Study On Action Targets Of Thomsen-Friedenreich Monoclonal Antibody (A78-G/A7) In Papillary Thyroid Carcinoma

Objective:Based on our previous findings that there is overexpression of Thomsen-Friedenreich antigen(TF-Ag)in Papillary Thyroid Carcinoma(PTC),To explore the action targets of Thomsen-Friedenreich antibody(TF-Ab)on PTC,and to provide theoretical and experimental basis for further exploration of the mechanism of TF-Ab in the therapeutic effect of PTC and the research and development of PTC targeted therapeutic drugs.Methods:(1)Exploring the potential role of Thomsen-Friedenreich monoclonal antibody(mA78-G/A7)in thyroid cancer cell lines:The thyroid cancer cell lines with high positive expression rate of TF-Ag were detected by immunofluorescence assay.BCPAP and 8305C cells were treated with 500?g/?L original mAb A78-G/A7 in accordance with the gradient concentration(1/10,1/20,1/40,1/60,1/80)and then the adhesion test was carried out to select the lowest effective concentration of mAb A78-G/A7 in the treatment of BCPAP and 8305C cells.After the intervention of BCPAP and 8305C cells with the minimum effective concentration of mAb A78-G/A7,CCK8 kit was used to detect cell proliferation,cell cycle and apoptosis were detected by flow cytometry,and cell invasion and cell migration were detected by Transwell assay,in order to observe whether the intervention of mAb A78-G/A7 had anti-cancer effect on PTC cell lines.(2)Study on the therapeutic effect of Thomsen-Friedenreich monoclonal antibody(mAb A78-G/A7)on nude mouse PTC xenograft tumors:Fresh tumor tissues from PTC patients were collected to construct nude mouse PTC xenograft tumor models.The growth characteristics,blood supply,HE staining and TF-Ag expression of different transplanted tumors from the same patient were contrastively analyzed to determine the reliability of the comparative study between them.After that,different nude mouse PTC xenograft tumor models derived from the tumor tissues of the same patient were randomly divided in the Ctrl group(in situ injection of 0.9%normal saline twice a week)and 0.5?g/?l treatment group(in situ injection of 0.5?g/?L mAb twice a week)A78-G/A7)and 2.5?g/?l treatment groups(2.5?g/?L mAb A78-G/A7 by in situ multi-point injection twice a week).Each group was further divided into 1-week treatment group and 2-week treatment group.Photos,measurements and records of the tumor were taken before each injection.To observe the therapeutic effect of mAb A78-G/A7 intervention on PTC xenograft tumor in nude mice.(3)Study on the effect of TF-Ag monoclonal antibody(mA78-G/A7)therapy on differential expression gene of PTC:The differentially expressed genes of PTC tumor tissues compared with paracancerous tissues were screened out by RT2 Profiler PCR Arrays(N=2).More tissue samples were collected for Q-PCR verification,and stable differentially expressed genes were found in PTC tumor tissues when compared with adjacent tissues(N=6).Bioinformatics technology mined TCGA database to analyze the differentially expressed genes in PTC tumor tissues compared with paracancerous tissues.The differentially expressed genes obtained by the two methods were combined and compared to screen out PTC differential genes with high probability.Illumina sequencing platform was used to analyze the effects of mA78-G/A7 intervention on the eukaryotic transcriptome of PTC cells,and the sequencing results were compared with the differentially expressed genes of PTC to screen out the possible gene targets of mA78-G/A7 for the treatment of PTC.Further,qRT-PCR,Western-blotting and immunohistochemical techniques were used to verify the possible gene targets of mAb A78-G/A7 for the treatment of PTC.(4)Clinical samples were analyzed to explore the expression characteristics of TF-Ag and natural TF-Ab in PTC patients,and to explore the evidence of TF-Ab targeting TF-Ag and playing a potential anti-cancer role:immunofluorescence was used to analyze the expression of TF-Ag in tissue chips which embedded PTC tumor tissues,and the correlation analysis was conducted with the characteristics of patients' case samples(multifocal,lymph node metastasis,pathological length diameter,TSH and TG expression levels).The expression characteristics of natural TF-Ab in serum were detected by ELISA and the correlation between TF-Ag and TF-Ab was analyzed to explore the evidence that TF-Ab could target TF-Ag.The correlation analysis of natural TF-Ab expression characteristics with some prognostic related clinical indicators and PTC differential genes with high probability was conducted to explore the potential value of natural TF-Ab expression characteristics in early non-invasive screening and prognosis judgment of PTC and to provide evidence for the anti-cancer effect of TF-Ab in PTC.Results:(1)The potential role of Thomsen-Friedenreich monoclonal antibody(mA78-G/A7)in thyroid cancer cell lines:BCPAP was selected as the experimental cell line as a PTC cell line with a relatively higher positive rate of TF-Ag expression(79.460±2.033).8305C,as ATC cell line with relatively higher positive rate of TF-Ag expression(30.880±0.699),was used as auxiliary control cell line to observe the therapeutic effect of TF-Ab on different pathological subtypes of TC cell lines.After mAb A78-G/A7 treatment,the adhesion index of PTC cell lines was significantly higher than that of Control group,and the cell line adhesion inhibition index decreased with the increase of antibody concentration,with a ratio of 1/10(BCPAP:0.411 ±0.008;8305C:0.635±0.065)and 1/20(BCPAP:0.407±0.022;8305C:0.735±0.007)antibody concentration showed the most significant inhibition effect.Thus,1/20 antibody concentration was selected.Immunofluorescence verification of neutralization efficiency of TF-Ag at 1/20 mAb A78-G/A7 showed that after treatment with 1/20 mAb A78-G/A7,the expression of TF-Ag in BCPAP and 8305C group was significantly lower than that in Control group.It is suggested that 1/20 mAb A78-G/A7 concentration can effectively neutralize the expression of TF-Ag.The results of CCK8 kit showed that the proliferation index of 8305C cells after treatment with mAb A78-G/A7 was significantly decreased compared with that of Control group(0.569±0.018 vs.0.7987± 0.029,P<0.05).The proliferation index of BCPAP cells treated with mAb A78-G/A7 was not significantly changed compared with Control group(0.357±0.009 vs.0.382±0.019,P>0.05).Cell cycle test results showed that after treatment with mAb A78-G/A7,the G1 cells of BCPAP and 8305C were significantly increased compared with those of Control group(BCPAP:48.300± 1.193 vs.42.056± 1.156,8305C:22.377± 1.159 vs.10.473±0.866,P<0.05).The apoptosis test results showed that the apoptosis rate of 8305C was significantly increased compared with Control group after treatment with mAb A78-G/A7(62.910± 2.311 vs.42.270 ± 2.284,P<0.05).The apoptosis rate of BCPAP cells treated with mAb A78-G/A7(27.090±0.584)was not significantly different from that of Control group(27.090± 0.584 vs.24.650 ± 0.879,P>0.05).Transwell test results showed that after treatment with mAb A78-G/A7,the number of invaded cells and migrated cells(73.670±4.667,16.670±2.404)of BCPAP and 8305C were significantly higher than those of Control group(invasion:147.700±4.910,85.670±3.528;migration:176.300±5.812,47.330±1.764)was significantly decreased(P<0.05).(2)The therapeutic effect of Thomsen-Friedenreich monoclonal antibody(mA78-G/A7)on PTC xenograft tumor in nude mice:There were no significant differences in the growth characteristics,blood supply,HE staining and TF-Ag expression of different transplanted tumors constructed from the same patient,which could be further studied by comparison.The results of further mAb A78-G/A7 intervention showed that the transplanted tumors in the mAb A78-G/A7 treatment group subsided more rapidly than that in the Ctrl group,and the regression was more obvious in the 2-week treatment group than in the 1-week treatment group.With the passage of time,the regression in the 2.5?g/?l treatment group was more significant than that in the 0.5?g/?l treatment group,but there was no statistical significance for the moment.Immunofluorescence detection showed that the expression of TF-Ag in the treatment group was significantly decreased compared with that in the Ctrl group.The 2-week treatment group compared with the treatment group for 1 week decreased more significantly(0.5?g/?l treatment group:0.013±0.004 vs.0.055±0.005,2.5?g/?l treatment group:0.001±0.000 vs.0.040±0.004,P<0.05),and there was also significant difference between the treatment group and the treatment group for 0.5(P<0.05).(3)Study on the effect of anti-TF-Ag monoclonal antibody(mAb A78-G/A7)treatment on differentially expressed genes of PTC:Nine possible PTC differential genes were screened out by using RT2 Profiler PCR Arrays combined with bioinformatics to mine TCGA database.Based on the sequencing results,PTC cells treated with mAb A78-G/A7 showed more differentially expressed mRNAs than PTC cells without special treatment.However,compared with the above 9 PTC genes with higher possibility,it was found that only three genes,DDIT3,DDB2 and E2F4,showed statistically significant changes after the intervention of mAb A78-G/A7.Furthermore,qRT-PCR,Western blotting and immunohistochemical detection confirmed that mAb A78-G/A7 intervention could effectively affect the expression of DIT3,DDB2 and E2F4 differently expressed PTC genes.(4)Clinical samples were analyzed to explore the expression characteristics of TF-Ag and natural TF-Ab in PTC patients,and to explore the evidence of TF-Ab neutralizing TF-Ag and playing a potential anti-cancer role:Immunofluorescence analysis of clinical tumor tissues of PTC patients showed that TF-Ag was expressed in all tumor tissues of PTC patients,and the expression of TF-Ag in patients with lymph node metastasis was higher than that in patients without lymph node metastasis.The corresponding clinical serum samples were collected and analyzed by ELISA.Compared with the healthy population,the expression characteristics of natural TF-Ab in PTC patients were significantly different,and the serum concentration of IgG subtype was significantly negatively correlated with the expression of TF-Ag in the corresponding tumor tissue,which provided certain evidence for the neutralizing of TF-Ab.In addition,the expression characteristics of natural TF-Ab were significantly correlated with the characteristics of some patients' clinical cases,which had a certain potential value in early non-invasive screening of PTC and prognosis judgment.More importantly,serum concentrations of natural TF-Ab IgG subtypes in PTC patients were significantly positively correlated with PTC differentially expressed genes DDIT3,DDB2,and E2F4,further confirming the possible molecular mechanism of TF-Ab treatment in PTC.Conclusions:mAb A78-G/A7 showed significant antitumor effects on PTC cell lines and xenograft in nude mice.The intervention of mAb A78-G/A7 could significantly affect the differentially expressed PTC genes DDIT3,DDB2 and E2F4.The serum concentration of natural TF-Ab IgG in PTC patients was negatively correlated with TF-Ag,which provided evidence for TF-Ab's neutralizing of TF-Ag.The analysis of the expression characteristics of natural TF-Ab subtype antibodies has certain potential value in early non-invasive screening and prognosis judgment of patients with initial PTC.The serum concentration of natural TF-Ab IgG subtype was significantly positively correlated with PTC differential genes DDIT3,DDB2 and E2F4,which further confirmed the possible molecular mechanism of TF-Ab treatment in PTC.