A Quick And Efficient Method For Isolating And Purifying Tumor-infiltrating Lymphocytes From Human Hepatocellular Carcinoma

Objective:One of the essential parts influencing its application to liver cancer is the quantity and condition of TILs generated in vitro.Therefore,it is necessary to establish an economical,efficient,high-quality and repeatable method for isolating and purifying tumor-infiltrating lymphocytes from human hepatocellular carcinoma nests.The expression of immune checkpoint molecules in TILs and its relationship with clinical stage and survival rate were analyzed by bioinformatics.Methods:(1)Existing procedures for isolating and purifying tumor-infiltrating lymphocytes will be simplified.The surgically removed tumor tissue was cryopreserved for the first time,and the tumor dissociation kit was used to prepare the single-cell suspension.TILs were produced using density gradient separation and cultured in vitro.(2)Flow cytometry was used to assess lymphocyte phenotype,and T cell function was identified.(3)TILs will be reprogrammed after magnet-activated cell sorting.(4)T cell single cell sequencing data were taken from the single cell data GSE98638,while hepatocellular carcinoma patient tumor tissue single cell sequencing data were obtained from GSE149614.The expression of immune checkpoint molecules and their relationship with clinical stage and survival rate of HCC were examined after data processing,cell clustering,and cell annotation.Results:(1)The total amount of tumor-infiltrating lymphocytes in human hepatocellular carcinoma nests isolated by improved density gradient separation was(5.54±2.36)×10~6 cells/g.The percentage of CD8+T cells in T cell population was(64.18±7.81)%,as determined by flow cytometry.And it had the ability to secrete related cytokines.(2)The reprogramming of HCC-TILs was unsuccessful.(3)The results of bioinformatics research demonstrated a correlation between the expression of immune checkpoint molecules or their ligands and HCC,as well as a correlation between common clinical classification and survival rate.Conclusions:(1)A stable density gradient separation system for HCC-derived TILs was successfully established.The overall separation steps are convenient,economical and reproducible.(2)The expression of immune checkpoint molecules and their ligands is closely related to HCC;the commonly used clinical classification criteria of HCC are almost unrelated to the expression level of immune checkpoints;immune checkpoint molecules with higher expression in TILs and lower expression of their ligands in HCC cells had improved survival rates.