Study On The Function And Mechanism Of S100A8 Deletion Of Hematopoietic Stem And Progenitor Cells Causing An Increase In MDSC And Promoting Tumor Growth

Research Background:S100A8 is an inflammation-related protein,which is involved in the initiation,amplification and maintenance of inflammation.In a variety of inflammatory diseases,such as inflammatory bowel disease,rheumatoid arthritis and other patients,S100A8 increased in serum,and the increased level of S100A8 was positively correlated with the severity of the disease.S100A8 is both an intracellular protein and a secreted protein.Secreted S100A8 interacts with Toll like receptor 4(TLR4),Receptor of advanced glycation endproducts(RAGE),CD68 on the surface of inflammatory cells such as monocytes and macrophages.In combination with other components,it activates downstream signaling pathways,enhances the activation and phagocytosis of inflammatory cells,and promotes the release of inflammatory factors.S100A8 not only plays an important role in inflammatory diseases,but also a star molecule in the field of tumor research.Studies have shown that among cancers with high incidence such as lung cancer and breast cancer,S100A8 is highly expressed in tumor tissues of cancer patients.In addition,the increased expression level of S100A8 indicates that the patient has a high clinical stage and a poor prognosis,which shows that S100A8 has played an important tumor-promoting effect.In the tumor microenvironment,tumor cells and myeloid cells which is one type of immune cells are the main cell components that express S100A8.On the one hand,S100A8 acts directly on tumor cells.Extracellular S100A8 binds to the RAGE receptor on the surface of tumor cells,chemotactic tumor cell migration;Intracellular S100A8 promotes tumor cells to highly express Matrix Metalloproteinase 2(MMP2)and Matrix Metalloproteinase 9(Matrix Metalloproteinase 9,MMP9),enhancing tumor Cell invasion ability;On the other hand,the effect of S100A8 on immune cells is mainly reflected in that S100A8 plays a role similar to chemokines,and binds to the surface receptors of Myeloid Derived Suppressive cells(MDSC)to activate The downstream NF-κB pathway chemoattracts and recruits MDSCs to tumor tissues and tumor metastasis sites,resulting in a large number of MDSCs accumulating in tumor tissues and tumor metastasis sites.By increasing the number of MDSCs in the tumor microenvironment,the tumor-killing effect of effector T cells can be inhibited or angiogenesis can be promoted,etc.,to promote tumor growth.The study found that knocking out S100A8 in tumor cells and using nude mice to construct a subcutaneous tumor model,that is,in the case of immunodeficiency,tumor growth was significantly slowed down.However,normal C57 mice were used to construct a subcutaneous tumor model,that is,under the condition of complete immunity,the tumor growth rate did not change significantly.The above results suggest that S100A8 acts on tumor cells themselves to promote tumor growth,but in the case of complete immunity,does it act on immune cells to regulate tumor immunity and exert anti-tumor effects,thereby inhibiting tumor growth? With such scientific questions,we have carried out research on this subject,aiming to explore the effects of S100A8 on immune cells and tumor growth.In order to achieve the research purpose of exploring the effect of S100A8 on immune cells and the effect of tumor growth,we first selected tumor cell lines(LLC,MC38)that did not express S100A8,and secondly,because hematopoietic stem progenitor cells(HSPC)are the precursor cells of all immune cells,we ordered a hematopoietic stem progenitor cells tissue-specific knockout mouse of S100A8 constructed with CRISPR/CAS9 combined with cre-loxp technology from Beijing Biocytogen,which can survive and reproduce.And there is no difference in various phenotypes from wild-type mice,which provides a good animal model for experiments.In this study,we first used hematopoietic stem progenitor cell S100A8 knockout mice and wild-type mice to construct tumor-bearing models,and explored the effect of hematopoietic stem progenitor cell S100A8 deletion on tumor growth and tumor biological characteristics;Secondly,explore the effect of the S100A8 deletion of hematopoietic stem progenitor cells on tumor immunity;Finally,we will further explore the cause and mechanism of the impact of S100A8 deletion of hematopoietic stem progenitor cells on immune cells;This study expands the understanding of the role of S100A8 in tumor immunity and tumor growth,and provides a theory for follow-up research basis.Method:1.Explore the effect of S100A8 deletion of hematopoietic stem and progenitor cells on tumor growth1.1 Construct hematopoietic stem progenitor cell S100A8 KO mice and WT mice Lewis Lung cell(LLC)tumor-bearing models,record the tumor growth curve,and measure the tumor weight;1.2 Construct hematopoietic stem progenitor cell S100A8 KO mice and WT mice MC38tumor-bearing models,record the tumor growth curve,and measure the tumor weight;2.Exploring the S100A8 deletion of hematopoietic stem and progenitor cells and the biological characteristics of tumors2.1 Explore the proliferation of tumor cells;2.2 Explore the apoptosis of tumor cells;2.3 Explore tumor angiogenesis;3.Detect the effect of S100A8 deletion of hematopoietic stem and progenitor cells on T cell ratio function under tumor-bearing state3.1 Detect and analyze the proportion of CD8+T cells and CD4+T cells in tumors,spleen,and draining lymph nodes;3.2 Detect and analyze the functions of CD8+T cells and CD4+T cells in tumors,spleen,and draining lymph nodes;4.Detect the effect of S100A8 deletion of hematopoietic stem and progenitor cells on the proportional function of MDSC under tumor-bearing state4.1 Detect and analyze the proportion of MDSC in tumor,bone marrow and spleen;4.2 Detect and analyze the function of MDSC in tumor and spleen;5.Demonstration that the lack of hematopoietic stem and progenitor cells S100A8,in the tumor-bearing state,MDSC induces T cell immune tolerance,which is the reason for promoting tumor growth5.1 Detection of S100A8 expression in mouse lymphocytes;5.2 Eliminate MDSC in S100A8 KO mice and WT mice,and carry out tumor-bearing,record tumor growth curve,and measure tumor weight;6.Explore the mechanism of the lack of S100A8 in hematopoietic stem and progenitor cells and the increase in MDSC under the condition of tumor6.1 Analyze the source of MDSC differentiation;6.2 Analyze the proliferation of MDSC;6.3 Analysis of MDSC maturation and differentiationResult:1.Loss of hematopoietic stem and progenitor cells S100A8 promotes tumor growth1.1 In the LLC tumor-bearing model,KO mice have faster tumor growth,larger volume and heavier weight than WT mice;1.2 In the MC38 tumor-bearing model,KO mice have faster tumor growth,larger volume and heavier weight than WT mice;2.Hematopoietic stem progenitor cells S100A8 is missing,and the biological characteristics of the tumor remain unchanged2.1 Compared with WT mice,KO mice have no changes in tumor cell proliferation;2.2 Compared with WT mice,KO mice have no change in tumor cell apoptosis;2.3 Compared with WT mice,KO mice have no change in tumor angiogenesis;3 The hematopoietic stem and progenitor cells S100A8 are missing,and the T cell immune tolerance in the tumor tissue in the tumor-bearing state3.1 Compared with WT mice,KO mice only had a lower proportion of CD8+ T cells in tumor tissues,but no change in the proportion of CD8+ T cells in the spleen and draining lymph nodes;3.2 Compared with WT mice,the proportion of CD4+ T cells in tumors,spleen,and draining lymph nodes in KO mice did not change;3.3 Compared with WT mice,KO mice have reduced CD8+ T cell function in tumor tissues,but no change in CD8+ T cell function in spleen and draining lymph nodes;3.4 Compared with WT mice,KO mice only have a reduced function of CD4+ T cells in tumor tissues,but there is no change in the function of CD4+ T cells in the spleen and draining lymph nodes;4.Hematopoietic stem progenitor cells S100A8 is missing,MDSC increases in tumor-bearing state,and there is no change in function 4.1 Compared with WT mice,KO mice have an increased proportion of MDSCs in tumors,bone marrow,and spleen;4.2 Compared with WT mice,KO mice have no change in MDSC function.5.The lack of hematopoietic stem and progenitor cells S100A8,the increase of MDSC in the tumor-bearing state induces T cell immune tolerance,which is the main reason for promoting tumor growth5.1 Mouse lymphocytes do not express S100A8;5.2 After clearing MDSC,there is no difference in tumor growth volume and weight in KO mice and WT mice;6.The lack of S100A8 in hematopoietic stem and progenitor cells,the mechanism of the increase of MDSC due to the myeloid deviation of cell differentiation in the tumor-bearing state6.1 The hematopoietic stem and progenitor cells S100A8 is missing,and the differentiation of hematopoietic stem and progenitor cells into MDSC increases in the tumor-bearing state;6.2 The hematopoietic stem and progenitor cell S100A8 is missing,and there is no change in the proliferation of MDSC under the tumor-bearing state;6.3 The hematopoietic stem progenitor cell S100A8 is missing,and there is no change in the downstream maturation and differentiation ability of MDSC in the tumor-bearing state;Conclusion:1.S100A8 is missing in HSPC,tumor growth is accelerated;2.S100A8 is missing in HSPC and the proportion of MDSC increases,leading to T cell immune tolerance is the reason for promoting tumor growth;3.The absence of S100A8 in HSPC and the myeloid deviation during HSPC differentiation is the mechanism for the increase of MDSC.