Studies On The Mechanism Of Radiation Resistance Using 3D Cultured Glioma Models

BACKGROUND&OBJECTIVE:Most of the in vitro models of current tumor research are based on traditional two-dimensional(2D)culture models.The 2D tumor model has limitations since it lacks of extracellular matrix(ECM),space-time structure,nutrient concentration gradient and other tumor microenvironment components.A growing body of research has confirmed that 3D tumor models are more prominent than 2D models in terms of tumor microenvironment construction,gene expression differences,drug treatment tolerance,and radiation therapy resistance studies.Our team has established 3D glioma model bioproduction,constructed a glioma stem cell microenvironment,and confirmed that the 3D glioma model is more tolerant to drug therapy than the 2D model.However,the differences in gene expression profiles between 3D glioma models and 2D models and their sensitivity to radiation therapy remain unclear.Therefore,we aim to study:1)In vitro biosynthesis of 3D glioma models using a gelatin/alginate/fibrinogen hydrogel system;2)Comparison of gene expression profiles in 3D and 2D glioma models;3)Screen for possible radiosensitivity-related genes;4)Verify radiosensitivity differences between 3D and 2D glioma models by in vitro radiotherapy studies;and 5)Explore the molecular mechanisms of radioresistance in 3D glioma models.Methods:(1)using the characteristics of chemical cross-linking of sodium alginate with calcium chloride,thrombin and fibrinogen,and gelatin as the main matrix to establish a suitable hydrogel system for cell growth;(2)fully mixing the glioma cells with good growth state with the above hydrogel system as bio-ink,using the principle of extruded biological 3D printing,Attaching a sterile syringe to a 3D printing needle for bio-manufacturing of a 3D mesh structure;After obtaining the 3D culture model,the culture is continued in the cell culture incubator;(3)In vitro biological evaluation of the growth state of 3D culture glioma cell model:including microscopic morphology observation,live/dead staining,confocal and electron microscopic morphology,and HE pathological section staining;(4)Collecting cells of 3D and 2D glioma models,performing RNA sequencing,KEGG database analysis,screening differential genes and radiosensitivity genes of interest;(5)The expression difference of the genes in 3D and 2D glioma models will be verified by qRT-PCR and Western Blot;(6)Irradiation of 3D and 2D glioma models with different doses of X-rays(0,2,4,6,8 Gy),comparison of clonal proliferation ability by clone formation experiments,confirming differences in radiosensitivity between 3D and 2D glioma models;(7)Quantification of differential gene expression levels by qRT-PCR and Western Blot to determine the relationship between these genes and radiotherapy resistance in 3D gliomamodels;(8)RNAi to knockdown the above genes and then perform radiotherapy to verify the role of the gene of interest in radiotherapy resistance of 3D glioma model,and further explore related signaling pathways.RESULTS:(1)The 3D hydrogel scaffold showed multi-level micropore structure under electron microscope and had good nutrient permeability;(2)The 3D hydrogel scaffold structure containing glioma cells can be cultured for more than 14 days,and the cells maintain high activity and gradually grow into tissue-like spheres;(3)Live/dead staining,microscopic and electron microscopic showed that 3D glioma model,cell survival rate was high(85.23±6.21)%;(4)A large number of differentially expressed genes were screened by RNA sequencing,in which the expression of the radiation resistance-related gene ITGA2 was significantly different;(5)qRT-PCR and Western Blot confirmed that ITGA2 was overexpressed in the 3D glioma model;(6)After radiotherapy,the results of cloning formation experiments showed that the 3D glioma model was more tolerant to radiation therapy than the 2D model(SER=0.92);(7)qRT-PCR and Western Blot confirmed that ITGA2 was overexpressed in the 3D glioma model and may regulate the radiotherapy resistance of the model;(8)Silencing of ITGA2 by RNAi showed that the radioresistance of the 3D glioma model was significantly lower than that of the control group(P<0.01);(9)KEGG data analysis drew the protein interaction network map showing that ITGA2 is directly related to the expression of AKT.Western Blot results suggest that ITGA2 gene can up-regulate the expression level of p-AKT.It is speculated that ITGA2 promotes the radiotherapy resistance of the 3D glioma model by up-regulating p-AKT.Conclusion:The 3D culture glioma model was successfully established by a sodium alginate/gelatin/fibrinogen hydrogel system.Compared with the 2D culture glioma model,the gene expression profiles of glioma cells are significantly different,and it is preliminarily proved that ITGA2 plays a role in the radiotherapy resistance of 3D cultured glioma models through the Pi3K-AKT pathway.