Construction Of Fiber Reinforced ECM-derived Hydrogel Tumor Model And Application In Drug Screening

It is possible to create and test new drugs as well as conduct mechanistic investigations of tumor biology by using tumor models that are generalized from patient cancers in vitro and in vivo.Two-dimensional(2D)cell cultures have been an important tool to help researchers understand the mechanisms of cell behavior in vivo,but they often fail to accurately reproduce the in vivo environment.Many studies have shown that three-dimensional(3D)cultures have a more realistic biochemical and biomechanical microenvironment,and they have also been shown to reproduce human tumor-stromal interactions in vitro.They have also been shown to generalize tumor cell signaling in vivo more accurately.Hydrogels are highly hydrophilic networks of polymers cross-linked physically or chemically and are well suited to mimic the extracellular matrix of tissues,but the lower and unstable mechanical strength and the failure to meet the complexity of the extracellular matrix(such as morphology and fibrous properties)limit their use as 3D tumor culture models.Based on this,this study introduces the fiber system into the gel scaffold and innovates the preparation process to create a fiber-hydrogel homogeneous interpenetrating composite scaffold that significantly resembles the fiber network structure of natural extracellular matrix(ECM)while also improving the mechanical properties of the hydrogel scaffold.The optimal preparation parameters of platelet-rich plasma(PRP)/sodium alginate(SA)composite hydrogel,an ECMderived composite hydrogel,were investigated,and the preparation parameters of electrospinning fibers were optimized.The effects of the mimetic ECM fiber-hydrogel composite scaffold on tumor cell proliferation,morphology and gene expression were investigated,and finally its effectiveness as an in vitro drug screening platform was verified by animal experiments.The main research contents and conclusions are as follows:(1)Optimized preparation of ECM-derived composite hydrogels.Composite hydrogels with different PRP concentrations(SAP-100,SAP-50,SAP-35,SAP-20)were prepared.Excessive PRP(SAP-100,SAP-50)concentration resulted in dense structure and poor pore characteristics which in turn affected tumor cell proliferation,while SAP-35 composite hydrogels provided the best cell culture conditions.(2)Optimized preparation of ECM-mimicked fiber-hydrogel interpenetrating composite scaffolds.In order to prepare PLGA electrostatic spinning fibers with similar scale to collagen fibers in ECM,different combinations of electrostatic spinning parameters were designed,and 10% PLGA,22 k V,1 m L/h was selected for subsequent experiments according to the scale of collagen fibers in ECM,and fibers with a diameter of 246.22 ± 58.33 nm were obtained.The composite scaffold has structural properties of tumor ECM with abundant fibrin network,which can provide adhesion sites for cells.The excellent porosity,pore size and swelling properties of the composite scaffold could provide sufficient space and channels for cell infiltration and proliferation,and the proliferation ability of breast and prostate tumor cells was increased by 33.48% and 39.71%,respectively.The fiber fraction effectively improved the elastic modulus of the hydrogel,with 0.66,1.20 and 3.84-fold increase in the elastic modulus of composite scaffolds with 5%,10% and 15% fiber content,respectively.Compression tests showed that the composite scaffolds with 10% and 15% fiber content were physiologically correlated with the stiffness of breast and prostate tumor tissues,respectively.The fibers also improved the abrupt release of growth factors from the hydrogel,facilitating the sustained release of growth factors for up to 18 days.(3)Study of the effects of ECM-mimicked fiber-hydrogel composite scaffolds on the proliferation,morphology and genetic phenotype of tumor cells.Tumor cells cultured on composite scaffolds exhibited similar proliferation rates and multicellular tumor spheroid characteristics of in vivo tumors.F-actin staining showed that tumor spheroids formed on composite scaffolds had more significant cell-cell interactions,implying the existence of cellmatrix interactions.PCR results showed that composite scaffolds promoted tumor cells to acquire a more aggressive phenotype(more than 10-fold increase)and increased tumor cell pluripotency,self-renewal(more than 2-fold increase)and angiogenic features(more than 4-fold increase).(4)Correlation between the ECM-mimicked fiber-hydrogel composite tumor model and the results of drug screening in animal models.Based on the therapeutic effects of the common antitumor drugs doxorubicin hydrochloride,paclitaxel and breast cancer-specific drug tamoxifen,tumor cells under composite scaffold culture exhibited higher drug resistance than2 D culture and single-component,which was related to drug action pathway,scaffold stiffness and tumor cell gene expression.Animal experiments showed that the results of drug screening in the fiber-hydrogel composite tumor model had better correlation with in vivo.In summary,the ECM-mimicked fiber-composite hydrogel prepared in this work contributes to cell-cell and cell-matrix interactions by mimicking the extracellular matrix structure and chemical components of tumors,reconstructing tumor structures in vitro and exhibiting in vivo-like characteristics,and making tumor cells in composite scaffolds more invasive and metastatic,with enhanced stemness and increased resistance to anticancer drugs compared to 2D cultures and single-component scaffolds.Tumor models based on mimic ECM fiber-hydrogel composite scaffolds are very valuable tools for in vitro tumor research and drug development.