Construction Of Lung Cancer Myeloid-derived Suppressor Cell (MDSC) Profile Based On Single-cell Transcriptome Technology

Malignant tumor patients generally have immune function defects.The inability of immune cells to effectively recognize,reject and kill tumor cells is an important reason for tumors.Studies have shown that the abnormal increase of myeloid-derived suppressor cells(MDSC)and the inhibitory cytokines produced by them in the tumor-bearing state are one of the important factors for the deficiency of anti-tumor immune response.On the one hand,MDSC can express high levels of arginase(Arg),i NOS,reactive oxygen species(ROS),etc.,or by secreting large amounts of inhibitory cytokines(TGF-β and prostaglandins),or by abnormally high expression Coinhibitory molecules(such as: PD-1)and other means inhibit the specific anti-tumor immunity mediated by T cells.On the other hand,MDSC can also inhibit the activity of macrophages and NK cells and other natural immune cells: For example,MDSC can down-regulate the production of IL-12 by macrophages by secreting IL-10,accelerate the polarization of macrophages to M2 type,and promote tumor growth.Development;TGF-β-mediated MDSC inhibits the production of IFN-γ by NK cells,which reduces its killing ability.The research on the heterogeneity of MDSC is still in its infancy.It is generally believed that MDSC is a collection of multiple immune cell precursors,but there are no reports on the in-depth study of its heterogeneous components.Therefore,an in-depth study of the composition of MDSC in the tumor-bearing state,clarification of its heterogeneous components and further clarification of its differentiation will be a breakthrough to solve the anti-tumor immune response defect,and will also provide ideas for the design of new anti-tumor immunotherapies.First,we use Lewis lung cancer cells to subcutaneously transplant c57BL/6 mice to construct a mouse lung cancer tumor-bearing model.On the day of tumor-bearing,7th,14 th,21st and 28 th,the spleen,peripheral blood and tumor tissue of the mice were taken,and the single cell suspension was prepared by enzymatic digestion.Through flow cytometry to detect the ratio of MDSC in different tissues in different periods,it is determined that the main site of MDSC aggregation is in the tumor.Through co-cultivation experiments with CD8+ T cells,the anti-T cell proliferation effect of MDSC was clarified.The MDSC obtained by flow sorting was used for single-cell transcriptome sequencing,and the data obtained was used for further bioinformatics analysis.By studying the characteristic genes of this group of cells,through Featureplot,Dimplot and other visualization methods,the heterogeneity between cell clusters is demonstrated.Analysis shows that MDSC is composed of a group of subgroups expressing fibroblasts,dendritic cells,erythroid cells,monocytes,neutrophils and T lymphocyte marker genes.In addition,the immunosuppressive molecules Arg1 and Arg2 are used to identify the monocytes and neutrophil subtypes in MDSC as M-MDSC and G-MDSC,respectively.CD274(PD-L1)and other immunosuppressive factors are highly expressed in G-MDSC,and they exert their immunosuppressive effects through reactive oxygen species(ROS)related pathways.M-MDSC has the highest proportion in MDSC,and we will conduct an in-depth analysis of it.We screened out M-MDSC from MDSC for further analysis.We know that M-MDSC can be divided into three subgroups,M-MDSC which routinely exerts immunosuppressive effect,Rp+ M-MDSC whose transcription and translation level and replication proliferation level are significantly increased,and tumor-associated macrophages which also exert immunosuppressive effect Cell(TAM).Further analysis showed that there is no difference between M-MDSC and TAM in the expression of most immunosuppressive molecules.The classic immunosuppressive molecule IL-10 in TAM has a higher expression,and it has almost no expression in M-MDSC.Functional analysis shows that both TAM and M-MDSC can exert immunosuppressive effects through the ROS pathway.Finally,according to the hints of the enrichment of the Notch signal pathway in M-MDSC,we performed a pseudo-chronological analysis of M-MDSC and TAM.The results show that M-MDSC can differentiate into TAM,and during the differentiation process,its phenotype gradually changes(such as F4/80),and the expression of certain immunosuppressive molecules will also change,leading to changes in its immunosuppressive function.In summary,through single-cell transcriptome technology,we have constructed a myeloidderived suppressor cell map in lung cancer,which provides a reference for solving anti-tumor immune response defects,and also provides new ideas for designing new anti-tumor immunotherapy programs.