| Lung cancer is the most prevalent form of cancer, and is the leading causeof death from cancer-associated disease. Prognosis and treatment options forboth small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC)depend on the stage of the tumor at diagnosis, and approximately75%ofpatients with lung cancer initially diagnosed have advanced-stage disease, soeffective treatment modalities are very limited. Therefore, more effectivetreatment methods are urgently required, tumor vaccine will be an alternativeapproach for lung cancer therapy in the future. Tumor vaccines requireabundant of the targeted tumor-associated antigens(TAA). However, the designof tumor vaccine is often limited for ignorance of the relevant immunologicallydominant antigens in lung cancer.Tumor cell lysates(TCL) are tumor antigen mixture prepared by lysingtumor cells with physical or chemical methods. Thus, although the targetantigens are not well defined in TCL, TCL as a source of antigens might beespecially advantageous for vaccination strategies in lung cancer, because theydo not require the identification of specific tumor antigens in each individualpatient and can potentially induce immune responses to multiple TAA. TCL canbe taken up by both MHC class I and II molecules on antigen-presenting cells,and can be cross-presented to T cells. However, TCL are poorly immunogenic,and fail to induce effcient anti-tumor immune responses by themselves, sosuitable adjuvant is necessary to increase the immunogenicity of the TCL.The adjuvants of tumor vaccines which should have minimal toxicity arenecessary to boost the desired immune response to weak antigens,andnecessary to overcome various tolerance mechanisms and facilitate induction of cytotoxic T lymphocytes (CTL) that can lyse malignant cells. Cimetidine(CIM),one of histamine receptor2antagonists, is commonly prescribed for gastric andduodenal ulcer disease. However, CIM also has an immunomodulatory effectby virtue of their interaction with H2receptors on cells in the immune system.The mechanisms proposed for the cell-mediated immunomodulation of CIMinclude the inhibition of suppressor T lymphocytes activity, stimulation ofnatural killer(NK) cells activity, an increase in cytokines production in helper Tlymphocytes such as interleukin-2(IL-2) and interferon-γ(IFN-γ), enhance ofthe antigen presenting capacity of dendritic cells. Hence, CIM has the potentialability to be an adjuvant of tumor vaccine.At present, the murine models for studying tumor vaccine aresubcutaneous transplantation models. However, comparing to subcutaneousmodels, orthotopic transplantation models have provided a unique advantage instudying the natural progression of the disease in-vivo,allowing researchers totest the effectiveness of tumor vaccine. Our research confirmed that TCLcombined with CIM could not significantly inhibit the growth of lung cancer,but CIM presented a favourable effect by itself. In preliminary study ourcollegue found that TCL plus mycobacterial heat shock protein65(MHSP65)had produced anti-cancer immunity in subcutaneous models, the anti-tumoreffect of TCL-MHSP65was demonstrated again in the orthotopic models.1.The preparation of TCLTo prepare TCL, the Lewis lung cancer cells were frozen at-70℃andthawed at37℃for several cycles. The results showed that the Lewis lungcancer cells after repeated freeze-thaw had been destroyed, and had beenstained into blue by trypan blue under a microscope. SDS-PAGE resultsshowed that the intracellular proteins had all been released, the concentration ofprotein in the TCL at2×107/ml was about7.54mg/ml, so there was none cellssurvival in TCL of Lewis lung cancer cells, which were prepared by repeated freezing and thawing method for five times, and contained abundant tumorantigens, in line with the requirements of subsequent experimental studies.2.Establishment of orthotopic lung cancer transplantation model in mouseWith small incision in chest wall, Lewis lung cancer cells were injectedinto the left lung through the intercostal muscle, successful establishingorthotopic transplantation model, the operation was simple with low mortalityin mice.39mice were divided into three groups inoculated with different dosesof lung cancer cells: Group1was inoculated with1×105per mouse; Group2was1×104per mouse; Group3was1×103per mouse. Three mice in each groupwere sacrificed on the7th day after tumor implantation, the positive rate ofinoculation in three groups was as follows:100%,66.7%,0%. Three mice ineach group were sacrificed on the14th day, the positive rate of inoculation wasas follows:100%,100%,66.7%. Alive mice were sacrificed on the32nd dayfor the survival analysis, survival time of Group1was17-24days, the averagesurvival time was20.6±0.8days.The first mice in Group2was died on the27th day after inoculation, and untill the32nd day four mice had been died, thesurvival compared to Group1was significantly prolonged(P<0.001), theremaining three mice burdened with tumors in left lung were comfirmed aftersacrifice. All of mice in Group3were sacrificed, survival time compared toGroup1(P<0.001) and Group2(P=0.022) were significantly prolonged, fivemice with tumors were found, but2mice without tumors, suggesting that miceinoculated with lower doses of cancer cells prolonged survival, but the positiverate of inoculation decreased. The subsequent experimental model choosed thedose of cancer cells5×103per mouse for inoculation. The weight of mice wasmonitored, the weight of mice in Group1on the last day compared to the4thday after inoculation decreased significantly (P=0.042).3.The effect of TCL combined with CIM on lung cancer mice modelOrthotopic transplantation models were established on the0day through inoculating with Lewis lung cancer cells at5×103per mouse, while two kindsof combinations verified that the antitumor effect generated by the same doseof CIM with different immunizing frequency: The first combination was GroupPBS, Group TCL, Group CIM and Group TCL-CIM (TCL mixed with CIM)with immunization on the-7th,1st,8th,15th day; The second combination wasGroup PBS, Group TCL, Group CIM Daily and Group TCL-CIM Daily,immunizing with PBS, TCL, CIM and TCL-CIM on the-7th,1st,8th,15th day,in addition to the four days mentioned above, CIM was injected to Group CIMDaily and Group TCL-CIM Daily each day during-7and21day. Moreover, anadaptive immunotherapy (Group TCL-CIM-SP) also had been confirmed onthe basis of the first combination: TCL-CIM was used on the-7th,1st,8th,15thday, the splenocytes stimulated by TCL-CIM in vitro was transfused to thebody through the tail vein of mice on the6th and13th day,which had producedanti-cancer effect.The experimental results are as follows:The first combination consisted of Group PBS, Group TCL,Group CIMand Group TCL-CIM. On the21st day after tumor cells inoculation a few micewere sacrificed in each group for evaluating the tumor volume, the timors inGroup CIM were obviously smaller than Group PBS(P=0.044) and GroupTCL(P=0.024). The survival of Group CIM compared to Group PBS(P=0.021)and Group TCL(P=0.029) was prolonged,while there was not significant longersurvival in Group TCL-CIM (P>0.05).The second combination consisted of Group PBS,Group TCL, Group CIMDaily and Group TCL-CIM Daily. On the21st day after tumor cells inoculationa few mice were sacrificed in each group for evaluating the tumor volume, thetimors in Group CIM(P=0.004vs PBS; P=0.003vs TCL) and GroupTCL-CIM(P=0.004vs PBS; P=0.003vs TCL) were significantly smaller thanGroup PBS and Group TCL, but there was no statistical difference between them(P>0.05). The suivival of Group CIM Daily compared with GroupPBS(P<0.001) and Group TCL(P<0.001) was significantly prolonged, survivalof Group TCL-CIM Daily compared with Group PBS(P=0.003) and GroupTCL(P=0.005) was aslo significantly Prolonged, but there was no significantdifference between the survival of GrouP CIM Daily and GrouP TCL-CIMDaily(P>0.05).Based on above two combination's experimental results, CIM is notsuitable to be TCL's adjuvant, but CIM itself had an antitumor effect in vivo,thesurvival of Group CIM was extended compared to Group PBS(P=0.021), whilesurvival of Group CIM Daily compared to Group PBS(P<0.001) and GroupCIM(P=0.01) was significantly prolonged.As the same dose of the CIM,increasing application of frequency could get Prominent effectiveness.The lungs with a primary tumour nodule were excised for histologicalexamination on the21st day after tumor cells inoculation. The results showedthat necrosis and a little lymphocytes infiltration could be seen in the centre ofthe tumors in Group CIM daily and TCL-CIM Daily.In order to verify whether CIM could inhibit the growth of Lewis lungcancer cells,2×104cells/pore was mixed with different concentrations ofcimetidine in vitro for48hours, MTT calorimetric assay showingdose-dependent inhibition of Lewis lung cancer cells growth after exposure tocimetidine.In order to verify whether CIM could activate NK cells in spleen of mice,mice were divided into three groups: Group PBS, Group CIM and Group CIMDaily. Group PBS and Group CIM were injected with PBS or CIM on day0,7and14, and Group CIM Daily were injected with CIM every day. Mice weresacrificed on the18th day for obtaining splenocytes, and the splenocytes werestained with FITC-NK1.1mAb, and then analyzed by flow cytometry. Theresults showed that Group CIM Daily with CIM injection for each day could significantly increase NK cells than Group PBS(P=0.016) and GroupCIM(P=0.025). CIM with daily injection could enhance the innate immuneresponse in C57CL/6mice.On the basis of the first combination with Group PBS and GroupTCL-CIM, additional Group TCL-CIM-SP was compared tieh them. Althoughthe survival of Group TCL-CIM compared with Group PBS hadn't showedobvious extension(P>0.05), but survival of Group TCL-CIM-SP wassignificantly prolonged compared to Group PBS grouP (P=0.037).4.MHSP65-TCL induced anti-cancer effect in mice40mice were divided into Group PBS,Group TCL,Group MHSP65andGroup TCL-MHSP65(TCL mixed with MHSP65),with immunization on the-7th,1st,8th,15th day.Orthotopic transplantation models were established on the0day through inoculating with Lewis lung cancer cells at5×103per mouse.Theresult showed that survival of MHSP65was no better than GroupPBS(P>0.05),TCL-MHSP65can significantly prolong the survival time oftumor-bearing mice in Group TCL-MHSP65(P<0.05).In conclusion, preparation of TCL by repeated freeze-thaw methodaccords with the standard for experimental application. Establishment oforthotopic lung cancer transplantation model in mouse is simple and reliable,and provides a good test platform for cancer vaccines.Tumor inhibitionexperiment by TCL combined with CIM showed that CIM as TCL's adjuvantcould not induce significant anti-tumor effect, but CIM itself had an prominentantitumor effect in vivo and vitro, which also show us a new way for thetreatment of lung cancer. The effectiveness of anti-lung cancer inducing byTCL-MHSP65was reconfirmed on orthotopic transplantation model, whichprovids a more adequate evidence for future clinical trials. In short, CIM andTCL-MHSP65may bring new hope for the lung cancer patients.
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