| Background:Head and neck squamous cell carcinoma(HNSCC)is the sixth most common cancer worldwide.Standard treatment for HNSCC is surgery,with or without chemoradiation. Despite advanccs in surgical technique and the institution of novel chemoradiation approaches,there has been little evidence of improvement in the 5-year survival rate over the past 30 years.Molecular targeted therapy appears to become one of the focuses of the researches on tumor therapy.Overexpression of epidermal growth factor receptor(EGFR) has been frequently observed in many malignancies,such as HNSCC,non-small cell lung cancer,breast cancer,colon cancer and pancreatic cancer,and the level of expression is considered associated with prognosis and nodal status of these patients.Therefore,EGFR has been recognized as an attractive target for tumor therapy.EGFR monoclonal antibodies and small-molecule EGFR tyrosine kinase inhibitors(TKIs)are two major therapeutic agents that target the EGFR.They have been approved by the U.S.Food and Drug Administration.However,their clinical activities are still limited.The approach using dendritic cells(DCs)to present tumor antigens and thereby generate tumor-specific immunity is one of the focuses of cancer immunotherapy. Different from the monoclonal antibodies and TKIs which inhibit tumor growth by block EGFR signal-transduction pathway,the cytotoxic T-lymphocyte(CTL)responses elicited by DCs can kill the tumor cells directly.So this approach is more powerful.For this reason,using EGFR-pulsed DCs to induce active immunotherapy against EGFR-positive HNSSC may be a hopeful approach.For the moment,no such research has been reported. In the present study,GST-EGFR fusion protein-pulsed DCs was used to treatment HNSSC,and the antitumor effects mediated by CTL responses were observed in vitro and in vivo.We expect the results may provide a novel therapeutic strategy for HNSSC.Objects:To recombine GST-EGFR fusion protein;To generate DCs and pulse DCs with GST-EGFR fusion protein;To investigate whether GST-EGFR fusion protein-pulsed DCs could induce cellular immunity by measuring CTL responses and the secretion of IFN-γin vitro;To observe the ability of GST-EGFR fusion protein-pulsed DCs inducing active antitumor immunity against HNSSC in vivo.Methods:1.A prokaryotic vector was constructed for expression of GST-EGFR fusion protein, and this fusion protein was expressed and purified.2.DCs were generated and pulsed with GST-EGFR fusion protein.3.T cells were isolated from the spleen cells of the mice vaccinated with GST-EGFR fusion protein-pulsed DCs,then CTL assay and the secretion of IFN-γwere measured in vitro. 4.The in vivo antitumor activity elicited by GST-EGFR fusion protein-pulsed DCs was observed in mice bearing HNSSC.Results:1.Construction of a prokaryotic vector for expression of GST-EGFR fusion protein, and expression and purification of this fusion proteinThe cDNA corresponding to the ectodomain of mouse EGFR was amplified by reverse transcription followed by polymerase chain reaction(RT-PCR)using total mRNA from EGFR overexpressing murine HNSSC cell line SCCⅦas template.The cDNA was verified by DNA sequencing and aligned with sequence data in GenBank database. Then it was inserted into the pGEX-4T-2,a prokaryotic expression vector,and induced by IPTG.After purification,we obtained an expected 87kDa GST-EGFR fusion protein. Meanwhile the pGEX-4T-2 was also induced by IPTG,and an expected 26kDa GST protein was obtained after purification.The purity of either GST-EGFR fusion protein or GST protein was＞90%.2.DCs generation and pulsed with GST-EGFR fusion proteinThe bone marrow was collected from the tibias and femurs of the C3H mice and bone marrow-derived DCs were generated.On day 5,the degree of expression of CD11c on DCs' surface was measured by flow cytometry and CD11c-positive cells were 59.86%. On day 7,DCs were measured to detect CD40,CD80,CD86 and MHCⅡ(I-A~k)-positive cells.The positive cells among the DCs pulsed with GST-EGFR fusion protein were 58.82%,59.88%,89.42%and 90.28%respectively.The positive cells among the DCs pulsed with GST protein were 54.32%,55.02%,88.36%and 89.04%respectively.The positive cells among the DCs unpulsed with protein were 17.87%,42.84%,81.07%and 80.36%respectively.Obviously,CD40,CD80,CD86 and MHCⅡ(I-A~k)-positive cells among the DCs pulsed with either GST-EGFR fusion protein or GST protein were more than the DCs unpulsed with protein.3.CTL assay and the secretion of IFN-γmeasured in vitro3.1 CTL assayCTL assay was performed at different effector-to-target ratios as 200:1,150:1,100:1 and 50:1.T cells isolated from spleen of GST-EGFR fusion protein-pulsed DCs immunized mice exhibited higher cytotoxicity against tumor cells with 81.71±7.21%, 64.05±5.9%,72.21±5.73%and 70.51±15.73%specific lysis respectively than those from DCs alone immunized mice with 7.69±2.32%,10.47±2.75%,8.93±0.67%and 9.13±1.09%specific lysis respectively(P＜0.05).Whereas,the cytotoxicity of T cells from GST-pulsed DCs immunized mice were 9.47±4.42%,10.22±4.28%,6.76±3.53% and 13.55±8.54%respectively;the cytotoxicity of T cells from control group were 8.99±5.07%,10.74±6.19%,8.24±3.55%and 9.93±1.4%respectively.There was no significant difference(P＞0.05).3.2 IFN-γresponseThe secretion of IFN-γwas significantly higher in the T cells from the mice that had been vaccinated with GST-EGFR-pulsed DCs(250.36±4.83pg/ml)than in the T cells from the mice that had been vaccinated with DCs alone(66.17±18.49pg/ml)(P＜0.05). However,the secretion of IFN-γin the T cells from GST-pulsed DCs immunized mice was 84.47±12.96pg/ml;the secretion of IFN-γin the T cells from control group was 102.64±20.36pg/ml.There was no significant difference(P＞0.05).4.To explore the in vivo antitumor activity elicited by GST-EGFR fusion protein-pulsed DCs in mice bearing HNSSC.4.1 To explore therapeutic activityBefore immunized with GST-EGFR fusion protein-pulsed DCs,GST-pulsed DCs,or DCs alone,mice were challenged with tumor cells.The tumor grew progressively in mice treated with GST-pulsed DCs or DCs alone.On day 28 after tumor inoculation,the tumor volume in these two groups was 17345.18±2916.22mm~3 and 15692.16±4410.53mm~3 respectively.There was no significant difference(P＞0.05).However,the tumor grew slowly in mice treated with GST-EGFR-pulsed DCs.On day 28 after tumor inoculation, the tumor volume was 8257.1±4254.2mm~3(P＜0.05,compared with GST-pulsed DCs,or DCs alone immunized group).Otherwise,the mean survival time of the mice immunized with GST-EGFR-pulsed DCs(44±6.38 days)was significantly longer than that of the mice treated with GST-pulsed DCs(37.17±3.13 days)or DCs alone(36.43±4.24 days)(P＜0.05).4.2 To explore preventive activityMice were challenged with tumor cells on day 7 after the third immunization. Similar with above results,the tumor grew progressively in mice treated with GST-pulsed DCs or DCs alone.On day 26 after tumor inoculation,the tumor volume in these two groups was 8453.35±2049.26mm~3 and 9309.32±3493.99mm~3 respectively.There was no significant difference(P＞0.05).However,the tumor grew slowly in mice treated with GST-EGFR-pulsed DCs.On day 26 after tumor inoculation,the tumor volume in this group was 3911.5±1656.45mm~3(P＜0.05,compared with GST-pulsed DCs,or DCs alone immunized group).The mean survival time of the mice immunized with GST-EGFR-pulsed DCs(52.75±3.3 days)was significantly longer than that of the mice treated with GST-pulsed DCs(41.6±5.37 days)or DCs alone(43.75±5.74 days)(P＜0.05).Disscusion1.CD11c is a special molecule expressed on mouse DCs.The expression of CD11c indicates the purity of DCs.In our study,the purity of DCs was similar to other researches.MHCⅡmolecule and costimulatory molecules such as CD40,CD80 and CD86 increase with maturation of DCs.Our results show that these molecules increased when DCs were pulsed with protein,which indicated that the DCs matured.2.The results of CTL assay and IFN-γresponse in our study indicated that GST-EGFR fusion protein-pulsed DCs could mediate CTL activity to target HNSCC cells specifically,and increase the secretion of IFN-γ.However,these antitumor activities were not associated with GST protein.3.The in vivo studies show that GST-EGFR fusion protein-pulsed DCs could provide therapeutic and preventive antitumor activities against HNSSC,and prolong the survival time of the mice bearing HNSSC.Whereas,these antitumor activities were also not associated with GST protein.Conclusions:1.High purity GST-EGFR fusion protein was obtained.2.DCs was generated and pulsed with GST-EGFR fusion protein successfully.3.GST-EGFR fusion protein-pulsed DCs could provide antitumor immunity aganist EGFR overexpressing HNSSC cells.4.GST-EGFR fusion protein-pulsed DCs could provide therapeutic and preventive antitumor activities against EGFR overexpressing HNSSC in vivo. |
