Preliminary Study Of The Construction Of Individualized Tumoroid And Screening For Tumor Neoantigens

Objective:1.The genomic mutation results which obtained from the whole exon sequencing analysis of ovarian cancer patients combined with HLA typing results to predict and screen potential tumor neoantigens or antigenic epitopes.2.The synthesized new antigen is tested for the antigenicity in vitro,screenning new antigenic polypeptides that can stimulate immune response.It'll provide a feasible research method and experimental basis for the application of new antigen in the treatment of ovarian cancer.3.We initially explored the culture system of ovarian orgnoids and provided an individualized specific model for the subsequent experiments that screened new antigenic peptide-induced CTL killing effectence.Methods: 1.The DNA of tissues and whole blood of patients with ovarian cancer was subjected to whole exon sequencing.HLA I typing was predicted by Polysolver.Somatic mutation was detected by Mutect,Mutect2 and edico.ANNOVAR(v2015Jun16)was uesd for SNV/InDel annotation.Combing mutation and HLA typing results,we used theNetMHC4.0,NetMHCpan4.0,PSSMHCpan,PickPocket and SMM to predicte neoantigen.We set the threshold as IC50<20nM and filtered the results.The neoantigen which at least three of the above five softwares predicted an IC50 <20 nM were the final candidate antigetic peptides.2.We synthesized the predicted antigen peptides and then used them to stimulate the human peripheral blood mononuclear cells.Finally,we tested the antigenicity of the new antigen peptide by ELISPOT assay in vitro.3.After being removed within 2 hours of surgical resection,the biospecimen were separated into individual cells,mixing into matrigel and formed a three-dimensional model.we cultured them with new organoids medium.Results:1.We found a total of 783 non-synonymous mutations and predicted 362 new antigen peptides based on these non-synonymous mutations.2.Predicted peptides of Patient 5 were synthesized by taking the top 20 HLA affinity.The ELISPOT experiment showed that the thirteen peptides of P5P3?P5P4?P5P5?P5P7?P5P8?P5P9?P5P10?P5P12 ? P5P13 ? P5P15 ? P5P16 ? P5P17 ? P5P20 demonstrate strong antigenicity in vitro.3.Primary cells cultured in normal medium died within 1 month gradually,but can survive for more than 2 months in organoids medium in 2D.They can also continue to proliferate after standard cryopreservation and recovery.The primary cells cultured in 3D with the organoids medium formed an organ-like sphere at about 3-4weeks.Conclusions:1.By setting the screening conditions and based on DNA mutations and HLA typing information,we successfully predicted HLA high-affinity neoantigens.2.The predicted HLA high-affinity neoantigenpolypeptide can detected strong antigenicity in vitro,and can be uesed for the immunogenic study of personalized ovarian cancer.3.The newly used organoids culture system prolongs the culture time of primary cells of ovarian cancer,and can form an organoids sphere under three-dimensional conditions.