Ionizing Radiation Promotes The Infiltration And Anti-tumor Activity Of Chimeric Antigen Receptor (CAR) Modified NK Cells On Colorectal Cancer

Background and purposeColorectal cancer is a common malignancy,and globally,colorectal cancer(CRC)incidence and mortality are ranked in the 3rd and 2nd place,respectively.In our country,both the incidence and mortality of colorectal cancer are ranked in the 5th place.Moreover,its incidence rate shows an increasing trend year by year,and about 190 000 people die from CRC every year,which seriously threatens people’s health and life.Over the past 20 years,some progress has been made in colorectal cancer treatment with multidisciplinary comprehensive therapies dominated by minimally invasive surgical procedures,however,bottlenecks remain in the improvement of patient outcomes with these treatments.In recent years,adoptive immune cell therapy has gained much attention,especially chimeric antigen receptor(CAR)-modified immune cell therapy,which has achieved remarkable efficacy in the treatment of hematological tumors,and is expected to be a new and breakthrough direction in the treatment of malignancies.Our research group previously introduced a CAR gene targeting carcinoembryonic antigen(CEA)into pluripotent stem cells to establish a CAR-CEA carrier modified pluripotent stem cell line,which was further induced to differentiate into CAR-CEA-NK cells against colorectal cancer,and verified in vivo and in vitro experiments that CAR-CEA-NK cells have better anti-tumor activity on colorectal cancer cells compared to NK cells of other sources.However,our vivo experimental results found that the application of CAR-CEA-NK cells for solid tumor therapy such as colorectal cancer needs to be addressed for their poor infiltration ability into the solid tumor microenvironment.Radiotherapy has a wide range of applications in human solid tumor treatment,and the applications of ionizing radiation can change the tumor microenvironment from an immunosuppressive to an immune activated state.Alteration of the tumor microenvironments promotes the activation of immune cells and the function of their infiltration into solid tumor and antitumor effect.In addition,studies have reported that ionizing radiation also elevates the expression of NKG2D ligands and CEA on the surface of malignant tumor cells,such as lung,prostate,and colorectal cancer,and can further increase the tumor killing efficacy of cytotoxic T cells specific for CEA.Therefore,to further enhance the anti-tumor efficacy of CAR-CEA-NK cells against colorectal cancer,this study aimed to investigate the effects of ionizing radiation on colorectal cancer cells and the tumor microenvironment in combination with CAR-CEA-NK cells and further explored the effects of ionizing radiation on solid tumor infiltration and anti-tumor effect by CAR-CEA-NK cells.Methods1.Detect the expression of CEA and NKG2D ligands and the proliferative activity of colorectal cancer cells under different doses of ionizing radiation;2.CAR-CEA-NK cells were generated in vitro by induction with a previously established serum-free culture system using CAR gene modified pluripotent stem cells;3.Verification the in vitro anti-tumor effect of CAR-CEA-NK cells on irradiated colorectal cancer cells at different doses and determine the optimal irradiation dose;4.Establish murine xenograft model to verifying the tumor killing effect of CAR-CEA-NK cells combined with ionizing radiation;5.After the in vivo experiment,the transplanted tumor of mice was taken for paraffin section,and the expression of transplanted tumor molecules and the infiltration of NK cells in the tumor were detected by immunohistochemistry.Result1.There was no significant change in the expression of CEA or NKG2D ligands detected by flow cytometry in SW837 or Caco-2 colorectal cancer cell lines exposed to different doses of ionizing radiation,however,in LS-174T colorectal cancer cell line,CEA expression was found to increase significantly after irradiation in both flow cytometry and Western blot analysis;2.CAR gene carrier modified pluripotent stem cells induce differentiation to NK cells,obtaining functional CAR-CEA-NK cells;3.low-dose irradiation did not significantly improve tumor killing of CAR-CEA-NK cells,medium and high-dose irradiation could enhance the in vitro killing of LS-174T cell line by CAR-CEA-NK cells to some extent;4.Ionizing radiation promotes infiltration and anti-tumor effect of colorectal solid tumors by CAR modified pluripotent stem cell-derived NK cells in vivo.ConclusionIonizing radiation can promote CEA expression in colorectal cancer cell lines,and can promote the infiltration and killing of colorectal solid tumors by pluripotent stem cell-derived NK cells targeting CEA.