Construction And Application Of In Vitro Three-dimensional Tumor Model Based On Hydrogel

Cancer is one of the leading causes of death in various diseases in China.Cholangiocarcinoma(CCA)is the second most prevalent primary malignancy in liver cancer.A deep understanding of the biological characteristics of CCA is vital for developing more effective treatments.Recently,three-dimensional culture models have made rapid progress in tumor pathogenesis research and drug screening research in vitro.As a new research model,the three-dimensional tumor model can recapitulate the complexity of the tumor microenvironment in vivo,maintain the heterogeneity of cancer cells,and make the tumor cells closer to the internal environment in terms of morphology and signal pathways.The critical step of constructing a three-dimensional tumor model is to select a suitable bioscaffold material.Hydrogel biomaterials have been widely used in the construction of three-dimensional model in vitro,regenerative medicine,drug screening,drug delivery and other fields due to their similar mechanical properties to native tissue and excellent biocompatibility.For instance,Matrigel has been widely used in 3D tumor model.In the molecular targeted therapy of CCA,TKI inhibitors have obtained extensive attention.For example,Sorafenib and Regorafenib have invested in clinical trials.However,many TKI inhibitors used in clinical trials of CCA have failed.As a small molecule multi-target tyrosine kinase inhibitor,anlotinib has many advantages of anti-tumor angiogenesis and inhibits tumor growth,and is expected to be a potential targeted drug in the treatment of CCA.The development of decellularized technology provides an opportunity for the construction of three-dimensional tumor models in vitro.The researchers removed all cellular substance from tissues or organs by different decellularized methods,then acquired the acellular matrix without distruction of structural integrity and biological activity.As a biological scaffold,acellular matrix can provide three-dimensional network,structure and functional support of macromolecules for surrounding cells,and is increasingly used in tissue engineering research.Acellular matrix scaffolds can more closely mimic the microenvironment in vivo.Compared with other models in vitro,tumor cells have biological properties closer to those in the body tissue in their derived tissue or metastatic tissue decellularized scaffolds,thus ECMs are expected to become an ideal material for the construction of 3D tumor models.We focus on the following two aspects:Part ?:Construction and application of an in vitro three-dimensional model of cholangiocarcinoma based on MatrigelOBJECTIVE:To engineer a 3D model of CCA based on Matrigel and utilize as a model to detect the response of CCA treated with anlotinib.METHODS:Three CCA cell lines QBC939,RBE and HCCC9810 were seeded into Matrigel for 5 days to establish three Matrigel-based three-dimensional models in vitro of CCA.Histology,CellTiter-Blue assay and real-time quantitative PCR were used to study the histomorphology,cell proliferation,vascularization and metastasis-inducing gene expression,reactiveness of anlotinib in a three-dimensional model of CCA.RESULTS:The CCA cell lines were cultured in Matrigel for 5 days to form cell clusters with a diameter of 70?120?m.It mainly contained major extracellular matrix proteins and polysaccharides,and the content of collagen I and collagen IV was higher than collagen III.The growth rate of QBC939 in 3D-Matrigel model is lower than that of 2D model,while RBE and HCCC9810 are higher than of 2D model.Compared with 2D model,the vascularization and the MMP family related genes in 3D-Matrigel model have shown significant differences.The result of drug treatment indicated that anlotinib could inhibit the proliferation of CCA cells in 3D-Matrigel model with a dose-dependent manner,and 3D-Matrigel model was less sensitive to anlotinib than the 2D model.CONCLUSION:The Matrigel-based three-dimensional model of cholangiocarcinoma was successfully established.This model differs from the two-dimensional model in tumor cell growth,vascularization and invasion-related gene expression and reactivity to anlotinib.This part of the study provides experimental support for the feasibility of anlotinib for the clinical treatment of cholangiocarcinoma,and also affords a(?)comparison and reference for the second part.Part ?:Construction and application of an in vitro three-dimensional model of cholangiocarcinoma based on decellularized liver matrix hydrogelOBJECTIVE:Preparing decellularized liver matrix hydrogel and constructing a 3D model of CCA based on decellularized liver matrix hydrogel,and evaluating its feasibility as a biological study and drug screening model for CCA in vitro.METHODS:The whole liver cells were eluted by the chemical elution method with Triton X-100 and SDS to prepare the decellularized liver matrix hydrogel.The CCA cell lines QBC939,RBE and HCCC9810 were seeded into the decellularized liver matrix hydrogel.The 2D culture model and the 3D-Matrigel model were used as control to study the histomorphology,cell proliferation,vascularization and metastasis-inducing gene expression,the response to anlotinib and the related signaling pathways for the inhibition of vascularization with anlotinib in a 3D model of CCA based on decellularized liver matrix hydrogel.RESULTS:The rat liver was decellularized by Triton X-100 and SDS and retaind the main protein components,polysaccharides and some growth factors of the natural extracellular matrix.The acellular matrix was digested by pepsin and could obtain biocompatible and temperature-sensitive decellularized matrix hydrogel(DLM);CCA cells cultured in DLM hydrogel for 5 days can form cell clusters with 50?100 ?m diameter which contains major extracellular matrix proteins and polysaccharides.Among the biomacromolecules,we found collagen I was strongly expressed,and collagen III and collagen IV were weakly expressed,indicating that the three-dimensional model of CCA based on DLM hydrogel was different from Matrigel three-dimensional model in terms of collagen distribution patterns;The growth rate of CCA cells in 3D model of DLM is significantly higher than those in the Matrigel 3D model.Compared with the 2D model and the Matrigel 3D model,we found that the vascularization of CCA cells and the mRNA expression of MMP family-related genes are significantly increased in the 3D-DLM model,indicates that 3D-DLM may have stronger vascularization and invasion ability;drug treatment results show that two kinds of three-dimensional models have the reduced sensitivity to anlotinib compared with 2D culture models,but the cholangiocarcinoma cells in the 3D-DLM model were more sensitive to anlotinib than 3D-Matrigel model.Western Blotting further confirmed that anlotinib inhibits PDGF-BB and FGF-2-induced phosphorylation of ERK and Akt in the 3D-DLM model of CCA,thereby exerting vascularization inhibition.CONCLUSION:A three-dimensional model of cholangiocarcinoma based on liver acellular matrix hydrogel in vitro was constructed.Compared with the three-dimensional model of Matrigel,the expression of angiogenic and vascularized and invasion-related genes of cholangiocarcinoma cells in the acellular matrix of the liver and the drug reactivity of anlotinib was also significantly increased.This study is not only expected to provide a new three-dimensional model of decellularized liver matrix hydrogel for the study of cholangiocarcinoma in vitro,but also provide guidance and basis for the design and development of new scaffold materials in the construction of three-dimensional tumor models.