Construction Of 3D Tumor Model And Research On Drug Resistance Mechanism Using Multi-omics Integrated Analysis

Cervical cancer is a serious health problem in women around the globe,with nearly 600 thousand new cases each year,according to the statistics from the International Agency for Research on Cancer(IARC).5-Fluorouracil(5-FU)is a commonly used clinical drug for the treatment of cervical cancer.However,some patients with advanced cervical cancer eventually develop resistance to 5-FU.The development of drug resistance seriously limits the use of clinical drug,which is largely related to metabolic reprogramming and heterogeneity in tumor cells.Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application research.Multicellular tumor spheroid(MTS)can in a way recapitulates tumor traits in vivo,such as gradients of oxygen,nutrients and metabolic wastes,as well as enhanced cell-cell interactions and cell-extracellular matrix interactions.Hence.MTS could be used as a powerful transitional tumor research model between 2D monolayer cell culture and 3D xenograft.In this study,based on the liquid overlay method,a rapid and efficient protocol was established to achieve the high-throughput production of the MTSs with diameter of about 500 μm,a roundness greater than 0.9 and a necrotic core.This protocol has been experimentally verified to be suitable for the construction of 3D models originating from a variety of tumor cells(e.g.,HeLa,HCT116,MCF-7),and can be further extended to co-culturing model with stromal cells(such as fibroblasts UCF,immune cells PBMC)to construct multi-component MTSs,thus achieving more complex tumor traits in vivo.Experimental results showed that cell proliferation and division in MTSs were inhibited.As expected,the results showed that there was enhanced 5-FU resistance in MTSs than monolayer cells with a resistance index of 5.92.With supplement of 5 mM glutathione,the 3D derived cells maintained partial phenotypes of 3D MTSs under 2D culture condition with the resistance index of 2.91,which was reflected in blocking the cell cycle,ur-regulating non-mitochondrial respiration,etc.,and similar resistance gene expression.However,HeLa cells derived from 3D MTS had limitations such as slow proliferation,which is necessary to further optimize the culture conditions to achieve normal culture in 2D monolayer.Furthermore,the mechanism of 5-FU resistance of HeLa cells in 3D culture was explored by using multi-omics techniques.Our data revealed a global rewiring of the proteome and metabolome,along with differential transcript levels of tumor resistance-associated genes.The effect of 5-FU on MTSs was significantly weaken than that of 2D cluture,especially in the protein expression profile.There were 133 different proteins in 2D culture before and after 5-FU treatment,while only 27 proteins were changed in 3D MTSs.The differences in the protein synthesis,processing,and transportation between 2D culture and 3D MTSs were significant,mainly in the heat shock protein family,with the upregulation of protein folding function in endoplasmic reticulum(ER)which promoted the maintenance of ER homeostasis.Compared with 2D culture,proteins related to mitochondrial function were significantly down-regulated in 3D MTSs,including mitochondrial translation,transcription and electron transport,etc.Meanwhile,we found that non-mitochondrial respiration of MTSs was significantly up-regulated under both control and 5-FU treatment conditionds,which may caused by the shifted main energy source from mitochondrial oxidative phosphorylation to glycolysis.The intermediate metabolites of the tricarboxylic acid cycle(TCA cycle)were significantly decreased under control conditions.Under the 5-FU treatment,intermediate metabolites of the TCA cycle were decreased in 2D culture,while the increase of glutamine and glutamate in 3D MTSs to some extent supplemented TCA cycle flux.In addition,at the transcriptional and protein level,the expression of extracellular matrix proteins including laminin was up-regulated in the MTSs,which enhanced the physical barrier of drug penetration and signal transduction pathways,including integrin-mediated pathways.Furthermore,hypoxia-induced changes in signal transduction pathways and increased stemness of tumor cells were also associated with increased 5-FU resistance towards MTSs.In conclusion,this study formulates a rapid,scalable and reproducible in vitro model of MTS for drug screening purposes,which provides a model basis for anticancer drug screening,tumor development and progression research,and personalized tumor therapy,etc.The phenotypic differences between 2D culture and 3D MTSs were systematically analyzed by combining transcriptome,proteome,and metabolome data,and the findings establish a critical role of glycolytic metabolism,ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.