Monoclonal antibodies(mAbs)to human TRAIL receptors(TRAIL-Rs)have been developed in tumor targeted therapy and currently used in clinical Ⅰ~Ⅳtrials.MAbs can bind death-receptors(TRAIL-R1 and TRAIL-R2)and then results in formation of the death inducing signaling complex (DISC)and apoptosis of tumor cells.TRAIL -Rs mAbs have become one of the research and development hotspots of anti -tumor drugs.These mAbs have achieved remarkable results in combination with radio -or chemo-therapy drugs or oth-er agents and immunotherapy in the treatment of tumor and offer a new therapeutic strategy for clinical tumor treatment.

This study was performed to explore the impact of glutamine(Gln)on the anti-tumor immune response of CD8+ T cells and its mechanism.TCGA database was used to analysis the relationship between tumor Gln metabolism and the quantity and functionality of infiltrating CD8+ T cells.CRISPR/Cas9 was employed to knock down GLS expression in mouse MC38 cells,and a mouse tumor model was established.Flow cytometry was conducted to assess tumor proliferation,apoptosis,and the quantity and functionality of tumor-infiltrating immune cells.Lymphocytes isolated from health individuals were treated with Gln-deficient media,complete media or media supplemented with GSH,RSL3 in vitro.Then the apoptosis,the expression levels of GPX4,Lipid-ROS,and effector function protein of CD8+ T cells were detected by flow cytometry.Furthermore,RNA-seq was performed to analyze the differential gene expression on the Gln-depleted CD8+ T cells.Data showed that tumor Gln metabolism was inversely associated with the quantity and functionality of tumor-infiltrating CD8+ T cells.Low expression of GLS in MC38 cells could inhibit C57BL/6 tumor growth,decrease Ki-67 expression,promote casepase-3 expression,increase the amount of tumor-infiltrating immune cells,suppress PD-1,TIM-3,and LAG-3 expression,and enhance CD137,CD107a,IFN-γ and TNF-α expression in tumor-infiltrating CD8+ T cells.RNA-seq results indicated an upregulation of ferroptosis genes TFRC,HMOX1,CYBB and SLC7A11 in CD8+ T cells following glutamine deficiency.Gln deficiency led to lower CD137,CD107a,IFN-γ,GSH,GPX4 expression,increased Lipid-ROS level,and caused cell death in CD8+ T cells.Supplementation of GSH upregulated GPX4 expression,downregulated Lipid-ROS level,and increased IFN-γ secretion in CD8+ T cells.In conclusion,Gln deficiency inhibits the effector function of CD8+ T cells by inducing ferroptosis,and promotes tumor growth.

This study intends to establish a colorectal cancer(CRC)organoid model,expand and isolate CRC-reactive tumor-infiltrating lymphocytes(TILs),screen tumor-specific T cell receptors(TCRs)and perform functional verification,in order to provide a technological platform and research foundation for the clinical transformation of individualized adoptive T-cell immunotherapy for colorectal cancer.An organoid model derived from colon cancer patient tissues was constructed using in vitro 3D culture techniques,which then subjected to HE staining and immunohistochemistry for detecting morphological characteristics and representative molecular expression.Subsequently,CRC organoids were co-cultured with TILs for sorting reactive TILs using flow cytometry,and the characteristics of reactive TCR clones was analyzed through single T cell receptor gene cloning technology.Furthermore,the function of TCRs was verified through cytotoxicity experiments.Morphological analysis and representative molecules(CK20 and CDX2)expression indicated that there is high similarity between colorectal cancer organoids and patient tumors.In the in vitro expanded and cultured TILs,colorectal cancer-reactive T cells with upregulated CD137 expression and increased IFN-γ secretion were screened out successfully,among which TCR2-T cells demonstrated superior tumor reactivity and in vitro tumor killing function.In conclusion,a platform for screening and function validation of reactive TCRs based on CRC-Org has been established,providing a technological platform for the translational application of individualized T-cell therapy for colorectal cancer.