Vitexin Reduces Tumor Growth By Reprogramming Tumor-Associated Macrophages To An M1 Profile

Tumor associated macrophages(TAMs)are important components of stromal cells in tumor microenvironment and have the characteristics of strong plasticity and versatility.TAMs can be affected by the tumor microenvironment and polarized into two subtypes of cells with completely different functions,namely the classically activated Ml type and the alternative activated M2 type.M1 type TAMs mainly express iNOS and can recognize malignant tumor presenting antigens to effector cells in the immune system to exert antitumor effect;while M2 type TAMs marker is CD206,which can promote tumor cell migration and angiogenesis and inhibit T cell-mediated anti-tumor immune response accelerating tumor progression.Due to TAMs play an important role in the malignant progression of tumors,they are biomarkers for tumor diagnosis and prognosis as well as important targets for cancer therapy.At present,there are three main anti-tumor strategies through targeting TAMs,namely:clearing TAMs,inhibiting the recruitment of TAMs into the tumor microenvironment,and regulating the polarization of TAMs.Inhibition of TAMs proliferation and maturationrelated signaling pathways,such as CSF-1R inhibitors,preclinical and clinical studies have confirmed that it can reduce the total number of TAMs infiltrated in tumor tissue and inhibite the growth of a variety of tumors.Another class of compound bisphosphonate which can directly kill macrophages and has also been shown to inhibit growth and metastasis of tumor cell by reducing the total number of TAMs.To reduce the recruitment of macrophages,such as small molecule inhibitors of CCL2,which has been shown to inhibit tumor proliferation and migration in a variety of tumor models,and many of those have entered to the clinical research stage.Agonists that regulate TAMs polarization,such as TLRs(Toll-like receptors)and CD40,have been shown to promote M1 polarization of TAMs,activate anti-tumor immunity,and inhibit tumor progression.The above studies not only clarified the important role of TAMs in tumor progression,but also confirmed the feasibility of targeting TAMs in tumor therapy.However,there are some side effects in the application of some small molecule compounds,such as adverse reactions caused by systemic clearance of TAMs,and increased rebound of TAMs after drug discontinuation,as well as systemic activation of TLRs and CD40-induced adverse reactions.Therefore,it is of great practical significance to find more safety and effective immunotherapeutic drugs for TAMs.The latest research shows that paclitaxel,an anti-tumor natural product,has a new mechanism of action to regulate the polarization of TAMs to against tumors,indicating its potential as a tumor immunotherapy drug.But the growth of Taxus chinensis(Pilger)Rehd,a raw material for paclitaxel,is very slow resulting in expensive price of paclitaxel and limiting its wide application.Therefore,it is particularly necessary to find safe and effective natural products with abundant resources which can regulate the polarization of TAMs.Vitexin has a wide range of resources with low toxicity,and many studies have shown that it has a wide range of tumor suppressor effects in a variety of cancer cell models in vitro.Our previous study found that it can alleviate the tumor progression of colitis-associated colorectal cancer mice and increase the content of NO in tumor tissues.Since NO is abundantly derived from Ml type macrophages,this suggests that in addition to directly promoting tumor cell apoptosis in vitro,may be vitexin also could regulate TAMs in the tumor microenvironment.Based on this,this study aims to explore the anti-tumor effects and mechanism of vitexin on the transplanted tumor mice model from the perspective of regulating TAMs and further to provide more data for the further development and application of vitexin.ObjectiveVitexin can inhibit the proliferation of a variety of cancer cells in vitro and can also promote tumor cell apoptosis to inhibit tumor growth in a nude mouse xenograft model.However,the anti-tumor effect of vitexin on the transplanted tumor model of normal mice has rarely been reported.Therefore,this thesis will use two tumor type which are more prevalent in clinically to induce xenograft models in normal mice to evaluate the anti-tumor effect of vitexin and further to explore its underling mechanism of regulating TAMs and Tlymphocyte immunity.Methods1.Therapeutic effect and mechanism of vitexin on subcutaneous CT26 and ortho topic 4T1tumor modelBALB/c male and female mice were used to construct a colon cancer allograft mice model by subcutaneous inject CT26 colon cancer cells and an orthotopic breast cancer allograft mice model by injecting 4T1 cells subcutaneously into the second mammary gland.Control group was given distilled water and vitexin administration group were given 20 and 40 mg/kg of vitexin once a day.Mouse tumor volume,body weight,food and water intake were recorded during the administration.After the treatment,the anti-tumor effect of vitexin and its effect on the overall state of the mice were evaluated by weighing the tumor and the main organs as well as by histopathological examination.Then the macrophages in the tumor microenvironment,and the T lymphocytes in the tumor microenvironment,spleen and peripheral blood were analysised by flow cytometry.The expression of F4/80 and iNOS in tumor tissues was determined by immunofluorescence.The content of nitric oxide(NO)in tumor tissue homogenate was detected by grisses regaents.2.Regulation and Mechanism of Vitexin on Macrophage PolarizationMouse mononuclear macrophage cell line RAW264.7 and mouse bone marrow drived macrophage(BMDM)were used to evaluate the regulation of vitexin on macrophage polarization in vitro.First,the dose of vitexin in vitro study was determined by MTT assay and preliminary experiment.Further,on the two cell models:vitexin alone,vitexin combined with 100 ng/ml LPS,vitexin combined with 100 ng/ml LPS and 50 ng/ml IFN-y as well as combined with 10 ng/ml IL-4 were used to intervene the polarization of macrophage.Then the NO levels in the supernatant of cell culture medium were detected by grisses reagents and the numbers of iNOS+or CD206+macrophages were counted by flow cytometry to investigate the regulate effect of vitexin on macrophage polarization.Finally,by detecting the expression of iNOS,STAT1 and p-STAT1 proteins,the mechanism of vitexin on macrophage polarization was investigated.3.Vitexin combine with iNOS inhibitor 1400W to verify its anti-tumor mechanismOn the allograft mice model of colon and breast tumor,1400W an inhibitor of inducible nitric oxide synthase(iNOS)was used to systematically block the activity of iNOS in vivo.And by observing the control group,the 1400W inhibitor group and the 1400W inhibitor plus vitexin treatment group:(1)body weight,food and water inatke;(2)organ coefficient and histopathological examination of major organs;(3)tumor volume change,tumor weight,pathological examination of tumor tissue;(4)macrophage profile in tumor tissues and lymphocyte profile in tumor tissues,spleen and peripheral blood;and finally(5)immunofluorescence to determine the expression of F4/80 and iNOS in tumor tissues and to detect NO level in tumor tissue of control group,the 1400W inhibitor group and the 1400W inhibitor plus vitexin treatment group to evaluat the anti-tumor targets of vitexin whether achieved by acting on the regulation of TAMs further to worked on CD8+cells.Results1.Therapeutic effect of vitexin on subcutaneous CT26 and orthotopic 4T1 tumor modelThere was no significant change in body weight,food intake and water intake of three groups in CT26 model.In 4T1 model,there was no significant difference between the food and water intake in each group of mice.The high dose vitexin can alleviate the weight loss of female mice in the early stage of tumor growth While tumor volume and tumor weight were both inhibited in a dose-dependent manner after vitexin administration in two models.On the immune organs of two model,vitexin dose-dependently reduced the spleen coefficient and increased the thymus coefficient;The pathological examination of the spleen and tumor tissue of each group of two model mice showed that the spleen of the control group appeared swollen,and the structure of the spleen was not clear.Although the splenic corpuscle was still atrophied after the administration of vitexin,but the structure of the spleen corpuscle red and white pulp was clearly visible.Tumor histological examination showed that there was a multi-necrotic area in the tumor tissue after vitexin administration;Besides,the pathological examination of the liver of 4T1 model showed that the tumor metastases appeared in the central venous of the liver,vitexin reduced liver central venous tumor metastasis.The lung pathological results of three groups of 4T1 mice showed that the interval of pulmonary alveoli was widened,and after vitexin administration the interval of pulmonary alveoli was narrowed.In two tumor mice models,the macrophages and its subtypes in the tumor microenvironment showed that the vitexin dosedependently reduced the total number of TAMs,increased the iNOS+ macrophages ratio,and decreased the ratio of CD206+macrophages.The iNOS expression and NO level in tumor tissue homogenate was significantly increased after vitexin administration.The results of T lymphocyte counts in tumor microenvironment,spleen and peripheral blood showed that,compared to control group,the vitexin treatment groups can at different levels increased the number of CD3+CD8+T cells and CD45+CD8+cells.2.Regulation and mechanism of vitexin on M1/M2 polarization of macrophagesAfter vitexin(5?M and 10?M)intervention in two macrophage cell line,the iNOS'macrophages ratio decreased and the NO level in the cell supernatant were also decreased in a dose-dependent manner;And after vitexin(5?M and lO?M)combined with LPS(100 ng/ml)intervention in two cell model,NO content in cell culture medium and iNOS+macrophages ratio were both significantly increased in LPS-stimulated group,while LPS plus vitexin(5 ?M and 10 ?M)dose-dependently decreased NO content and iNOS+macrophages ratio compared to LPS-stimulated group.The high dose of vitexin(10 ?M)significantly inhibited the NO content in two cell line;On RAW264.7 and BMDM,iNOS+macrophages ratio and NO content in the culture medium was significantly increased after 24 hours interaction with LPS+IFN-? when compared to control group.After the combination of vitexin with LPS and IFN-?,the iNOS+ macrophages ratio was increased compared to the LPS+IFN-y group.The NO level in the culture medium increased in a dose-dependent manner.In RAW264.7 and BMDM,IL-4 significantly increased the CD206+macrophaegs ratio compared to control group.After administration vitexin with IL-4,vitexin could decrease CD206+macrophages ratio in a dose-dependent manner compared to IL-4 group.Western blot results showed that vitexin combined with LPS+IFN-? can promote M1 polarization of macrophages by increased the expressions of STAT1/p-STAT1 on macrophages.3.Vitexin combine with iNOS inhibitor 1400W to verify its mechanismIn CT26 tumor model,there was no significant difference in body weight,food and water intake in three group;And in 4T1 tumor model there was no significant difference of the three groups in the water and food consumption,while the body weight of the 1400W group was significantly decreased when compared with the control group,when compared to the 1400W group,the 1400W plus vitexin significantly increased the body weight of the mice.In two models,the tumor volume and tumor weight of the 1400W group were higher than the control group.And the tumor volume and tumor weight were decreased after the 1400W plus vitexin administration,while when compared to control groups,there were no obvious changes.Compared with the control group in CT26 model,the heart,spleen,lung and kidney coefficient of the 1400W group were all increased,while the liver coefficient was significantly increased.Compared to the 1400W group,the 1400W plus vitexin could decrease the heart,spleen,lung coefficient,and increase the thymus coefficient,the 1400W plus vitexin significantly reduced the liver coefficient compared to the 1400w group.Compared with the control group in the 4T1 model,the 1400W group had a significant higher heart and liver coefficient,and the spleen,lung,kidney and thymus coefficient were also increased but without statistically significant.Compared with the 1400W group,the heart,liver,spleen,lung,kidney and thymus coefficients of the 1400W plus vitexin group were decreased,and the kidney coefficient was significantly decreased.In two tumor models when compared to the control group,the spleen stracture of the 1400W group were severely changed,and vitexin improved the spleen pathogeny structure.When compared with the control group,the tumor cells in the 1400W group were denser in vascular density,and the tumor cells and vascular density were decreased after the addition of vitexin.Besides,in the 4T1 model,liver and lung tissue of the 1400W group were accompanied with more tumor metastases,and 1400W plus vitexin treatment could improve it.In CT26 and 4T1 model,the TAMs in the tumor environment of 1400W group increased when compared with that of control group,the iNOS+macrophages ratio decreased significantly,the CD206+macrophages ratio increased.While when compared with the 1400W group,vitexin administration significantly increased iNOS+macrophages ratio,and decreased the CD206+macrophages ratio.Whiel when compared with the control group,1400W plus vitexin lower the total number of TAMs,while the iNOS+macrophages ratio and the CD206+ macrophages ratio had no obvious changes or had slightly reduced.In two models,the expression of iNOS in tumor tissue and the content of NO in the tumor tissue homogenate of 1400W group were reduced at different levels than those of the control group.And these indicators were significantly increased after 1400W plus vitexin administration when compared to 1400W group.While iNOS expression and NO level of mice of 1400W plus vitexin group did not increase or increased sightly when compared to the control group.The T lymphocyte counts in the tumor microenvironment showed that in the two models the ratio of CD3+CD8+T cells and CD45+CD8+cells in the 1400W group decreased;the 1400W plus vitexin administration significantly increased the CD3+CD8+T cells and CD45+CD8+cell counting compared to the 1400W group,while there was no obvious changes when compared to the control group;The CD3+CD8+T cells and CD45+CD8+cells in peripheral blood and spleen of 1400W groups were decreased compared to the control groups.When compared with the 1400W inhibitor group,the inhibitor plus vitexin can increase the ratio of CD3+CD8+T cells and CD45+CD8+cells at different levels.While in CT26 model the ratio of CD3+CD8+T cells and CD45+CD8+cells were lower in spleen and slightly higher in peripheral blood than control group.And in 4T1 model the ratio of CD3+CD8+T cells and CD45+CD8+cells in spleen and peripheral blood were both increased slightly than control group.Conclusion1.Vitexin inhibits the malignant progression of tumors in colon cancer allograft and orthotopic breast cancer allograft normal mice models,and the inhibition is achieved by regulating TAMs in the tumor microenvironment,activating iNOS+TAMs,and further activating the CD8+ T cells.2.Vitexin can regulate the polarization of macrophages in vitro,can synergize with LPS+IFN-y to promote M1 macrophages polarization,and antagonize the action of IL-4 to inhibit M2 polarization of macrophages,and the actions of vitexin on promoting M1 macrophages polarization is achieved through activing STAT1/p-STAT1.3.The iNOS inhibitor 1400W can antagonize the anti-tumor effect of vitexin.It is proved that anti-tumor effect of vitexin is achieved by modulating TAMs to increasing iNOS+macrophages further works through NO and CD8+cells,but in addition to acting on iNOS+TAMs,vitexin maybe can also directly activate CD8+cells.