM Tris,pH 7.4,1%NP-40,1mM Na3VO4,1mM EDTA and 1mM PMSF.The supernatants were electrophoresed on 10%SDS-PAGE and transferred to a PVDF membrane.After blocking with 5%BSA and 0.05%Tween-20 in Tris-buffered saline,the membrane was incubated with mouse monoclonal anti-GPC-3 and anti-β-actin antibodies(both from Santa Cruz,USA),finally subjected to chemiluminescence detection using goat anti-mouse IgG(Zhongshan,China),using a DAB kit(Maixin,China).Proliferation of responder cells.The responder cells described above were adjusted at the concentration of 1×103 cells per well for all experiments.Matured DCs with various treatments were incubated with 25μg/ml mitomycin C(Sigma,USA)at 37℃for 30 min and subsequently mixed with responder cells at various DC-to-responder ratios of 1:10,1:50 and 1:100.DCs cultured in the absence of responder cells served as controls for background proliferation.Cells in triplicate wells of a 96-well plate were cultured for 48h and subsequently pulsed with 20μl WST-1 (Roche,Switzerland)during the last 4h of culture.The absorbance for measuring wavelength(450nm)and the reference wavelength(650nm)were measured.Stimulator index(SI)represented proliferation of responder cells and was calculated as follows: SI=A experiment/(A responder cells+A DCs).IFN-γsecretion.Supernatants of the cultures were harvested and assayed in triplicate by ELISA using commercially available reagents(BD,USA),which were performed according to the manufacturer's instructions.The concentrations of IFN-γin the supernatants were determined by regression analysis.Cytotoxicity assay.The responder cells stimulated by DCs with various treatments mentioned above were used as effector cells in the cytotoxicity assay using LDH cytotoxicity detection kit(Roche,USA).HepG2,THLE-3 and SW620 were used as target cells in this assay.Briefly,target cells and effector cells were resuspended in assay medium(RPMI 1640 supplemented with 1%BSA),and then target cells(103 cells per well)were cocultured with effector cells at different effector-to-target ratios of 10:1,50:1 and 100:1 in 96-well plate at 37℃.After 24h of incubation,the plate was centrifuged and the supernatant was removed.100μl per well LDH detection mixture was then added and incubated in the dark for 30 min at room temperature.After adding 50ul stop solution per well,the absorbance of each sample was measured at 490 nm with 650 nm as reference.The spontaneous release of LDH by target cells or effector cells was assessed by incubation of target cells in the absence of effector cells and vice versa.The maximum release of LDH was determined by incubation of target cells in 1%TritonX-100 in assay medium.The percentage of specific cytotoxicity was determined as follows:cytotoxicity(%)=[(effector&target mixture-effector spontaneous-target spontaneous)/(maximum-target spontaneous)]×100.Statistical analysis.The SPSS 13.0 software was applied to complete data processing.Independent-samples t-test was used to evaluate the differences between DCs with various treatments in responder cells proliferation and IFN-γsecretion,as well as cytotoxicity against target cells.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.ResultsThese cells exhibited irregular shapes,and many fine processes at their edges.The expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immuture DCs were examined by immunocytochemistry.Positive cells were defined as cytoplasm or membrane staining.Compared with empty vector,the intense expression of GPC-3 was observed in GPC-3 transfected DCs,which suggested an effective transfection.In HepG2 lysate pulsed DCs,GPC-3 immunoreactivity was occasionally detected and poorly distributed.Western blot was also performed to evaluate the expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immature DCs.GPC-3 transfected DCs expressed GPC-3,which was detected at a much lower level in HepG2 lysate pulsed DCs, demonstrating a successful uptake of GPC-3 antigen by DCs.These date indicated that GPC-3 transfected DCs might have the ability to effectively present the GPC-3 antigen to responder cells.To evaluate the effects of exogenous GPC-3 or HepG2 lysate on the phenotypes of immature DCs,we analyzed the cell surface markers of DCs transfected with GPC-3 or pulsed with HepG2 lysate by flow cytometry.The intensity of expression of surface markers remained stable in GPC-3 transfected or HepG2 lysate pulsed DCs,compared with that in control immature DCs.These results suggest that both GPC-3 and HepG2 lysate had no apparent influence on DC phenotypes.We also investigated whether GPC-3 or HepG2 lysate treated DCs could promote the activation of effector cells,which is represented by proliferation and IFN-γsecretion.Compared with the GPC-3 transfected DCs,HepG2 lysate pulsed DCs had more power to induce the proliferation of effector cells at the ratio of 1:10(4.05±0.08 vs 3.46±0.11,p=0.002).However,the proliferations of effector cells promoted by either GPC-3 transfected or HepG2 lysate pulsed DCs were no significant difference at the ratio of 1:10 and 1:50,respectively(p＞0.05).After a culture period of another 48h,the levels of IFN-γin the supernatants of effector cells sitmulated by GPC-3 transfected and HepG2 lysate pulsed mature DCs,as well as the control cells were measured by ELISA.The effector cells stimulated by HepG2 lysate pulsed DCs producted more IFN-γthan GPC-3 transfected DCs at the ratio of 1:10(708.45±11.81 pg/ml vs 643.43±22.11 pg/ml,p=0.011),indicating that HepG2 lysate was efficient in promoting IFN-γproduction of effector cells to some extent.However,the IFN-γsecreted by effector cells in GPC-3 transfected DCs was observed nearly the same as that in HepG2 lysate pulsed DCs at the ratio of either 1:50 or 1:100(p＞0.05).To identify the specificity and validity of GPC-3 modified DCs involved in the process of HepG2 cells being lysed,we selected another HLA-A2+human tumor cell line SW620,except for HepG2 cells,as target cells for cytotoxicity assay.HepG2 cells exhibit overexpression of GPC3,SW620 cells are GPC3 negative.The cytotoxicity against the two tumor cell lines were assessed by a standard LDH release assay.The effector cells stimulated by HepG2 lysate pulsed DCs showed intensive lytic activity of HepG2 and SW620 cells with no statistical significance(p＞0.05).However,effector cells stimulated with GPC-3 transfected DCs effectively lyse HepG2 cells,at the ratios of both 50:1 and 100:1(16.6%and 34.1%,respectively).On the other hand,the GPC3-null SW620 cells were only observed minimally lysed at the ratio of 100:1 (12.0%),yet the lytic activity of effector cells stimulated with HepG2 lysate pulsed DCs was significantly elevated at different ratios(p＜0.05).Conclusions1,IFN-γcan enhance the properties of DCs,and provide a better method of culturing DCs in vitro to increase the immune activity.2,DCs modified with full-length GPC-3 could generate specific cytotoxicity against HepG2 cells,which may provide a new method of treating HCC.3,Compared with lysate pulse,GPC3 genetically modified DCs induced specific cytotoxity against HCC cell in vitro,which have the potential to serve as novel vaccine for HCC with less nonspecific cytotoxicities.