Generation of reproducible 3D MTSs using an optimized liquid overlay method
Up to now, researchers have established a variety of methods for the preparation of MTSs (scaffold-based or scaffold-free), among which the liquid overlay method based on non-adhesive surfaces has been the most popular technique for the MTS production due to its generality and low cost (Costa et al., 2018). Different culture methods could be combined with each other, and external forces, such as centrifugation, magnetic fields, electric fields, mechanical vibrations, acoustic waves, are conducive to cell aggregation (Anggayasti, Imashiro, Kuribara, Totani, & Takemura, 2020). Based on the liquid overlay method, in this study we optimized the MTS culture process and established a rapid, efficient and reproducible MTS generation method (Table S2 ). The tightness and compactness of MTSs reflect the strength of the interaction between cells and the density of cells accumulation, which directly affects cell function, drug penetration and drug response (Koudan et al., 2020). The introduction of the whole well-plate centrifugation and Matrigel™ additives significantly improved the roundness and particle size uniformity of MTSs, facilitating the formation of tighter and more regular 3D structures. A single MTS was formed in each well of the plates, which allows to conveniently monitor the growth dynamics of MTSs, improves the reproducibility, facilitates multi-omics analysis and high-throughput screening of antitumor drugs. The diameter of MTSs could be controlled at about 500 μm, above which they would stratify to form a necrotic core (Oldham et al., 2015). The reduced proliferative activity (Figure. 2F, Figure. S3 ) and blocked cell cycle in 3D MTSs (Figure. 3B ) may contribute to the development of resistance to 5-FU (Ijichi et al., 2014; Mehta, Hsiao, Ingram, Luker, & Takayama, 2012). This method was suitable for the 3D culture of a variety of tumors, and could also be used for co-culturing with stromal cells and immune cells to construct more complex 3D tumor models. HeLa cells cultured in 3D MTSs showed enhanced 5-FU resistance, with a drug resistance index of about 5.72. Therefore, this study provided a scalable method for 3D MTS generation, which benefits the study of tumor drug resistance mechanism and the application of high-throughput drug screening.
Metabolic  shift toward glycolysis in 5-FU resistant Hela cells
It has been reported that the increase of glycolytic pathway in melanoma cells promotes the growth and invasion of tumor, mediates stronger drug resistance, and impaired T cell killing of tumor cells (Cascone et al., 2018). Mitochondrial dysfunction and hypoxia are two important factors that induce the Warburg effect (Xu et al., 2005). In the 3D MTSs, the utilization of glucose was increased compared with 2D monolayer cultures (Figure. 3E ) and the expression of GLUT1, SLC2A1 and LDHA were also up-regulated (Figure. 4 , Figure. 5A ), which indicated the enhanced glycolytic flux. The non-mitochondrial respiration of MTSs was significantly improved (Figure. 3C ). Through comparative proteome and metabolome analysis (Figure. 5A-D , Figure. 6A, B ), we found that in 3D MTSs the biological processes related to the mitochondrial function were significantly down-regulated, and the intermediates involved in TCA cycle were decreased in 3D MTSs, but there were no significant differences in the enzymes and intermediate metabolites of the glycolytic pathway. Ruprecht et al. have shown that post-translational modifications of glycolysis, especially phosphorylation, lead to glycolysis addiction and mediate drug resistance (Ruprecht et al., 2017). The existence of hypoxic regions of 3D MTSs restricted mitochondrial respiration, leading to incomplete oxidation of nutrients, and forcing tumor cells to up-regulate the glycolysis as the primary pathway for energy supply. Previous study has demonstrated that mitochondrial function as oxygen sensors and releasing ROS can stabilize the signal hypoxia induced factors such as HIF-1α and HIF-2α (Guzy et al., 2005). Consistent with this, the ROS levels are significantly higher in the 3D MTS than in the 2D monolayer cultures (Figure. 3D ). Also, we observed that 5-FU significantly induced the formation of the ROS under both 2D monolayer cultures and 3D MTSs (Figure. 3D ). The continuous accumulation of ROS would further cause damage to mitochondrial DNA and electron transport chain, aggravating mitochondrial dysfunction and dependence on the glycolysis (Pelicano, Carney, & Huang, 2004). Tumor cells relying on glycolysis produce large amounts of lactate, which was indeed observed especially after the 5-FU treatment (Figure. 3H ). Lee et al. identified an oxygen-regulated protein NDRG3 encoded by N-myc downstream regulated gene, which can bind to lactate so as to be protected from destruction under hypoxic conditions, thereby promoting angiogenesis and cell growth(Lee et al., 2015). Apicella and colleagues found that the secretion of lactate contributed to activate the MET-dependent signal transduction pathway in tumor cells, resulting in the resistance to tyrosine kinase inhibitors (Apicella et al., 2018). Therefore, the metabolic shift of 3D MTSs to glycolysis may contribute to the increased resistance to 5-FU.