Isolation And Characteristics Of Tumor-infiltrating Lymphocytes(TILs) From Hepatocellular Carcinoma

Background and objective:Primary hepatocellular carcinoma(HCC)is a kind of malignant tumors with high morbidity.Every year,the number of deaths from liver cancer in China exceeds120,000,accounting for about 44%of the deaths of liver cancer worldwide.The mortality rate ranks third cause among all types of tumors.Therefore,immunotherapy,especially cellular immunotherapy,can activate the body's immune function,change the tumor microenvironment(TME),and induce specific tumor immunity to achieve the goal of treating liver cancer and reducing the recurrence and metastasis of liver cancer,which has become the fourth treatment method after surgery,chemotherapy and radiotherapy.Tumor Infiltrating Lymphocyte(TILs)is a tumor antigen-specific lymphocyte population that isolated from tumor tissue.It contains T lymphocytes,B lymphocytes,and NK cells.Lymphocytes can be divided into CD4+T cells and CD8+T cells according to the cell phenotype.Among them,CD8+T cells can play a tumor-killing role,and they are 50-100 times more effective than lymphokine-activated killer(LAK)[2,4-6].Studies have shown that the amount of CD8+TILs is positively correlated with the objective response rate of tumors[4-6].At present,there are relatively few domestic studies on TILs,especially CD8+TILs for the treatment of liver cancer.In this study,we will explore the distribution of CD8+TILs in liver cancer tissues,as well as efficient methods for extracting,cultivating and expanding CD8+TILs.Further,we will verify the immunological characteristics of CD8+TILs and lay the foundation for the future application of autologous CD8+TILs immunotherapy for advanced HCC.Methods:1.Hematoxylin-eosin staining(HE)and immunohistochemical staining(IHC)of liver cancer tissue specimens:take cancer nests and adjacent tissues and make sections,and perform HE staining and IHC towards cell surface CD8 antigen to study the distribution of CD8+TILs in liver cancer and adjacent tissues;2.Isolation and identification of tumor infiltrating lymphocytes(TILs):cancer nests and adjacent tissues of the liver cancer were extracted by enzyme digestion,the viability of TILs was determined by acridine orange(AO)staining,and identification was detected by flow cytometry Expression of markers CD3,CD4,and CD8 antigens and study of the proportion of CD4+TILs and CD8+TILs cells;3.In vitro amplification of TILs and sorting of CD8+TILs:Use the immunomagnetic bead method to sort CD8+TILs among the expanded TILs in vitro;4.CD8+TILs cytotoxicity test:CD8+TILs is compared with non-CD8+TILs,they both co-cultured with hepatoma cell lines(HepG2,Hep3B)respectively according to the effecor-target ratio(E/T)of 1,5,and 10.CCK8 method was used to detect the tumor suppression rate of CD8+TILs and to explore the most effector-target ratio;5.Early exploration of three-dimensional culture:293T cells were transfected with lentivirus carrying the green fluorescent protein(GFP)gene,and the successfully transfected 293T cells were coated with sodium alginate to form microcapsules.Three-dimensional structure culture was established.Then the growth of 293T cells in the capsule can be observated by fluorescent microscope.Further more,TILs were transfected with the same kind of lentivirus;6.Establishment of PDX model for liver cancer:The model was established by subcutaneous transplantation of tumor tissue mass and subcutaneous inoculation of liver cancer tumor cell suspension.The tumor formation method of tumor tissue block is to directly embed the tissue block under the skin;the tumor formation method of tumor cell suspension is to make the tumor cell suspension from the specimen of liver cancer patient after operation by mixed enzyme digestion method,and inoculate the two sides of the nude mouse Subcutaneous inguinal,observe subcutaneous tumor formation.Results:1.Hematoxylin-eosin staining(HE)and immunohistochemical staining(IHC)of liver cancer tissue specimens:Compared with cancer nest tissue,CD8+TILs are mainly distributed in the adjacent tissues,and it is mainly concentrated in the portal area of adjacent tissues.2.Isolation and identification of tumor infiltrating lymphocytes(TILs):AO staining(Day14)showed that the cell viability was(90.5±3.0)%.The proportion of CD3+cells detected by flow cytometry was(77.53±16.37)%,the proportion of CD3+CD4+was(27.08±21.56)%,and the proportion of CD3+CD8+was(44.55±12.73)%.CD3 cell epitope antigen expression was detected by flow cytometry,and the proportion was high,indicating that most of the extracted cells were TILs,and most of them were CD8+TILs.3.TILs was amplified in vitro and CD8+TILs were sorted:the number of expanded cells was(1.2?2.5)×10~8,and the expansion fold was 17-50 times.The amount of CD8+TILs sorted by immunomagnetic beads was consistent with the results of flow cytometry approximately.4.CD8+TILs cytotoxicity experiment:In non-CD8+TILs,when the effector-target ratio(E/T)is 1,5,and 10,correspondingly,the inhibition rates were(0.53±2.21)%,(0.30±1.67)%,(75.00±4.90)%(Hep G2)and(1.29±2.83)%,(1.94±2.04)%,(43.54±1.98)%(Hep3B),while in CD8+TILs,the effector-target ratios is1,5,and 10,the inhibition rates were(1.60±0.23)%,(85.16±3.66)%,(77.74±5.81)%(HepG2)and(3.35±2.59)%,(81.34±2.32)%,(91.03±2.41)%(Hep3B),SPSS software was analyzed by Bonferroni method,and the difference between E/T=5 or 10 and E/T=1 was statistically significant(p<0.05),while E/T=5 and E/T=10 comparison,the difference was not statistically significant(p>0.05).The results suggest that CD8+TILs can play a better tumor suppressive effect when E/T=5.5.Preliminary exploration of three-dimensional culture:Transfected 293T cells can survive in the sodium alginate microcapsules,and the number of cells cultured for 72 hours is the largest.At the same time,TILs can be transfected with the same kind of lentivirus.6.Establishment of PDX model of liver cancer:Subcutaneous inoculation of liver cancer tissue mass was unsuccessful,but subcutaneous inoculation of tumor cell suspension showed signs of subcutaneous tumor formation after about 3 months.Conclusion:This study confirms that CD8+TILs are mainly concentrated in the portal area of adjacent tissues.Compared with tissue block culture and small needle puncture culture,mixed enzyme digestion can efficiently extract CD8+TILs from human liver cancer tissue The improved method can efficiently extract CD8+TILs from human liver cancer tissue and achieve a large number of amplifications.Experiments in vitro have shown that the extracted CD8+TILs has a relatively strong tumor suppressive effect.A preliminary attempt was made to establish liver cancer PDX model of nude mice,moreover,preliminary experiments have initially verified the feasibility of using alginate to coat cells for three-dimensional culture and explansion.