Study Of Immunotherapy Responses On Human Breast Cancer Cells And Transplantation Tumor Of Naked-mice After Vaccination With MAGE-A3 Peptide Tumor Vaccine

Breast cancer is one of the most common malignant tumors in women.Like many other malignant tumors, breast cancer derived from mutation of itsown cells with a weak antigenicity. The cancer cells can escape from themonitoring system of the body with variety of blocking factors. At the same time,the immunorepressive predominance and the low immunoreaction of somecancer cells, make the development of knubs out of control. Even more,general treatments such as radiotherapy and chemotherapy, do obvious harm tothe immune system. Therefore, experts put eyes on the new immunotherapy ofbreast cancer that can wake up patients' defensive system so as to improve theirimmune function. Dendritic Cell (DC) vaccine shows an attractive foreground asa new immunotherapy. However, the percentage of DC in peripheral blood isjust 0.1% of leucocyte, and cancer cells always excrete some cytokines whichrestrains DC, it means that DC cultured in vitro would be very important forexciting anti-cancer immunological effect when infused in vivo. The key point ofdesigning oncotherapy vaccine is to find out the specific target antigen, and sendit to the immune system, so as to be recognized as the foreign object by immunesystem, that the activated immune system would kill the cancer cell with tumourantigen at last by some very mechanism. Antigen albumen or polypeptide is very important for presentation of antigen and induction of specific immunoreaction,so transfecting antigen to DC for preparing the tumour vaccine by carrier hasbeen one of the hotspots of tumour biotherapy. Because of the immunity reactionand security, making full use of viral carrier is restricted. Taking exogenousDNA for example, when infused in vivo, it could be integrated into the hostgenome, which make the anti-cancer genes of host cells inactive, or cancer genesactive, then would be transformed into cancer cells. Working as in-viral carrier,liposome can be degraded naturally in vivo, without any immunogenicities,obvious toxicities,or side effects, but can be cycle using, which makes the uniqueeffects in cancer antigen carrier vaccine.Antigens encoded with Melanoma associated antigen gene-3 (MAGE-A3)is the HLA restricted tumour-rejection antigens which express just in malignanttumor. They also express in none of the normal tissues but testis and placentas.Cytotoxic T lymphocyte (CTL) didn't have any lethal effects because of noexpression of HLA-I in testis and placentas. At the same time, the close connectsbetween trophocyte in testis and placentas which express CD95 ligand, cominginto a physical and functional barrier, which prevent the entry of CTL and causethe apoptosis of T-cell. All of these shows the potential in target antigen of cancerremedial vaccine.So, the RT-PCR(Reverse Transcription Polymerase Chain Reaction) andthe SSP-PCR(Sequence Special Primer Polymerase Chain Reaction) were firstused apart to observe the expression of MAGE-A3 and histocompatibilityleukocyte antigen-A2 (HLA-A2) in human breast cancer cell line MCF-7,ovarian cancer cell line SK-OV3 and normal vascular endothelial cell lineEC-304. It showed that Mage-A3 and HLA-A2 genes were positive-expressed inbreast cancer cell MCF-7. In ovarian cancer cell line SK-OV3, MAGE-A3 geneswere positive-expressed while HLA-A2 negative-express. The complete reverseresult showed in the normal vascular endothelial cell line EC-304.Then we separated the mononuclear cell from peripheral blood of healthyvolunteers who expressed HAL-A2-positive, amplified in the 1640 culture medium with GM-CSF, IL-4, a-TNF and the other cytokines. The resultsuggested that detected by flow-cytometer, the percentages of CD1a, HLA-DR,CD80, CD83 and CD86 increased step by step, from 2.3%, 46.2%, 18.4%, 8.36%,19.8% by the first day to 14.3%, 57.8%, 42.7%, 9.2%, 47.8% by the third daythen 21.8%, 99.0%, 63.4%, 18.9%, 80.6% the sixth day. It showed that CD83and HLA-â…¡molecules both increased which stood for the maturity of DCs. DCswith specific shape can be seen in high-multi-microscope and electric microscope.The yield of DCs increased from 2.12% by the first day to 9.89% by the thirdday then to 16.31% the sixth day, while purity of DCs increased from 2.08% bythe first day to 61.25% by the third day then to 90.83% the sixth day. Itconcluded that DCs with high purity can be amplified with mononuclear cellfrom the peripheral blood.For preparing MAGE-A3 polypeptide vaccine, 9-peptide amino acidsequences with MAGE-A3 epitope which searched in net, synthesized artificiallyand then marked by fluorescein, analysed by reverse phase high performanceliquid chromatography (RP-HPLC) and mass spectroscopy(MS), were foundthat the purity of polypeptide reached more than 95%, the molecular weight is1292.05. Then we transfected MAGE-A3 polypeptide to the third-day-DCs withBioPORTER as the carrier. The distributing of MAGE-A3 mainly in cellularmembrane and intracytoplasm of DCs were detected by laser scanning co-focalmicroscope(LSCM). The transfection efficiency of MAGE-A3 in DCs weredetected by fluorescence microscope and flow cytometer by the fourth, eighth,and twenty-fourth hour. And the transfection reagent infection to DCs' activitywas observed by MTT (4,5-dimethyiazol-2-diphenyl tetrazolium bromide).Wefound that BioPORTER can transfect MAGE-A3 polypeptide to DCs effectively.Transfection efficiency detected by fluorescence microscope and flow cytometewas positive correlation, correlation coefficient was 0.796(P＜0.01). Flowcytometer is convenient, quickly, well repeatability and nicety compared withconventional microscope. In the condition that PH of reaction system was 7.5,density of the DC suspension was 5×10⁵/ml, proportion of polypeptide and transfection reagent was 4/1, higher transfection efficiency was obtained by thecomposite cultivated 8 hours with DCs. The average transfection efficiency ofMAGE-A3 polypeptide with BioPORTER was more higher than withoutBioPORTER(14.21%±2.83% versus 10.37%±2.25%, P＜0.01) under thisconditions. Even more, it did no obvious cytotoxicity to DCs, the average celllivability was not different from each other (92.58%±4.38% versus93.74%±1.94%, P＞0.05) under the same conditions. All the results provided anapplied experiment method for DC vaccine based on MAGE-A3 antigenpolypeptide.To observe the infection of DC immunity function transfected withMAGE-A3 polypeptide, we added the tumor necrosis factor to the DCssuspension in 8 hours of MAGE-A3 polypeptide transfection, cultivating asstimulus cells for 24 hours. On the other hand, T lymphocyte as effect cells,mixed with 1/20 stimulus cells to cultivate for 6 days, it indicated by flowcytomete that MAGE-A3 polypeptide could accelerated the DC maturation, DCswith transfection reagent would more mature than that with free antigen load(P＜0.05).To detect the affection of MAGE-A3 polypeptides on secretary functionof T lymphocytes which derived from peripheral blood mononuclear cells, the Tlymphocytes were purified with nylon wools firstly. And then the T lymphocyteswere mixed with DCs which delivered polypeptides for 7 days. The content ofIL-12 were detected in the first day, third day and the fifth day respectively byELISA. And the content of IFN-r were detected in the second day, the fourthday and the sixth day respectively also by ELISA. The results showed that theIL-12 contents in supernatant of each culture media increase by degrees,contents of DC group with polypeptide antigen was more than that withoutpolypeptide antigen (P＜0.01), contents of DC group with transfection reagentwas more than that with free antigen load (P＜0.05). The IFN-r contents insupernatant of each group were also increased gradually. Till the fourth day,compared the DC/MAGE3-BIO group with the DC/MAGE group, the DC group without MAGE-A3 polypeptide and single T cells, the IFN-r content insupernatant of culture media increased obviously(P＜0.05). Till the sixth day, thedisparity enlarged (P＜0.01), while the last three group had quiet disparity(P＞0.05). It means that DCs which activeited by MAGE-A3 antigenpolypeptide, can stimulate T cell to excrete IL-12, IFN-r which can excite thespecific immunoreaction. BioPORTER can accelerate the secretion of cytokine toenhance immunoreaction by improving the transfection efficiency of MAGE-A3antigen polypeptide.To evaluated stimulating effects of DCs on the allogeneic T lymphocytes,density gradient method was used to separate the lymphocytes of the healthymen's peripheral blood which had the different origin from the DCs. The96-pore plate disposed with ultraviolet radiation, added apart with the DCsuspension of transfected polypeptide, 1×10⁴, 5×10³, 2×10³ and 1×10³ cells perpore, compared with the self T-lymphocytes, 3 ambo-pores in one. Variantlymphocytes of the same race were added to the ambo-pores(1×10⁵ per pore),that meant the proportions of DCs and T cells were 1/10, 1/20, 1/50 and 1/100,end with 200ul in volume. Cultivated in incubator for 90 hours, 37℃, 5%CO₂,the MTT showed that all transfected polypeptides were more better stimulatingT lymphocyte to proliferate (P＜0.01). DC group with transfection reagent wouldbe more proliferous than that with free antigen load (P＞0.05). The more DCsconcentration was, the more obvious the stimulate would be. MAGE-A3 antigenpolypeptide would enhance the presentation of DC polypeptide. BioPORTERwould enhance that even more while accelerate the proliferation of Tlymphocyte.Observing the colony fission alleosis of CD4⁺ and CD8⁺ T lymphocytesaffected with DC, T lymphocytes which were the homologies of DCs, worked aseffector cells, mixed with 1/20 stimulus cells to cultivate for 6 days. It indicatedby flow cytometer that the contents of CD4⁺ T lymphocytes of theDC/MAGE-BIO group and DC/MAGE group reduced slightly, while that of CD8⁺ T lymphocytes increased sharply. Contents of CD4⁺ and CD8⁺ Tlymphocytes without polypeptide reduced by some degrees. It clued us that DCsstimulatived with MAGE-A3 polypeptide would accelerate the differentiationand proliferation of CD8⁺ T lymphocytes so as to induce the specificityimmunoreaction. Because the main ingredient of eliminating tumor cells withcytotoxic immunological effects was CD8⁺ T lymphocytes, while CD4⁺ Tlymphocytes worked on the excitation moment of immunoreaction but noteffector moment.For finding out that how the MAGE-A3 polypeptide vaccines killstumour target cells, MTT was used for evaluating the infection that each effectorcell group worked on proliferation of target cells. The apoptosis of target cellsinduced by each effector cell group were determined by flow cytometer (kitAnnexin V-FITC, PI-PE). The groups of T-DC/MAGE-BIO, T-DC/MAGE, T-DCwere prepared for 2×10⁷/ml, 1×10⁷/ml, 5×10⁶/ml and 2×10⁶/ml concentration,100ul target cells were mixed with 100ul different effector ceils in 96-pore plate,which meant the proportions of effector cells and target cells were 100/1, 50/1,25/1 and 10/1, three pores in one group. T cells without DCs stimulation mixedwith target cells at the same concentration gradient as the control group, 200uleach pore, three pores in one group. Cultivated in incubator for 12 hours, 37℃,5% CO₂, it showed that lethal effects of MCF-7 with MAGE-A3 andHLA-A2-positive, which affected by effector cells transfected with MAGE-A3polypeptide, was more distinct than the SK-OV3 with MAGE-A3-positive,HLA-A2-negative and EC-304 with MAGE-A3-negative, HLA-A2-positive.Result demonstrated the significant difference (P＜0.01). Lethal effects enhancedby the proportion of E/T. Lethal effects of MAGE-A3 polypeptide group wasmore significance than the control groups of without polypeptide, while therewas no significant difference in the two control groups(P＞0.05). Lethal effects ofpolypeptide group with transfection reagent would be more significant than thatwith free polypeptide (P＞0.05). While the Lethal effects of each effector cell group which acted on SK-OV3 and EC-304 was too low to show the significantdifference (P＞0.05). When tumor vaccine of MAGE-A3 antigen polypeptidemediated with BioPORTER in the proportion of 100/1 and 50/1, lethal effectswere more significant difference than that of free MAGE-A3 antigen polypeptide(P＜0.05). While there was no significant difference in the proportion of 20/1and 10/1 (P＜0.05). There was no significant difference between the two controlgroups (P＞0.05).According to the results hereinbefore, we detect the target cells apoptosisinduced by effector cell group alone in the proportion of 100/1. It showed by flowcytometer that mortalities of MCF-7 cell line affected by MAGE-A3 polypeptidevaccine were distinct higher than that of SK-OV3 and EC-304 cell lines(P＜0.01).Mortalities of MCF-7 cell lines induced by polypeptide groups were higher thancontrol groups without polypeptide (P＜0.01). Significance of polypeptide groupsmediated by transfection reagent were higher than the free polypeptide groups(P＜0.01). Fluorescence microscope also suggested that MAGE-A3 polypeptidevaccine would accelerate the apoptosis of MCF-7. There was no significantdifference between the mortalities of SK-OV3 and EC-304 induced by three DCvaccines (P＞0.05), the same with that of the three cell lines induced by the groupwithout antigens (P＞0.05). These findings demonstrate that lethal and apoptosiseffects of tumor cells induced by MAGE-A3 polypeptide vaccine showed theMAGE-A3 antigenic specificity and HLA-A2 gene restriction. It meant that theeffects were indistinctive for the normal cells and other tumor cells whichexpressed no MAGE-A3 genes or HLA-A2 genes. MAGE-A3 polypeptidemediated by transfection reagent would more induce the lethal and apoptosiseffects of tumor target cells.Feasibility and mechanism of breast cancer treated by MAGE-A3polypeptide vaccine were studied further for founding a breast cancertransplantation tumor model of naked-mice by inoculating MCF-7 cell lines inexponential phase of growth to one-side back subcutaneously of 20 naked-mice (5×10⁶ /120ul/mouse). Production rate of tumor was 95% (19 in 20) after oneweek. The 19 naked-mice were divided into 4 groups in random that 5 mice withMAGE-A3 tumor vaccine mediated by BioPORTER in group A, 5 mice with freeMAGE-A3 tumor vaccine in group B, 5 with single DCs in group C and 4 in D asthe control group with normal saline, 1×10⁶/120ul/mouse DCs or normal salinewere infused into the opposite back of tumor subcutaneously in every seven days,for 4 weeks. Developments of tumor were under continuous dynamicobservations until the mice were executed at the end of the observation.The results shows that the bulk of knubs in group A and B both reducedobservably compared with group C and D (P＜0.01) ,while there was nosignificant difference between group A and B, the same with group C and D(P＞0.05) after one week. The condition changed after two weeks, the bulk ofknubs in group A reduced observably compared with group B(P＜0.01).and thechanges among other groups were the same as before. Such state was continueduntil the end of date. The weight of knubs in group A and B both reducedobservably compared with group C and D (P＜0.01). The weight of knubs ingroup A reduced observably compared with group B (P＜0.01), While there wasno significant difference between group C and D (P＞0.05). Which meant thattumor inhibition ratio increased observably in group A and B. The order oftumor inhibition ratio is: group A＞group B＞group C and group D.And then stained the knub slices with anti-monoclonal antibody of CD8⁺.The results of infiltration densities variety of CD8⁺ T cells in tumor tissuessuggested that lots of CD8⁺ T cells were infiltrated in tumor tissues by staining.Infiltration densities of CD8⁺ T cells were 72.6±9.76 in group A and 55.8±3.70 ingroup B, while 14.2±1.92 in C and 11.2±3.38 in D under per high power field.Infiltration densities of group A and group B were observably higher than C andD respectively by statistics (P=0.000), group A were higher than groupB(P＜0.01),but with no significant difference between group C and D (P＞0.05).Subsistences of the cancer bearing mice in four weeks were that 100% in group A, 100% in B, 60% in C(3 in 5) and 50% in D(2 in 4). After the treatment, twomice died in the third and fourth week respectively in group C, while the othertwo also died the third and fourth week respectively in in group D, whichinterrelated with the knubbly augment. The further statistics treatment didn'ttake because of the small sample size. The present studies have assessed theinhibition and lethal effects of MAGE-A3 polypeptide vaccine for the breastcancer transplanted tumor of naked-mice. Further research would be taken inlarge-sample mice to study the connection between therapeutics of polypeptidevaccine and subsistences.It concluded from the experiment that MAGE-A3 antigen polypeptide canaccelerate the DCs maturated, which came from mononuclear cells of peripheralblood, and submited the antigen to T lymphocyte via HLA-A2 and polypeptidecompounds. The active T lymphocytes then divided, proliferated, and excretedlots of cytokine (IL-12, IFN-r) to start the specificity immunoreaction. CD8⁺ Tlymphocytes educed the magistral specificity cytotoxic, which mechanism couldbe to restrain cells proliferating and accelerate cells to apoptosis. Theimmunological effects were MAGE-A3 antigen specific and limited by HLA-A2genes. It meant that the effects were indistinctive for the normal cells and othertumor cells which expressed no MAGE-A3 genes or HLA-A2 genes. We found inthe breast cancer tissues of naked-mice cured with MAGE-A3 polypeptidevaccine, infiltration density of CD8⁺ T cells increased sharply. The bulk andweight of knubs both reduced, which affected affirmative to the subsistences ofanimals bearing cancer. The increase of transfection efficiency of MAGE-A3polypeptide mediated by BioPORTER would enhance the specificityimmunological effects of effector cells, which acted on tumor target cells andbreast metastatic tumor of naked-mice. Experiments of curing breast cancerwith MAGE-A3 polypeptide vaccine could be based on these resultshereinbefore.