Study Of Cholesterol Metabolism And Antitumor Response Of CD8~+ T Cells In Tumor Microenvironment And CRISPR/cas9 Gene Editing Based Screening Library Construction Of Metabolic Genes

PART Ⅰ Study of cholesterol metabolism of CD8+T cells in tumor microenvironmentBackgroundAlthough T-cell-based immunotherapy has achieved good results in tumor treatment,there are still some limits due to the immunosuppressive effect and complex regulation mechanism of tumor microenvironment.Studies have shown that in addition to immunosuppressive cells and cytokines,metabolic regulation,which is an important part in T cell immune response and anti-tumor immune response,is also involved in the formation of immunosuppressive microenvironment.Cholesterol and its metabolism pathway play a key role in the process of regulating tumor microenvironment to remodel cholesterol metabolism of T cells and to mediate immune escape,but its mechanism is still not clear.The purpose of this study is to explore the changes of cholesterol metabolism of CD8+T cells in tumor microenvironment and its mechanism of regulating CD8+T cell function and anti-tumor activity,so as to explore new methods of tumor immunotherapy.MethodsIn order to understand the cholesterol metabolism in tumor microenvironment,we used immunohistochemistry(IHC)technology to detect Apolipoprotein B(APOB)content in colorectal cancer,lung cancer,breast cancer tissue sample and in tumor tissue of MC38 cell caecum implant model and B16F10 melanoma lung metastasis model.Then,the model of mouse MC38 cells subcutaneous tumor was constructed,free cholesterol in mouse spleen CD8+T cells and tumor infiltrated CD8+T cells were labeled with filipin III and then was detected by laser confocal imaging.In addition,spleen cells of OT-1 transgenic mice were cultured and differentiated into mature CD8+T cells(CTL).CTLs were transferred into tumor bearing mice by tail vein injection.Then tumor infiltrated CD8+T cells(CD8+TILs)were isolated on the 3 and 7 days post injection.Meanwhile,naive CD8+T cells were isolated from the spleen of OT-1 transgenic mice and then the transcription levels of genes related to cholesterol metabolism pathway were detected by quantitative PCR(qPCR).Meanwhile,the LDLR level of tumor infiltrating CD8+T cells and the immune response of CD8+T cells in LDLR knockout mice were detected by flow cytometry(FC).Finally,we measured the tumor growth of Rag2-/-immunodeficient tumor-bearing mice after adoptive transfer of LDLR-OE-CTLs in order to explore the effect of LDLR on the anti-tumor activity of tumor infiltrating CD8+T cells.ResultsLdl/cholesterol content in colorectal cancer,lung cancer,breast cancer sample,mouse MC38 tumor planting sample and B16F10 lung metastasis cancer sample was higher than that of normal tissues,but the level of cholesterol in tumor infiltrated CD8+T cell was significantly lower than that of spleen CD8+T cells.While the cholesterol synthesis in CD8+T cells was inhibited after infiltration into the tumor microenvironment,the transcription of LDLR in CTLs is also decreased.In addition to the result of lower clonal expansion and effector function,LDLR knockout induces the impairment of immunological synapse formation and the cytotoxicity of CD8+T cells.We also discovered that overexpression of LDLR significantly enhanced the antitumor activity of CD8+T cells both in vitro and vivo.ConclusionThe tumor microenvironment is rich in cholesterol while the cholesterol uptake of tumor infiltrating CD8+T cells is inhibited.LDLR can regulate the immune response and anti-tumor activity of tumor infiltrating CD8+T cells.PART Ⅱ CRISPR/Cas9 gene editing based screening library construction of metabolic genesBackgroundCRISPR/Cas9 gene-knockout technology has become a powerful and accurate tool which can directly link genetic factors corresponding to specific phenotypes with biological phenomena in forward genetic screening.The gene-knockout library formed by sgRNA targeting specific genes under the function of Cas9 protein is an effective platform for phenotype screening.Those various gene knockout libraries builted based on this technology have been widely used in gene screening of apoptosis,drug resistance,tumor function and so on.The purpose of this study is to construct a knockout library of genes targeting lipid and mitochondrial metabolism in colon cancer cells by CRISPR/Cas9 technology,and to prepare for the screening of genes related to tumor growth and drug resistance in colon cancer cells.MethodsThe sequence of blue fluorescent protein(BFP)is acquired by polymerase chain reaction(PCR),and it was clone into LentiGuide vector using seamless cloning technology.Then,the stable cell line of mouse colon cancer cell expressing Cas9 protein is constructed by virus infection.The knockout function is verified by Western blot and Pdcdl gene knockout.Finally the sgRNA oligo of genes targeting lipid and mitochondrial metabolism is cloned into the LentiGuide-BFP vector to build the screening library.ResultsWe constructed a screening vector of plasmid LentiGuide-BFP with labeled fluorescent protein and stable cell lines of mouse colon cancer cell expressing Cas9 protein.And we built a gene knokout library of genes targeting lipid and mitochondrial metabolism.ConclusionIn the experiment,we successfully clone the blue fluorescent protein gene into the recombinant vector,construct the stable cell lines of mouse colon cancer cells with Cas9 protein expression and build the plasmid gene knockout library.