| Hepatocellular carcinoma (HCC) is the third leading cause of cancerous deaths inthe world with killing over600000sufferers annually. Liver transplantation and tumorresection have been proved to be the standard therapies, and radiofrequency ablationand transarterial chemoembolization are the next preferred lines of treatment.Nevertheless, these therapeutic approaches usually could not offer a complete cure, asabout50%of the treated patients experience relapse within3years. Metastasis isusually the main cause in HCC recurrence, and lungs are the most common metastaticspots. Thus, it is extremely necessary to establish a complementary remedy inpreventing and treating HCC metastasis.Tumor growth, invasion and its metastasis are closely correlated with angiogenesis,which is defined as new blood capillaries engendered from pre-existing microvesselsand venules. Vascular endothelial growth factor (VEGF) and basic fibroblastic growthfactor (bFGF) have been proposed to be the crucial endogenous factors. They have a stimulative effect on angiogenesis, causing a series of signal transduction whichinduces endothelial cell (EC) proliferation and promotes EC migration. All theseactions ultimately lead to neovascularization. Microvessel density (MVD) isconsidered as golden standard in assessing tumor angiogenesis, and markers such asFactor VIII, CD31and CD34have been used in exhibiting MVD.At the early stage of angiogenesis, EC sprouting relies mainly upon the enzymaticdegradation of extracellular matrix (ECM). The tumor progressive cascades are alsomediated by the degradation of ECM and basement membrane (BM), which allowsmalignant cells to penetrate through tissue barrier. Up to now, Heparanase (HPSE) isthe only endoglycosidase found that can specifically degrade the heparan sulfate (HS)side chain of heparan sulfate proteoglycans (HSPG) in ECM or at BM, resulting indestructing ECM or BM, releasing multiple kinds of cytokines and facilitating cellularmovements. Some investigations have proved that HPSE is overexpressed in mostmalignancies, including in HCC, and plays a key role in cancer invasion and metastasis.While HPSE is expressed at a relatively low level in mammalian lymphoid organs,leukocytes and platelets, and it is either not expressed or expressed at very low levelsin other normal tissues. Recently, it was discovered that HPSE inhibitors couldeffectively suppress the invasion and metastasis of some malignant tumors. Therefore,HPSE could be regarded as an important tumor associated antigen (TAA) as well as atarget molecule in antitumor treatment. The HPSE precursor protein has a molecularweight of about65kDa. It is a hetero dimmer consisting of two subunits, with amolecular weight of50and8kDa, and the larger one represents the mature activatedform of HPSE. On the basis of human HPSE protein structure and its predictedB-lymphocyte epitopes via bioinformatics, we had designed and synthesized themultiple antigenic peptides (MAP) vaccine in our previous studies, and validated thatits polyclonal antibodies had an anti-invasion potency on HCCLM6cell lines in vitro.In this study, to investigate the in vivo immune impact on human HCC growth andmetastasis, purified antibodies induced by the B-cell MAP vaccine were administratedto tumor-bearing BALB/c nude mice through passive immunity. The results showed that the MAP vaccine could elicit a potent antigrowth and antimetastatic effect in vivo,by virtue of its anti-MAP polyclonal antibodies. This study probably provides newinsights into the immunological prevention of HCC growth and metastasis.【Objectives】(1) To isolat, purify and identify the specific anti-MAP polyclonal antibodiescontained in the rabbit immunized serum induced by HPSE B cell epitope vaccine,during the stage of active immunization.(2) To observe the impacts of the purifiedantibodies on the growth, HPSE enzymatic activity, invasive ability of HCC97H cells.(3) To investigate the antigrowth and antimetastasis effects of the specific anti-MAPantibodies on HCC-bearing nude mice, during the stage of passive immunization.(4)To investigate the anti HCC mechanism of this synthesized HPSE vaccine, and topreliminarily evaluate its in vivo safety.【Materials and methods】(1) The white hair black eye (WHBY) rabbit was immunized with the freshlysynthesized HPSE MAP vaccine to obtain the immunized serum containing specificantibodies against HPSE B cell epitopes.(2) The anti-MAP polyclonal antibodiescontained in the immunized rabbit serum were purified by caprylic acid/ammoniumsulfate (CA-AS) precipitation, and the quantity of the antibodies was determined usingCoomassie Brilliant Blue. Western blot assay was carried out to verify the specificityof the immunized serum; the primary antibody was the commercialized rabbit antihuman HPSE antibody or the purified rabbit anti-MAP antiserum, while thepre-immunized rabbit serum was used as a negative control.(3) The antigrowth impactof the antiserum on HCC97H cells was evaluated by MTT test.(4) The influence onthe HPSE enzymatic activity of HCC97H cells of this antiserum was investigated usingHPSE activity measuring kit.(5) The anti invasive ability on HCC97H cells of thespecific anti-MAP antibodies contained in the antiserum was assessed using woundhealing test and Transwell assay.(6) Tumor-bearing murine models of orthotopic implants using HCC-97H cell line were built in BALB/c nude mice, and Bultrasonography with ultrasonic elasto-test was carried out to confirm the existence ofthe implanted tumors and measure their sizes.(7) Sixty nude mice were randomlydivided into6groups, and the vaccine-derived polyclonal antibodies at the volume of0.2ml were used to immunize the mice in different groups with the antibody dosage of2mg/kg or4mg/kg, by caudal vein injection once or for2times with an interval of2weeks. For the negative control group,0.2ml unimmunized rabbit serum wasintravenously administrated, and Xeloda was intragastric administrated as positivecontrol.(8) Eight weeks after passive immunization, mice were sacrificed and thetumor volume was evaluated and pulmonary micro metastatic foci were counted.(9)Murine sera were separated and were assayed in ELISA cultures for VEGF or bFGFconcentration, referring to the user manual of the ELISA kit.(10) Histological sectionsof the HCC were manufactured and immunohistochemically stained for VEGF, bFGFand CD34, and the immunohistochemical results were evaluated referring to Image proplus6.0software and the method we described previously.(11) Histological sectionsof the spleens and lymph nodes were made and H&E staining was performed in orderto preliminarily estimate the possible impairments of the B-cell MAP vaccine oncertain HPSE positive organs and cells, and the amounts of certain HPSE positiveblood cells were counted. Peripheral blood smears were manufactured and possiblemorphological changes were observed.【Results】1. Active immunization and the isolation, purification and identification of theimmunized WHBY rabbit serumFifty days after the first active immunity in WHBY rabbits using B-lymphocyteMAP vaccine of human HPSE, the specific antibody titer could reach1:140000. Theisolated antiserum was purified successfully by CA-AS precipitation, and theconcentration of the anti-MAP polyclonal antibodies contained in the purifiedantiserum was15.2mg/ml, determined by Coomassie Brilliant Blue assay. In Western blot assay, chemiluminescence showed a very clear band of about50kDa and a clearband of about65kDa in the commercialized antibody group. In the anti-MAPantiserum group, both65kDa and50kDa bands were exhibited distinctly. The proteinof65kDa is most probably the precursor HPSE protein of HCC97-H cells, while the50kDa protein corresponds to its large subunit. In contrast, no band could be detectedin the corresponding locations for the negative control.2. The influence of the vaccine-derived specific antibodies on growth, HPSEenzymatic activity and invasive ability of HCC97H cells.The specific polyclonal antibodies derived from HPSE MAP vaccine had noinfluence on the growth of HCC97H cell line, according to the results of MTT assay.Compared with the unimmunized WHBY rabbit serum, the HPSE enzymatic activityof HCC97H cells decreased by51%(P<0.01) and15.8(P<0.05) after being incubatedwith the anti-MAP antibodies at the concentration of100μg/ml and50μg/ml,respectively. The results of the scratch assay demonstrated that the antibodies inducedby HPSE B cell MAP vaccine had no anti migration impact on HCC97H cellsthemselves without the existence of ECM, and the wound healing rates had nostatistical differences among different groups (P>0.05). However, Transwell assayillustrated that being compared with the unimmunized WHBY rabbit serum, the48htrans-membrane rate of HCC97H cells decreased by36.5%(P<0.05) and5.8(P<0.05)after being treated with the anti-MAP antibodies at the concentration of100μg/ml and50μg/ml, respectively.3. Results of passive immunizationBoth the pulmonary metastatic foci and the orthotopic HCC volumes in theanti-MAP antibody treating groups were much fewer/less than that in either negativecontrol group or positive control group (P<0.05). Moreover, the foci and the volume in4mg/kg groups were also markedly fewer/less than that in2mg/kg groups (P<0.05),which demonstrated a certain dose-dependent effect. However, the frequency ofpassive immunization had not affected the outcomes. To quantitatively evaluate theimpacts of the polyclonal anti-MAP antibodies on serous concentration of VEGF and bFGF, which were released mostly by HPSE enzymolysis, we analyzed the serumVEGF and bFGF level by ELISA as well. The mean concentration of VEGF and bFGFin experimental groups treated with anti-MAP antibodies at different concentrationsand different administrative frequency were much lower than that in either negativecontrol group or positive control group, with showing a certain dose-dependence effect.According to the scoring standard of the immunostaining and the help of Image proplus6.0software, the expression of VEGF, bFGF and CD34(representing the meanvalue of MVD) in experimental groups treated with anti-MAP antibodies at differentconcentrations and different administrative frequency were much lower than that ineither negative control group or positive control group (P<0.05), and their expressionswere also markedly weaker than that in2mg/kg groups (P<0.05), which showed acertain dose-dependent effect. However, the outcomes in groups immunized twice hadno statistical advantage compared with that in groups immunized only once (P>0.05).4. HPSE associated impairments had not been detectedNo obvious impairments were detected in H&E-staining pathological slices ofspleens and lymph nodes. Moreover, no morphological changes of rabbit or murineblood cells such as lymphocytes, neutrophils and platelets were observed in peripheralblood smears. The rabbit blood cells were counted in triplicates before immunizationand shortly after sacrifice, the results showed that there were no significant disparitiesin lymphocyte numbers (P>0.05), leukocyte numbers (P>0.05) and platelet numbers(P>0.05). Certain HPSE positive blood cells were also counted shortly after all micewere euthanized, there were no statistical differences in murine lymphocyte numbers(P>0.05), leukocyte numbers (P>0.05) and platelet numbers (P>0.05) among allgroups.【Conclusions】In conclusion, this study suggests that the synthesized MAP vaccine based on B-cellepitopes of human HPSE is capable of attenuating human HCC growth and metastasisin vivo, which is probably induced by suppressing HPSE activity and tumor associated angiogenesis, by virtue of the anti-MAP polyclonal antibodies. Furthermore, theseHPSE-specific antibodies do not cause obvious impairments in certain HPSE positiveimmune organs and cells. Therefore, our investigations provide theoretical evidencefor the clinical use of synthesized MAP vaccine based on B-cell epitopes of HPSE intreating HCC and preventing HCC metastasis. |
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