Construction Of Tumor Model Based On Derived Matrix/Composite Scaffold And Anti-breast Cancer Activity And Mechanism Of Astragalus Polysaccharide

Recently,great attention has been paid on the anti-tumor activity of plant polysaccharides due to their various advantages such as a wide variety of sources,multi-pathway and multi-target.Astragalus polysaccharide(APS)has attracted growing interests in the field of anti-cancer by direct killing effect and improving immune function.Anti-tumor activity of drugs relies on tumor models,among which three-dimensional(3D)tissue engineered tumor culture system is becoming an important part to study tumor biology because it can better mimic the in vivo tumor microenvironment.The choice of scaffolds is of paramount importance as one of the basic factors in the construction of tissue engineered tumor.The decellularized matrices have been widely used in tumor models as they can better retain the original extracellular matrix(ECM)components,biochemical and mechanical properties.In this study,a novel decellularized porcine lung derived scaffold was prepared for the construction of 3D tumor model.The anti-breast cancer bioactivity and mechanism of astragalus polysaccharides(APS)were comprehensively analyzed using various tumor models such as the in vitro 2D,3D culture and in vivo subcutaneously implanted tumor in mouse.Meanwhile,the other possible anti-cancer mechanism of APS was also evaluated using bioinformatics based on network topology in order to provide more experimental and theoretical basis for the clinical application of APS.In this study,the same batch of APS from Pharmagenesis Inc.was used since the structure and composition of polysaccharides are directly related to their bioactivity.APS had the characteristic absorption peak of polysaccharide,and the main unit was a-glucopyranoside bond.APS did not contain nucleic acid and protein.APS is mainly composed of rhamnose(Rha),galactoic acid(GalTA),glucose(Glu),galactose(Gal)and arabinose(Ara)following multiple assays including ultraviolet(UV)spectroscopy,infrared(IR)spectroscopy,nuclear magnetic resonance(NMR),and high performance liquid chromatography(HPLC).APS was spherical or spheroidal particles and stacked together irregularly.Most of APS particles were nanoscale which was directly associated with their macrophages activation and further anti-cancer bioactivity.Overall,APS used in this study is appropriate for the subsequent experiments because of their high purity and good properties.The anti-tumor mechanism of most polysaccharides mainly includes direct killing effect and indirect anti-tumor activity by improving immune function.In the present study,we firstly confirmed that APS had no direct cytotoxic effect on breast cancer cell lines(MCF-7 and 4T1)using traditional in vitro 2D culture model.Afterwards,the inhibitory effects and mechanism of the culture supernatant produced from APS mediated macrophages(hereafter simply referred to as conditioned medium,CM)on MCF-7 and 4T1 were evaluated based on the immunoregulated strategy.The results indicated that CM mediated by APS could significantly inhibit the growth of MCF-7 and 4T1 cells,which may be associated with the up-regulation of tumor necrosis factor-a(TNF-a)and nitric oxide(NO)induced by the macrophages activation after the binding of APS and Toll-like receptor 4(TLR4)on the surface of macrophages.The anti-cancer bioactivity of APS mediated CM was mainly achieved by the induction of cell cycle arrest(G1 and G2 phase)and cell apoptosis.CM promoted the apoptosis of breast cancer cells by regulating mitochondrial apoptosis pathway.The inhibitory effect of CM on MCF-7 cells in 3D culture model was further evaluated since 3D tumor model can better mimic the microenvironment of in vivo tumor growth and improve the efficiency and accuracy of anti-cancer drugs.In this study,we prepared the decellularized porcine lung derived scaffold and further fabricated the in vitro 3D tumor model.The cell behaviors and the expression of specific proteins of MCF-7 in 3D scaffolds were assessed.Decellularized porcine lung derived scaffolds possessed favorable biocompatibility.MCF-7 cells could progressively proliferate to multiple tumor spheres.Compared to 2D culture,3D decellularized porcine lung matrix significantly promoted the malignancy potential of breast cancer cells by decreasing cell growth rate and up-regulating the expression of breast cancer-specific proteins.In addition,3D decellularized porcine derived matrix markedly promoted the local hypoxia environment of MCF-7 cells.On this basis,the tumor model based on decellularized porcine lung derived scaffold may provide a more reliable platform for further study of the inhibitory activity of CM on 3D breast cancer cells.The results indicated that CM significantly decreased cell viability and the size of tumor spheres developed from MCF-7 cells.CM promoted the apoptosis of MCF-7 cells in 3D culture by regulating the expression of Bcl-2 and Bax proteins.In this study,chitosan/gelatin(Chi/Gel)and poly(L-lactide)(PLLA)were also prepared from natural and synthetic sources of biomaterials in order to evaluate the difference of tumor models based on decellularized porcine lung scaffold prepared in this study and other scaffolds.Moreover,cell behaviors,the expression of breast cancer proteins,drug sensitivity in vitro of MCF-7 cells and in vivo tumorigenicity of 4T1 cells were studied in 2D culture and 3D culture systems based on decellularized porcine lung,chitosan/gelatin and PLLA scaffolds.Compared to 2D culture,3D culture significantly decreased cell sensitivity towards 5-FU and promoted the malignancy potential in MCF-7 cells.The morphologies of MCF-7 cells were significantly different under 2D and 3D culture systems.MCF-7 cells showed similar growth patterns in three 3D scaffolds,and appeared to form tumor spheres stacked in the "grape bunch" style.The growth and proliferation of MCF-7 cells in vitro in PLLA scaffolds were the best due to the rough surface and the smallest pore size of PLLA scaffolds.In contrast,PLLA scaffolds presented the worst outcome on in vivo tumor development and angiogenesis due to the degradation of lactic acid after 4 weeks of subcutaneous implantation of 4T1 cells-scaffolds in BALB/c mice.The growth of MCF-7 cells in vitro on decellularized porcine lung derived scaffolds was superior to that on Chi/Gel scaffolds,while the development of 4T1 cells on decellularized porcine lung derived scaffolds was better than that on PLLA scaffolds.The results showed that the decellularized porcine lung derived scaffolds prepared in this study showed superiority in tumor development compared with natural and synthetic scaffolds,which was suitable for the study of tumor biology.The body's immune system is very complex involving a variety of immune cells and their secreted cytokines.Compared to the simplex macrophages activation induced by APS,we further investigated the anti-breast cancer effect of APS in BALB/c mice from the immune organs,immune cells and cytokine secretion after we succeeded to develop the tumor model by subcutaneous seeding of 4T1 cells.After 2 weeks of intraperitoneal injection,the tumors were significantly inhibited in APS(200 mg/kg),5-FU(20 mg/kg)and combined administration group(APS+5-FU).However,the effects on the structure and function of immune organs were obviously different.APS could markedly improve the thymus index and spleen index,and restore the structure of the damaged thymus and spleen tissue.On the contrary,5-FU significantly decreased the spleen and thymus index and aggressively damaged thymus and spleen structure.Meanwhile,APS could significantly improve the proliferation of spleen lymphocytes and increase phagocytosis of peritoneal macrophages in mice.Furthermore,APS could up-regulate the expression of IL-2,TNF-?and IFN-? in peripheral blood.All these findings indicated that the anti-cancer activity of APS may be achieved by restoring immune organs,regulating cellular immune responses and increasing cytokine levels.APS combined with 5-FU could improve the anti-tumor effect and alleviate the damage of 5-FU on immune system,which is suitable as an immune adjuvant for chemotherapy.In the present study,the possible in vivo anti-tumor mechanism of APS was further evaluated using bioinformatics analysis based on network topology.Seven gene expression profiles of human breast cancer were downloaded from EMBL-EBI database.A total of 201 differentially expressed genes(DEGs)were identified with the number of 189 uniformly regulated genes,of which 73 genes were up-regulated and 116 genes were down-regulated.Gene ontology(GO)analysis indicated that DEGs were mainly involved in the biological processes such as cell cycle,cell proliferation,chromosome segregation,and cell differentiation.Specially,protein-protein interaction(PPI)analysis was conducted of DEGs in the biological process items involving in cell cycle,proliferation and apoptosis,and the hub genes were screened based on the degree value considering the anti-cancer mechanism of APS on breast cancer cells in 2D and 3D culture previously obtained in this study.EGFR and ANXA1 were identified as the hub and common genes involving cell cycle,proliferation and apoptosis.The results of literature retrieval also showed that the two genes were abnormally expressed in most cancers.The results of immunohistochemistry(IHC)analysis revealed that APS had anti-regulatory effect on the expression of EGFR and ANXA1 in breast cancer,which may be one of the possible in vivo mechanisms of APS to inhibit the growth of breast cancer.In conclusion,a novel 3D decellularized porcine lung derived scaffold was prepared and proved to be feasible in tissue engineered tumor model.On this basis,APS exhibited the in vivo anti-breast cancer activity mainly through the improvement of immune function and also enhanced the chemotherapy effect,while APS activated macrophages in vitro inhibited the growth of breast cancer cells by inducing cell apoptosis and cell cycle arrest after comprehensive analysis using various models from in vitro 2D,3D culture,and in vivo implanted tumor.Bioinformatics analysis demonstrated that the down-regulation of EGFR and up-regulation of ANXA1 protein by APS may be another in vivo mechanism of anti-breast cancer.This study can provide more experimental and theoretical bases for the clinical application of APS and its adjunctive therapy in anti-breast cancer.