Study On The Anti-metastatic Effect And Mechanism Of Icaritin Based On The Lung Pre-metastatic Niche Induced By Microparticles

【Background】Tumor metastasis is an important cause to lead to cancer patients’death in clinical,and there is a lack of effective treatment methods for tumor metastasis.Recent studies have found that the primary tumor cells can secrete extracellular vesicles and some factors to induce adaptive changes in the microenvironment of the distant organ to form a pre-metastatic niche with being beneficial for colonization and growth of tumor cells.The hypothesis of the pre-metastatic niche provides a new direction for the prevention and treatment of tumor metastasis.At the same time,the excessively abnormally activated STING signal provides a key condition for tumor metastasis by promoting the formation of the inflammatory pre-metastatic niche.Therefore,intervention in the pre-metastatic niche is an effective strategy for the prevention and treatment of tumor metastasis,and it coincides with the traditional Chinese medicine theory of"prevention of disease prevention".The previous research of the research group found that the traditional Chinese medicine Epimedium and its active ingredients have anti-tumor metastasis effects,but the specific mechanism is still unclear.In order to confirm the anti-pulmonary metastasis effect of Epimedium and clarify its action mechanism,this project intended to investigate the effect and molecular mechanism of epimedium flavonoids in inhibiting tumor metastasis from the perspective of intervention in the inflammatory pre-metastatic niche,and based on the activation of STING signal.【Research contents and results】（1）Icaritin inhibited microparticles-induced inflammatory PMN.First,we screened out Icaritin（ICT）with the dose-dependent and strong anti-tumor metastasis effect,from the six common components of epimedium flavonoids,Epimedin A,Epimedin B,Epimedin C,Baohuoside I,Icariin and Icaritin on the malignant biological behaviors of tumor cells.Therefore,ICT was chosen to study its anti-metastasis mechanism.The microparticles（MP）derived from melanoma B16BL6 cells were further extracted and characterized,confirming the successful extraction of MP.Based on the uptake of MP by mouse lung epithelial cells MLE-12 and mouse alveolar macrophages MH-S,an in vitro PMN model of co-culture of MLE-12 and MH-S cells induced by MP was established.To investigate the in vitro intervention of ICT via inflammatory PMN indicators including Fibronectin（FN）,LOX and so on.At the same time,an in vivo model of inflammatory PMN induced by injection of MP into the tail vein of C57BL/6 mice was established.And using Fibronectin,SAA,S100A8/9 and vascular permeability as indicators to investigate the in vivo intervention of ICT.The results showed that ICT can reduce the markers of inflammatory PMN in vitro and in vivo in a dose-dependent manner,and inhibit the formation of inflammatory PMN.（2）ICT regulated MP-induced PMN on the malignant metastasis biological behavior of B16BL6.The scratch healing experiment and transwell experiment were used to investigate the effect of the constructed PMN on the migration and invasion ability of B16BL6 cells.MP-induced PMN promoted the migration and invasion of B16BL6 cells.And the intervention of ICT on PMN lead to the decline of the migration and invasion ability of B16BL6.The plate cloning experiment was used to investigate the effect of PMN on the proliferation of B16BL6 cells.The cell colony of the MP-ICT-CM group was significantly lower than that of the MP-CM group.The results indicated that ICT may inhibit the proliferation of B16BL6 cells by inhibiting the formation of the PMN.Construct an experimental metastasis model of B16BL6 cells to investigate the effect of ICT intervention PMN on tumor metastasis.The mice were sacrificed at 22 days after tumor inoculation.The results showed that ICT reduced the number of lung metastatic nodules in metastatic mice model,and reversed the increase in lung weight induced by MP and improve lung disease.（3）ICT regulated STING pathway in PMN.Elisa experiment was used to detect the release of type I interferon IFN-βin the culture supernatant of cell PMN model in vitro and the lung tissue and serum of PMN animal models in vivo.The results showed that extracellular vesicles derived from tumor cells can induce the increase in the expression and release of IFN-βin PMN and that ICT can reduce the release of IFN-β.Western blot experiment was used to detect the effect of ICT on the activation level of TBK1,STING and phosphorylation and the dimerization of STING in the cell PMN model.The results showed that ICT can inhibit the expression of STING,TBK1 and P-TBK1,and inhibit dimerization of STING in PMN.Western blot and immunohistochemistry experiments were utiliazed to detect the expression levels of TBK1,p-TBK1,STING,and p-STING in lung PMN tissue.The results showed that MP induced an increase in the expression of TBK1,p-TBK1,STING,and p-STING in lung tissue,while ICT could significantly reduce the expression of TBK1 and STING phosphorylated proteins.Observing the expression level and cell localization of STING and IRF3 protein in the nucleus of MP and ICT treated cells under a fluorescent inverted microscope for 24 hours.The results showed that after MP induced MH-S or MLE-12 cells,the fluorescence of STING and IRF3 protein basically coincided with the fluorescence of the nucleus,indicating that STING and IRF3 moved to the nucleus,while ICT inhibited the nuclear translocation of the protein.Western blot method was used to detect the protein and nuclear changes of IRF3 and NF-κB.Compared with the blank group,the expression of IRF3 in the MP group increased.In the ICT group,the expression of IRF3 protein in the nucleus of MH-S cells,MLE-12 cells and co-cultured cells was lower than that in the MP group.ICT had an inhibitory effect on the up-regulation of p105 expression in co-cultured cells and MLE-12 single cells induced by MP.（4）ICT inhibited the STING-IFN-βpathway by combining with STING.Using computer to simulate the molecular docking of ICT and STING,the results showed that ICT combined with the active site of STING.The cell thermal analysis experiment（CETSA）was used to detect the thermal stability of the protein,we found that ICT could increase the thermal stability of p-STING protein,which proved that ICT and STING have a combined effect.Simultaneously,the carboxyl group of Biotin was used for esterification reaction with the hydroxyl group of ICT to synthesize Bio-ICT.Western blot results showed that Bio-ICT inhibited the expression of STING,p-STING,and p-TBK1 in PMN.The FITC-labeled streptavidin was observed under a fluorescence microscope to track the co-localization of Bio-ICT and fluorescent-labeled STING.The experimental results showed that STING may be the target of ICT.The use of si RNA interference technology reversely confirmed that STING played a key role in ICT suppression of PMN.The experimental results showed that si RNA transfection reduced the expression of STING and IRF3protein in PMN,and inhibited the expression of PMN marker protein LOX.The scratch experiment was used to investigate the ability of ICT to interfere with the migration of B16BL6cells in STING-inhibted PMN.The results indicated that after STING interference,ICT inhibited the migration of B16BL6,indicating that ICT played an anti-tumor metastasis effect through STING.Western blot experiments investigated the effect of RTA-408 on the pathway proteins in PMN.The results showed that RTA-408 inhibited the expression of STING,TBK1,and p-STING proteins at a concentration of 40 nmol·L^-1,and MP could not promote the expression of IRF3 after inhibiting the expression of STING.Scratch tests were used to investigate the migration of tumor cell B16BL6 cells in ICT-interfered PMN after STING was inhibited by RTA-408.Experimental results showed that the cell migration ability was inhibited after inhibiting STING of co-cultured cells by RTA-408.While the effect of ICT inhibited the migration of B16BL6 cells was not affected,indicating that the mechanism of ICT intervention in STING may be different from RTA-408.【Conclusions and Significance】In this project,Chinese medicine for strengthening the body and invigorating deficiency is related to the pre-metastatic niche,seeking and revealing its anti-tumor metastasis mechanism from a new perspective.In this paper,tumor-derived extracellular vesicles were used to construct the PMN model in vivo and in vitro,and PMN forming marker proteins and pro-inflammatory factors to evaluate the effect of ICT intervention on PMN.The in vitro migration experiment induced by PMN and the experimental tumor metastasis model confirmed that ICT can inhibit the metastasis of B16BL6 cells by interfering with PMN.The mechanism of ICT intervention in PMN was discussed in depth,which confirmed that ICT intervention in PMN is related to the inhibition of STING pathway,and initially confirmed the possibility of STING as a target of ICT action.The implementation of this topic is not only a prominent frontier innovation in the current tumor metastasis mechanism and therapeutic research,but also a modern pharmacological research on the scientific connotation of the clinical treatment of tumor metastasis with traditional Chinese medicine for strengthening the body and strengthening the deficiency and"prevention of disease prevention".This study provides new ideas and methods for clinical prevention and treatment of tumor metastasis.