The Application Of Agrocybe Aegerita Lectin In Tumor Therapy And Tumor Diagnosis

Lectins are non-immunoglobulin proteins which selectivily bind to specific monosaccharides or oligosaccharides.A variety of plant and fungi derived lectins have been widely used as important tools for glycobiology.During the tumor progression,glycosylation patterns on the surface of tumor cells significantly changed,and closely related to the functional state of immune system.Therefore,lectins have received extensive attention in the research of cancer diagnosis and treatment.Part 1:AAGL can be used as a potential cancer immunotherapy drug.In the thesis,we proved that AAGL has antihepatoma activity in vitro.In orthotropic transplantation hepatocellular carcinoma mouse model,AAGL significantly inhibited tumor growth,reduced the number and diameter of tumor nodules,prolonged the survival period of tumor-bearing mice.In-depth analysis of liver transcriptome data of tumor-bearing mice showed that AAGL significantly upregulated the expression of cytokine genes related to various immune cells migration and chemotaxis,especially the expression of various chemokines such as CCL2,CCL3,CCL5 and CXCL9.In addition,AAGL also significantly promoted the expression of genes associated with macrophage and T cells activation.In vitro experiment indicated that the profile of chemokines(CCL2,CCL3,CCL5 and CXCL9)in peritoneal macrophage and RAW264.7 cell induced by AAGL consistanted with the expression profile of chemokine in the liver of tumor-bearing mice in the transcriptome data,which indicated that liver macrophages may be the main source of the increased chemokines induced by AAGL.Recruiting lymphocytes is one of most important physiological functions of chemokines.Flow cytometry analysis showed that the amounts of liver T cells,NK cells and macrophages all have significantly increased after the treatment of AAGL.Meanwhile,the number of T,NK and macrophages in spleen have significantly decreased.The results of the splenocyte transplantation experiment validated that AAGL could promote the infiltration of immune cells from spleen to liver.In addition,The treatment of Cs A,anti-CD4 or anti-CD8 also could completely offset the antitumor effect of AAGL,which suggested that CD4⁺T and CD8⁺T cells both play a crucial role in this case.Flow cytometry results showed that AAGL significantly increased the number of IFN-?⁺CD4⁺T,IFN-?⁺CD8⁺T and IFN-?⁺NK cells in liver of tumor-bearing mice.What's more,western blot results showed that AAGL increased the expression of granzyme B,perforin,Fas L and TRAIL in tunor-bearing mice liver,which indicated that AAGL increased the cytotoxicity of T cells by inducing the apoptosis of tumor cells.PD-1 is a member of TCR co-stimulatory molecule family,which is mainly expressed on the surface of activated T cells,NK cells,and some macrophages and DCs.In the tumor microenvironment,the interaction of PD-1 and PD-L1 rapidly induced the apoptosis of T cells.The clinical effect of PD-1/PD-L1 blockade have shown significant therapeutic effects in a variety of cancers,especially in melanoma and renal cell carcinoma patients which complete response rates have exceeded 20%,while the response rate in liver cancer is only 17%.In the development of cancer,low infiltration of immune cells such as effector T cells is one of important factors.Combination treatment of AAGL and anti-PD-1 significantly prolonged the survival period of tumor-bearing mice compared with AAGL or anti-PD-1 treatment alone.And the number of CD4⁺T cells and CD8⁺T cells in combination treatment group were both dramatically higher than anti-PD-1 and control group,which means AAGL significantly improved the response rate of anti-PD-1 therapy.Finally,these results not only provided the theoretical basis of the development of AAGL as a potential anti-hepatic tumor drug,but also provided a new strategy to promote the combination therapy of PD-1immunotherapy for HCC.Part 2:The application of lectins in the identification of tumor biomarkersThe aberrant glycosylation of tumor cells can be as a new biomarker for clinical diagnosis,so screening early tumor specific biomarkers through glycan-binding activity of lectins has great application potential.Previous study have found that serum ORM1 in early stage of lung cancer patients have significantly decreased compared with health controls,which identified by AAGL and AANL.Further transcriptome analysis of peripheral white blood cells in early stage of lung cancer patients found that immune genes associated with neutrophil activation such as ORM1,CD177,MMP9,ARG1 have significantly decreased in m RNA level.And ORM1 acting as an early stage of lung cancer biomarker was validated by western blot and BN*?/BN Pro Spec System.The results of ROC curve showed that ORM1significantly distinguished early stage lung cancer patients from health(AUC=0.839)and benign samples(AUC=0.821),ELISA result shows that MMP9 also decreased in the early stage lung cancer patients,the AUC value between health and early stage lung cancer patients is 0.854,and the AUC value of the combination diagnosis of MMP9and ORM1 increased to 0.964,which means that ORM1 and MMP9 have great application prospects as biomarkers of lung cancer at eraly stage.It is reported that ORM1 in serum was predominantly produced by hepatocytes,in early stage of mouse LLC tumor model,the serum ORM1 also have obviously decreased,but the m RNA and protein level of ORM1 in liver have not changed.This indicated that the decrease of serum ORM1 was mediated by peripheral blood leukocyte in the early stage of tumor not hepatocyte.And the flow cytometry result showed that ORM1 also expressed in 94%CD15⁺neutrophils,which indicated that reduced ORM1was mediated by neutrophils.We found that TGF-?not only significantly inhibited the expression of ORM1,CD177,MMP9 and ARG1 in peripheral WBC,but also inhibited the expression of that in peripheral neutrophils in vitro.The level of serum TGF-?in early stage lung cancer patients were much lower than healthy samples,which consistented with the concentration we used in vitro experiment.These data indicated that the immune system inhibited the expression of ORM1 and MMP9 in the peripheral neutrophils by lowering the concentration of TGF-?in the blood.Therefore,ORM1 and MMP9 may be useful diagnostic biomarkers for early stage lung cancer.