Human Esophageal Cancer Stem Cells Resistant To Natural Killer Cell-Mediated Cytotoxicity Via Down-regulation Of ULBP-1

Background and PurposeEsophageal cancer is a common malignant tumor of the digestive tract and one of the major cancer-related deaths all over the world.About half of the diagnosed cases of esophageal cancer worldwide occured in China.The pathological types of esophageal cancer are mainly divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma.The predominant pathological type of esophageal cancer that occurs in China is ESCC.Due to the lack of obvious symptoms at the early stage and the invasive of esophageal cancer,most patients are in the locally advanced or late-stage when they are first diagnosed.Furthermore,traditional therapies cannot effectively improve the prognosis of patients.Therefore,it is necessary to further study the key mechanism of the occurrence and progression of esophageal cancer and develop new strategies to improve the prognosis of patients with esophageal cancer.Increasing studies have shown that there exist cancer stem cells(CSCs)subgroup with ability of self-renewal and multi-differentiation potential in tumor cells,which are involved in tumor metastasis,recurrence,resistance to traditional radiotherapy,chemotherapy,and immune escape.CSC was firstly isolated from the peripheral blood of patients with acute myeloid leukemia.It was subsequently confirmed in a variety of cancers including breast cancer,colon cancer,melanoma,esophageal cancer and so on.The CSC theory provides new ideas and potential therapeutic targets for anti-tumor therapy.However,there is currently no effective clinically method to remove CSCs in clinical.Natural Killer(NK)cells belong to the innate immune system and play a vital role in the process of anti-tumor.In recent years,With the continuous remarkable progress have been made in immunotherapy,increasing researches focus on the cytotoxicity of NK cells against CSCs,however,there has no agreement at present.Most importantly,there has little researches focus on the sensitivity of esophageal CSCs to NK cells.It will be helpful to remove esophageal CSCs with the further analysis of the sensitivity of esophageal CSCs to NK cell cytotoxicity.This project aims to explore the sensitivity of esophageal cancer stem cells to NK cells cytotoxicity and explore the main molecular mechanism,so as to provide new theoretical basis for the elimanation of esophageal cancer stem cells.Methods1.We enriched esophageal CSCs using stem cell culture medium,determined the expression of stemness-associated markers and ligands for NK cell receptors on CSCs by qPCR.2.Healthy donor peripheral blood-derived NK cells were expanded in vitro,the proportion of expanded NK cells and the expression of CD 107a,IFN-γ,and Granzyme B were detected by using flow cytometry.3.NK cells were incubated with fluorescent dyes CFSE labeled target cells(both esophageal cancer cells and esophageal CSCs)at various effector:target ratios for 6 hours,the apoptosis of target cells and the cytokines secreted by NK cells in the co-culture supernatants were detected respectively.4.The surviving esophageal cancer cells were collected after co-incubating with NK cells for 6 hours and the stemness features were detected.5.We constructed ULBP-1 stable knockdown and overexpression esophageal cancer cell lines to verify whether ULBP-1 is involved in the process of NK cells killing esophageal cancer cells and esophageal CSCs.6.ULBP-1 stable knocdown and overexpression esophageal cancer cells were cultured in stem cells culture medium.The cytotoxicity assay was performed to determine the sensitivity of target cells to NK cells cytotoxicity.7.The expression of stemness associated markers on the all-trans retinoic acid treated esophageal CSCs were tested by qPCR,flow cytometry was used to test the sensitivity of treated CSCs against NK cells cytotoxicity.Results1.qPCR showed that,as compared with adherent esophageal cancer cells,tumor spheres have higher expression of stemness-associated genes,suggesting that the sphere formation culture could enrich esophageal CSCs.The expression of ligands for NK cells receptors in spheres were changed.2.We performed flow cytometry to test the percentage of NK cells and the expression of cytokines on NK cells suggesting that the NK cell amplification and cluture system is sufficient to expand activated NK cells.3.Cytotoxicity assay showed that human esophageal CSCs resistant to NK cell cytotoxicity by doregulating ULBP-1,the ligand of NK cells activating receptor NKG2D.4.The results showed that tumor cells survived from incubation with NK cells with increased stemness features and downregulated ULBP-1,these further varified the cells resistant to NK cells cytotoxicity are CSCs.5.Cytotoxicity assay showed that knockdown of ULBP-1 on esophageal cancer cells resulted in resistance to the cytotoxicity of NK cells.Inversely,the overexpression of ULBP-1 on esophageal cancer cells makes esophageal cancer cells more sensitive to NK cell-mediated cytotoxicity.Suggesting ULBP-1 participate in the process of NK cells killing.6.The results of cytotoxicity assay showed that the decrease of ULBP-1 induced the resistance of esophageal CSCs to NK cells.7.All-trans retinoic acid induced the re-differentiation of esophageal CSCs and reversed the resistance of esophageal CSCs to NK cell-mediated cytotoxicity,the inversion may be associated with the upregulation of ULBP-1.ConclusionThis study firstly reveals that human esophageal CSCs resisistant to NK cells cytotoxicity via downregulation ULBP-1.The addition of all-trans retinoic acid induced the differentiation of human esophageal CSCs and may inversed the resistance of human esophageal CSCs to NK cells cytotoxicity by increasing the expression of ULBP-1.These findings reveal a new mechanism by which esophageal CSCs escape from NK cells immune surveillance and provide a theoretical basis for the combination of immunotherapy and stem cell targeted therapy.