The Function And Mechanism Of ADCY7 Screened By CRISPR/Cas9 Library On T Cell-Mediated Immunity In Hepatocellular Carcinoma

Background and PurposeHepatocellular carcinoma（HCC）is the third leading cause of cancer-related deaths worldwide,characterized by low early detection rates,high recurrence and metastasis rates after surgery.Despite significant advances in surgical and drug treatments in recent years,most HCC patients still have poor prognosis with a five-year survival rate of only 15%.Immunotherapy has shown powerful anti-tumor activity in various cancer treatments,such as PD-1/PD-L1 antibodies which have demonstrated significant efficacy in advanced melanomaand non-small cell lung cancer patients.Current immunotherapy mainly targets CD8⁺T cells in the tumor microenvironment,by activating or enhancing their tumor-killing ability to achieve tumor inhibition.However,it is disappointing that immunotherapy is only effective in a small proportion of HCC patients,and most patients still experience disease progression over time.This is partly due to the fact that tumor cells can constantly evolve to escape immune recognition,and partly because tumor cells can reshape the microenvironment to suppress CD8⁺T cell infiltration and function,for example by recruiting regulatory T cells（Treg）and myeloid-derived suppressor cells（MDSCs）to inhibit CD8⁺T cell function,thereby accelerating tumor progression.Therefore,in-depth research on the molecular mechanisms of HCC cell regulation on T cell immunity and the discovery of new drug targets have important clinical significance and social value for improving the effectiveness of HCC immunotherapy.The whole-genome CRISPR/Cas9 library screening technology has been successfully applied in vitro or in vivo experiments to identify common essential genes for survival and specific cancer type-dependent survival genes in different types of tumors.By combining clinical therapeutic drugs or anti-tumor immune cells,such as sorafenib or CD8⁺T cells,key genes resistant or sensitive to these treatments can be screened,providing important theoretical basis and specific targets for cancer treatment.However,in vitro cell screening models lack a complete immune microenvironment and cannot effectively screen key genes regulating the immune microenvironment.In this project,we used CRISPR/Cas9library screening technology and bioinformatics analysis to systematically screen candidate genes regulating T cell immunity in HCC based on three mouse models with different immune backgrounds:wild-type immune-competent C57BL/6J mice（wild type,WT）,T cell immune-deficient mice(Rag1^null),and severely immunodeficient mice（NSG）.We explored the regulatory effects and molecular mechanisms of key genes through clinical samples and in vitro and in vivo experiments,aiming to provide new targets for immunotherapy of HCC.Methods1.Utilizing Cas9 lentivirus to infect Hepa1-6 cells and establish stable cell lines that express Cas9.Subsequently,we infected the Hepa1-6 stable cell line with the mouse Ge CKO v2 library,and screened for positive cells using puromycin selection.These cells were then subcutaneously implanted into three types of mice with different immune backgrounds,including WT,Rag1^null,and NSG.Tumor tissues were collected 14 days later,and genomic DNA was extracted and subjected to deep sequencing after PCR amplification.We used the MAGe CKFlute algorithm to screen for differentially expressed genes and identified candidate genes that may regulate T cell immunity in HCC.2.Using publicly available HCC transcriptome expression profile data and clinical prognosis data from the GEO and TCGA databases,the potential key genes were identified that are significantly positively correlated with T cell-mediated anti-tumor immune responses（Pearson coefficient>0.5,P<0.05）and differentially expressed in HCC patients with a significant impact on prognosis（P<0.05）.3.Utilizing single-cell transcriptome data on HCC from the GEO public database（GSE149614）to analyze the potential impact of the key gene adenylyl cyclase 7（ADCY7）on the immune microenvironment of HCC.4.Using a constructed chip array containing 155 pairs of HCC tissues and corresponding adjacent tissues to validate the expression level of ADCY7 protein in HCC,its impact on patient prognosis,and its relationship with CD3⁺T and CD8⁺T cell infiltration.5.We constructed stable cell lines overexpressing ADCY7 using lentivirus,and assessed the impact of ADCY7 on HCC cell proliferation.CD8⁺T cells were isolated from peripheral blood of healthy individuals and co-cultured with HCC cells overexpressing ADCY7 or control cells to evaluate the effects of ADCY7 on CD8⁺T cell migration using Transwell migration assay.6.Using HCC cells overexpressing ADCY7 or control cells,we established a mouse xenograft model to examine the impact of ADCY7 on HCC growth and immune microenvironment using immunohistochemistry and flow cytometry,in order to determine the important role of ADCY7 in T cell-mediated immunity.7.Utilizing the HCC dataset from the GEO public database,KEGG,GO,and GSEA enrichment analyses were performed to explore the key signaling pathways influenced by ADCY7.QPCR and western blot experiments were conducted to investigate the effect of ADCY7 on the expression of the key gene CCL5 in enriched pathways.8.Immunohistochemistry,cellular fraction protein extraction（cell membrane,cytoplasm,and nucleus）,and western blot experiments were performed to determine the localization of ADCY7 protein in HCC tissue and cells.Chromatin immunoprecipitation experiments were conducted to investigate the binding of ADCY7 to the upstream promoter region of the CCL5 gene.The importance of CCL5 in the anti-tumor immune response mediated by ADCY7 was explored through subcutaneous tumor implantation in immunocompetent mice and humanized mice with intact immune systems.9.Co-immunoprecipitation experiments,cellular fraction protein extraction（cell membrane,cytoplasm,and nucleus）,and nuclear transport protein inhibitors were used to explore the molecular mechanism of ADCY7 protein translocation from the cell membrane to the nucleus.10.Using ultracentrifugation,exsomes secreted by HCC cells with ADCY7overexpression and control cells were separated（named OE-exo and NC-exo,respectively）.Through transmission electron microscope,nanopartical tracking analysis,liquid chromatography-tandem mass spectrometry and western blot experiments to verify whether ADCY7 is present in exosomes was performed.11.Tumor cells were co-cultured separately with NC-exo and OE-exo in order to determine whether exsomal ADCY7 could enter into tumor cells using immunofluorescence.12.A mouse tumor model was established using Hepa1-6 cells to investigate the effects of exosomal ADCY7 on HCC growth by intratumoral injection of NC-exo and OE-exo.The impact of exosomal ADCY7 on the HCC immune microenvironment was evaluated using flow cytometry and immunohistochemistry in order to highlighting the significant role of exosomal ADCY7 in HCC immune regulation.13.Potential factors and therapeutic drugs influencing ADCY7 low expression in HCC were explored using the TCGA and GEO public databases.QPCR and western blot experiments were conducted to verify whether ADCY7 expression in HCC cells was affected by potential therapeutic drugs.A mouse subcutaneous tumor model was established using Hepa1-6 cells to investigate whether the combination of potential therapeutic drugs and PD-1 treatment could significantly inhibit the progression of HCC.ResultsThe first part of the project is bioinformatics analysis of the sequencing data screened by the genome-wide CRISPR/Cas9 gene knockout library to preliminarily identify key genes involved in regulating the T cells-mediated immunity in HCC.We performed genome-wide CRISPR screening in vivo in three mouse models with different immune backgrounds,including WT,Rag1^nulland NSG,and preliminarily screened out potential key genes using the MAGe CKFlute algorithm.Then,combined with the reported T cell infiltration and T cell-mediated anti-tumor immune response-related gene sets,TCGA and GEO public database HCC transcriptome data and survival prognosis data,the key gene ADCY7 was screened.Furthermore,the HCC tissue chip cohort verified that ADCY7protein was low expressed in HCC tissues,and suggested poor prognosis in patients.Subsequently,a closer relationship between ADCY7 and CD8⁺T cells infiltration was observed using the single-cell transcriptome data of HCC（GSE149614）and the HCC tissue chip cohort also confirmed that ADCY7 protein expression levels were highly positively correlated with cytotoxic CD8⁺T cells.We found that compared with the control cells,the proliferation ability of HCC cells itself was not significantly affected after ADCY7 was overexpressed.ADCY7 overexpression group can attract more CD8⁺T cells to infiltrate.In vivo animal results showed that ADCY7 overexpression did not affect the growth of HCC in severely immunodeficient mice,but ADCY7 overexpression in immunocompetent mice could significantly inhibit the growth of HCC,and the infiltration and killing function of CD8⁺T cells were significantly increased,but the infiltration level of other immune cells such as CD4⁺T cells and NK cells did not change significantly.These results suggest that ADCY7 inhibits tumor progression mainly by participating in regulating CD8⁺T cell infiltration and killing ability in the immune microenvironment of HCC.The second part of this project is to study the molecular mechanism by which ADCY7affects the anti-tumor immune response of T cells.We first performed KEGG,GO and GSEA enrichment analysis through HCC public transcriptome data,and the results showed that ADCY7 high expression group was significantly correlated with chemokine signaling pathway.We found that ADCY7 could positively regulate the expression of CCL5 m RNA and protein,a key gene in the chemokine signaling pathway.In vivo experiments confirmed that CCL5 is the key influencing factor of ADCY7’s anti-tumor immune response.Mechanically,ADCY7 entered into the cytoplasm from the cell membrane through the caveolae-mediated endocytosis pathway,and then completed the nuclear translocation with the assistance of the endoplasmicreticulum protein LRRC59 and nuclear transporter protein.In the nuclei,ADCY7 assisted the transcription factor CEBPA to bind to the upstream promoter region of the CCL5 gene to promote its transcription,thereby recruiting a large number of CD8⁺T cells for infiltration.Additionally,we have demonstrated that ADCY7 protein exists in exosomes secreted by tumor cells through immunoblotting and liquid chromatography-tandem mass spectrometry and exosomal ADCY7 could enter tumor cells to promote the expression of CCL5.In clinical application,we discovered that introtumoral injection of exosomal ADCY7 significantly inhibited the progression of HCC by promoting CCL5 expression to recruit more CD8⁺T cells to infiltrate and enhance their killing function.Moreover,we also found that the low expression of ADCY7 in HCCwas related to epigenetic modifications such as gene hypermethylation and histone deacetylation.The epigenetic drugs decitabine（methylation inhibitor）and vorinostat（histone deacetylase inhibitor）could significantly increase the expression level of ADCY7,and vorinostat combined with PD-1 therapy demonstrated stronger tumor suppression.ConclusionIn this study,ADCY7 was identified as a key molecule in the regulation of T cells-mediated anti-tumor immunity in HCC through in vivo genome-wide CRISPR screening.ADCY7 could enter the cytoplasm from the cell membrane in caveolae-mediated endocytosis,and then translocated to the nucleus with the help of endoplasmic reticulum protein LRRC59 and nuclear transporter protein KPNB1.Subsequently it acted as a transcription co-factor of CEBPA to bind to the upstream promoter region of CCL5 for inducing its transcription,and then attracting CD8⁺T cell infiltration.Meanwhile,ADCY7was secreted in the form of exosomes and entered into tumor cells,promoting CCL5expression to repress HCC progression.In summary,ADCY7 plays an important role in the regulation of the immune microenvironment of HCC and is a potential target for HCC treatment.