3D-bioprinted Glioma Microenvironment:a Glioblastoma Model To Study Cellular Interactions And Therapeutics

Background:Glioma is the most common primary intracranial tumor of the central nervous system,accounting for more than 50%of the central nervous system.It has a high degree of malignancy,strong invasiveness,and high recurrence rate and mortality,especially glioblastoma(GBM).At present,the standard treatment of glioma is the maximum surgical resection,combined with the Stupp program of postoperative radiotherapy and chemotherapy,but the treatment effect is still not satisfactory.Most of the traditional preclinical studies on glioma only focused on glioma cells themselves,but ignored the mutual regulation between the glioma microenvironment and tumor cells,and at the same time lacked of 3D space and orderly arrangement in vivo.Our previous study found that in vitro 3D bioprinted brain tumor models enhanced the sternness of tumor cells,and also enhanced the resistance to chemotherapeutics,suggesting the great potential of 3D bioprinted tumor models for the study of tumor cell behavior and tumor drug resistance.This study aimed at the two major non-neoplastic cell components in the glioma microenvironment,mesenchymal stem cells and immune cell components,and discussed the mutual regulation of glioma cells and non-neoplastic cells in different microenvironments and the distinguished effects of the overall glioma microenvironment on the progression of glioma in detail.Our study purposes to provide new ideas for the establishment of in vitro 3D glioma microenvironment and the diversification of in vitro glioma models,and achieves the purpose of studying the mutual regulation mechanism of tumor cells and microenvironmental cells and drug screening by in vitro 3D models.Part Ⅰ:Coaxial bioprinted microfibers with mesenchymal stem cells for study of the interaction between glioma cells and mesenchymal cellsObjective:The tumor microenvironment is composed of tumor cells,tumor stem cells,immune cells and mesenchymal cells,etc.The current research results indicated that glioma-associated macrophages have important regulatory effects on the progression of glioma,but they are basically all related preclinical studies completed in 2D co-culture conditions or in PDX models,which cannot simulate the tumor microenvironment in vivo.We established an in vitro glioma microenvironment model based on 3D bioprinting and filled with multi-cell and multi-biological materials to achieve the purpose of simulating the glioma microenvironment in the body,so as to carry out the research of interaction between neoplastic cells and non-neoplastic cells in vitro.Methods:(1)First,a custom-made coaxial extrusion 3D bioprinting system has been set up and then the shell-alginate/core-GelMA cell line can be fabricated by it with high-density cell suspension.The outer shell is 1%sodium alginate solution encapsulating mesenchymal stem cells or 1%sodium alginate solution,and the inner core is GelMA wrapped U87MG glioma cells,which are printed and cross-linked to form a shell-MSC/core-U87MG or shell-none cell/core-U87MG hydrogel microfiber;(2)Use live/dead reagents and Alamar blue reagent to evaluate the activity,proliferation and resistance to chemotherapy drugs of the printed cells.Observe the cell performance,changes in size and morphology through an inverted microscope and a confocal microscope;(3)Analyze the CD133,CD105,OCT4,GFAP and Nestin protein expression of U87MG cells in different culture conditions by flow cytometry and immunofluorescence;(4)Extract U87MG cell proteins in different culture conditions and use Western blot detection of MMP2 related to tumor invasion,Notchl related to tumor progression and N-cadherin and Vimentin related to tumor EMT(epithelial-mesenchymal transition);(5)Inject cell suspension into nude mice to verify the difference in tumorigenesis ability of U87MG in different microenvironments.Results:The shell/core hydrogel microfibers constructed by the coaxial bioprinting platform were supported by mesenchymal stem cells,and the U87MG cells in the inner core were obviously spread out,showing a good spatial network structure,and the mesenchymal stem cells increased the survival,proliferation,and resistance of glioma cells to chemotherapy drugs,made U87MG cells express more Nestin,MMP2 and Notchl at the same time.With the increase of MSC cell density,tumor cells express more N-cadherin and Vimentin,and the EMT capacity is positively correlated with the concentration of MSC.Finally,we found that in comparison of tumor formation ability,the ability of the U87MG group supported by MSC was significantly stronger than the group without MSC.Conclusion:In this study,the shell-MSC/core-U87MG hydrogel microfibers were prepared by coaxial extrusion bioprinting technology to simulate the glioma microenvironment composed of mesenchymal stem cells and extracellular matrix.In this microenvironment,mesenchymal stem cells can promote tumor cell invasion without contact with U87MG.Compared with the core-U87MG hydrogel microfibers without the blessing of mesenchymal stem cells,mesenchymal stem cells can improve the proliferation,EMT,activity,invasion,tumorigenesis and drug resistance of U87MG cells,and maintain their stemness of the shell-MSC/core-U87MGi microfibers.At the same time,we discovered that GelMA is a biomaterial suitable for observing the morphology,migration and metastasis of tumor cells in the inner core,and can establish a co-cultured tumor microenvironment in vitro.This model provides a new platform for the study of co-culture of tumor cells and stromal cells in vitro to reveal the mechanism of action of non-neoplastic cells in the cell matrix in vitro.Part Ⅱ Bio-fabrication of 3D glioma model in vitroObjective:3D glioma model was fabricated by 3D bioprinting method to evaluate the feasibility of 3D bioprinting glioma cells to establish a brain tumor model.Preliminary compare the differences in biology characteristics and chemotherapy sensitivity of various biomaterials for the establishment of 3D bioprinted glioma models and 2D glioma models.This study aims at laying a solid foundation for the subsequent establishment of a diversified and multi-component glioma microenvironment.Methods:(1)The gelatin/sodium alginate souble hydrosol system was established,and use of tranglutaminasen(TGase)/calcium chloride(CaCl2)as cross-linker.TGase cross-linked gelatin and CaC12 cross-linked sodium alginate;(2)Establish a hydrosol system using methacrylate gelatin(GelMA)containing photoinitiator(LAP)as raw material,and use blue light with a wavelength of 405nm to crosslink after molding.(3)Establish a hydrosol system containing photoinitiator(LAP)-containing methacrylate gelatin(GelMA)/methacrylate hyaluronic acid(HAMA)as raw materials,and use blue light with a wavelength of 405nm after molding(4)Use Medprin Medical’s multi-jet 3D bioprinter(LivPrintTM)to print 6*5*4 mm3 brain-like hydrogel scaffolds;(5)Use fluorescent live/dead staining reagents to evaluate the activity and proliferation ability of glioma cells on the hydrogel scaffold,use scanning electron microscope to observe the morphological structure of the cells in the hydrogel scaffold;(6)Flow cytometry analysis of glioma stem cells in a 3D environment in different biomaterials and in 2D conditions of glioma stem cell surface markers CD90,EMT-related marker CD44,and tumor angiogenesis-related CD105;(7)Use label free to analyze the difference in types and quantities of tumor secreted proteins in 3D and 2D conditions;(8)Next-generation sequencing(NGS)for bioinformatics analysis.Results:(1)The 3D printed biomaterial hydrogel scaffold system,whether it is gelatin/sodium alginate system,Ge1MA system or GelMA/HAMA system,can see porous structure,among which the glioma stem cells in GelMA/HAMA system may glioma cells can achieve "early escape",that is,tumor cells can digest extracellular matrix(ECM)by secreting matrix metalloproteinase(MMP)or other substances to achieve tumor cell invasion;(2)U87MG cell proliferation ability in GelMA/HAMA system is stronger than gelatin/alginate system and GelMA alone;(3)Glioma stem cells(GSC23)in GelMA/HAMA have a higher expression of stemness marker CD90 and the angiogenic ability marker CD 105 than GSC23 in suspension culture and gelatin/alginate system;(4)Compared with the 2D model,the 3D bioprinted glioma model secreted more cytokines and proteins such as POSTN,CSF-1,TGF-b1 and Vimentin,and up-regulated 82 kinds of cytokines.19 kinds of secretion of proteins and factors were down-regulated at the same time;(5)3D bioprinted glioma cells have increased expression levels of hypoxia-related genes,EMT-related genes,cell migration,connection,angiogenesis and other related genes.The expression of genes related to replication,cell cycle regulation,and proliferation was weaker than that in 2D culture environment.Conclusion:With the help of 3D bioprinting technology and printing by hydrogel systems of different components,this "brain-like" 3D glioma model formed in vitro can better simulate the tumor microenvironment in vivo,in which GelMA/HAMA is used as the 3D tumor model constructed by the matrix biomaterial has stronger abilities of proliferation,invasion,EMT,etc.for glioma cells,and provides a new research platform for studying the microenvironment of 3D glioma cells and the in vitro biological behavior of 3D tumor cells.Part Ⅲ 3D bioprinting glioma immune microenvironment and study of the interaction between tumor cells and tumor-associated macrophages in the 3D microenvironmentObjective:The tumor microenvironment is composed of tumor cells,tumor stem cells,immune cells and mesenchymal cells.The current research results indicate that glioma-associated macrophages have important regulatory effects on the progression of glioma,but they are basically all related preclinical studies completed under 2D co-culture conditions or in PDX models,which cannot simulate the tumor microenvironment in vivo.We established an in vitro glioma microenvironment model based on 3D bioprinting and filled with multi-cell and multi-biomaterials to achieve the purpose of simulating the glioma microenvironment in the body,so as to carry out the study of interaction between neoplastic cells and non-neoplastic cells.Methods:(1)Through the comparison of the experimental results in the second part,GelMA/HAMA was selected as the extracellular matrix biomaterial,and Medprin Medical’s multi-jet biological 3D printer(LivPrintTM)was used to print a 6*5*4mm3 brain-like hydrogel scaffold,in which the bottom layer is the substrate on which macrophages are planted,and the rest are glioma cells or glioma cells and glioma stem cells;(2)qPCR determination of gliomas cell/glioma stem cell hypoxia,stemness,transforming factor,and the expression of related genes that reflect the invasion ability in different culture conditions;(3)Western blot,flow cytometry,qPCR detection of M2 macrophages in different culture conditions;(4)Label free analysis of the differences in protein types and quantities of different groups;(5)Elisa kit detection to determine the reasons for the different ability of macrophages to polarize M2 type;(6)Second-generation sequencing(NGS)carry out bioinformatics analysis;(7)Observe the sensitivity of different glioma microenvironments to different chemotherapy drugs.Results:(1)The 3D glioma microenvironment using multi-jet 3D bioprinter can maintain a stable morphology and fixed structure in the cell culture environment through light curing.Glioma cells(U87MG)/glioma stem cells(GSC23)/Glioma-associated macrophages(GAM)have a high survival rate(greater than 80%)on the 5th day after printing;(2)Western blot detection of markers reflected by the polarization ability of M2 macrophages Arginase-1(Arginase-1),U87MG/GSC23/GAM>GSC23/GAM>U87MG/GAM>GAM.qPCR detection of Arg-1,IL-10,CD 163 three M2 type macrophage expression markers is the same conclusion.The results of flow cytometry CD206 are basically the same;(3)After adding GAM to the 3D microenvironment,qPCR detected that tumor cells/tumor stem cells express more TGF-b1,STAT3,IL-1b,and FGF2 genes that positively related to tumor progression,high expression of MMP2 and MMP9,two main matrix metalloproteinase genes,high expression of VEGFR angiogenesis-related genes;(4)In the detection of CSF-1 and TGF-b1 cytokine secretion,we got the same conclusion,the more complex the microenvironment components,the more cytokines are secreted;(5)By comparing the protein secretion by label free proteomics mass spectrometry,we found that after adding GAM,the overall protein secretes more Vim,SPP1,CXCL8,etc.,suggesting that the EMT and angiogenesis ability of tumor cells is further enhanced under the support of macrophages;(6)The 3D microenvironment has more obvious chemotherapeutic effects on BCNU and TMZ than DOX,and the more complex the composition,the stronger resistance to chemotherapy drugs.Conclusion:The more complex of the components the 3D glioma microenvironment are,the stronger the ability to polarize M2 type macrophages they have.At the same time,the polarized M2 type glioma-associated macrophages further enhance the proliferation,invasion,epithelial-mesenchymal transition,vascularization and other abilities of the glioma cells and glioma stem cells in the microenvironment,and the complex microenvironmental components have higher biomimetic properties in vitro.The 3D bioprinted tumor microenvironment model provides a new idea and a good platform for studying neoplastic cells with non-neoplastic cells interaction and drug development and screening in a 3D microenvironment.