Establishment And Application Of Zebrafish Model For Identification Of Colon Cancer Neoantigens

The mortality and morbidity of colorectal cancer(CRC)is currently increasing year by year and CRC has become the third malignancy tumor in humans.In addition to traditional surgery and chemoradiation methods,individual tumor vaccines based on tumor neoantigens are emerging anti-tumor therapies.Tumor neoantigens are derived from somatic mutations and are tumor-specific because they are not expressed in normal tissues.At present,there are relatively few studies on colon cancer neoantigens at home and abroad.Compared with preclinical immunodeficient mouse models of current cancer research,zebrafish(Danio rerio)has the advantages of high homology to the human genome,short research period,high throughput,dynamic visualization in vivo,no adaptive immunity in pre-development and low cost,and it has gradually evolved into another relatively fast and cost-effective in vivo research model for human cancer.Based on the above,this study aims to construct a simple and effective platform for the screening and identification of neoantigens for colon cancer,and it is expected to apply this model to obtain candidate target antigens.We rely on the ability of zebrafish xenografts to reveal the immunogenicity and killing activity of effector cells and target cells in vivo,so we(1)established zebrafish xenografts,microinjected CM-Dil-labeled T2-A24 cells into 2 dpf zebrafish embryos,continuously cultured for 4 days,found that T2-A24 cells have the ability to proliferate in the zebrafish;(2)In order to determine the appropriate effector-target cells ratio,we microinjected CM-Dil-labeled different numbers of PBMC into 2 dpf zebrafish embryos,cultured continuously,observed that PBMC stably existed in zebrafish,and the effectortarget cells ratio was determined to be 2.5:1;(3)collected peripheral blood from healthy donors of HLA-A*24:02 typing,isolated PBMC,added IL-2 and AKF9(it has been reported in the literature and verified again by us that it is strongly bound to HLAA*24:02 molecules),after 12 days of co-culture,together with AKF9-loaded T2-A24,microinjected into 2 dpf embryos on the same day / every other day to establish zebrafish simultaneous / separate injection-in vivo immune intervention xenograft models,cultured for 3 days,and then observed that compared with about 40% killing efficiency in separate injection,simultaneous injection has a higher killing efficiency,which is about 50%;(4)Based on the neoantigen prediction analysis of colon cancer samples in the TCGA database and exome and transcriptome sequencing datas of peripheral blood,paracancerous tissues,and cancer tissues from 20 clinical colon cancer patients,we selected,designed and synthesized 40 pairs of HLA-A*24:02 and 12 pairs of HLAA*02:01 restricted peptides.We verified immunogenicity in vitro by using T2-Binding,ELIspot,LDH cytotoxicity assays and selected 3 pairs of HLA-A*24:02 and 2 pairs of HLA-A*02:01 restricted peptides with well immunogenicity;(5)Aiming at the above 5 pairs of representative peptides,we constructed simultaneous injection zebrafish models to verify their killing activity in vivo.It was found that 2 pairs of antigen peptides,such as 3-6 and 5-6,compared with their wild type,the mutant peptides can induce T cell activation and kill target cells loaded with corresponding antigens,suggesting that these new antigens have certain in vivo immunogenicity.In summary,this study successfully established the zebrafish immune intervention xenograft model for the first time to identify the immunogenicity and killing activity of neoantigens,which is expected to become a new platform for immunogenicity verification.At the same time,we also applied it to the screening of colon cancer neoantigen peptides for the first time,providing candidate target neoantigens for therapeutic vaccines and cell therapy.