Conclusion
In this study, based on the liquid overlay method, we established a rapid and efficient method to produce uniform size and high reproducibility of 3D MTSs. Based on multi-omics data, the phenotypic differences of HeLa cells under different culture conditions were explored based on the established 3D MTS model to investigate the resistance mechanism of 5-FU and discover the key regulatory genes and related reprogrammed metabolic pathways. We found that in 3D HeLa carcinoma cells the metabolic shift towards glycolysis, the maintenance of ER homeostasis and the up-regulation of ECM may contribute to the formation of increased resistance to 5-FU. In addition, the changes in ABC transporters, HIF-1 signaling pathway and the improvement of tumor cell stemness were also related to the increase of 5-FU resistance of 3D MTSs. Overall, this study demonstrates that resistant 3D Hela MTS can be a promising tool for testing drug delivery and efficacy for cervical cancer treatment, and a multi-omics analysis can reveal metabolic addictions which can be manipulated to restore susceptibility to chemotherapy drugs, since this model is readily compatible with the methodologies and techniques that are already used for the analysis of conventional 2D monolayer cultures.