Physiological characterization of 3D MTSs
To monitor the growth dynamics of MTSs, the diameter, roundness and cell growth of MTSs were recorded and assessed every day. In the initial stage of MTSs formation, cells grew slowly and were easy to distinguish individual cells (Figure. 2A ). Driven by the interaction and contact between the cells, cells gradually gathered into clusters. After 2 days of culture, the cells entered the logarithmic growth phase, proliferated rapidly and gradually formed smooth surfaces. On the 6th day, MTSs reached the plateau stage, with the largest diameter of 532.21 ± 21.42 μm and 592.54 ± 13.64 μm, respectively, when the seeding densities were 1000 and 2000 cells/well (Figure. 2B ). The roundness of obtained MTSs exceeded 0.9 (Figure. 2C ), with the margin of error no more than 10%. The number of living cells in a single MTS reached the maximum on day 6, approx. 17 times the initial amount (Figure. 2D ). After that, the boundaries of MTSs began to wrinkled and blurred, and the spheroids gradually disintegrated.
The morphology and microstructure of MTSs cultured for 6 days were observed by the SEM, and the MTSs showed a good 3D structure and regular spherical shape (Figure. 2E, Figure. S2 ). Interestingly, MTSs of different cell types had different surface structures. The surface of the single-component Hela cell spheroid was relatively smooth (Figure. 2E ), while the multi-component MTSs retained the more obvious tissue structures (Figure. S2 ). When co-cultured with fibroblasts such as UCF, the fibroblasts extended outwards MTSs. Obvious vesicles were observed in the MTSs co-cultured with immune cells such as PBMC. This indicates that the more complex 3D model would preserve more completely biological characteristics of tumor tissues in vivo .
Compared with 2D monolayer cells, the cell proliferation rate in 3D MTSs was reduced. The specific growth rate (µ) of cells cultured for 2 days under 2D conditions was 0.0455 h-1, while the µ of cells in MTSs cultured for 2, 4, and 6 days were 0.0263 h-1, 0.0246 h-1, 0.00522 h-1, respectively, with the margin of error no more than 13.6 % (Figure. S3 ). The cellular viability of 3D MTSs that were cultured for 2 days was similar to 2D monolayer cells, but the percentage of living cells and apoptotic/ necrotic cells in MTSs are gradually decreased and increased, respectively, with the culture time (Figure. 2F ). Compared with 2D monolayer culture, the cell cycle in 3D MTSs was blocked throughout the cultivation. In addition, cells in the 3D MTS are accumulated and decreased over the culture age in the G1 and S phase, respectively (Figure. 2F ). However, there was no significant change in the proportion of cells in G2 phase between 2D monolayer culture and 3D MTSs.
Chemosensitivity of Hela tumor spheroids following 5-FU treatment
The cytotoxicity of 5-FU to Hela cells cultured under both 2D monolayer and 3D MTSs was evaluated (Figure. 3A ). 3D MTSs showed stronger resistance than 2D monolayer cells, and after the 5-FU treatment for 48 h the IC50 of MTSs (93.88 μM, 95 % confidence interval: 64.88 ~ 148.5 μM) was approximately 5.72 times that of 2D monolayer culture (16.42 μM, 95 % confidence interval: 12.73 ~ 21.14 μM). Therefore, we treated the HeLa carcinoma cells from both 2D and 3D models with 16 μM 5-FU for 48 h in the following study. Next, the changes of cell apoptosis and cell cycle were compared before and after the 5-FU treatment (Figure. 3B ). The effect of 5-FU on cells in 3D MTSs was significantly reduced compared with 2D monolayer culture. After the 5-FU treatment, the percentage of living cells and apoptotic cells was decreased and increased, respectively, in 2D monolayer, while there were no significant changes in 3D MTSs. As is known, 5-FU mainly targeted S-phase cells (Ijichi, Adachi, Ogawa, Hasegawa, & Murakami, 2014). After the 5-FU treatment, the cell cycle was blocked in G1 phase in both 2D monolayer and 3D MTSs. Meanwhile, we observed a decrease in the proportion of S phase cells and a constant proportion of G2 phase cells in the 2D monolayer after the 5-FU treatment, while no significant changes were observed in the 3D MTS.
To adapt to rapid metabolic requirements of tumor cells, the metabolic pathways have significantly changed to metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production (Vander Heiden et al., 2009). Mitochondrion is the energy factory and the main position of oxygen consumption, and the abnormal mitochondrial metabolism in tumor cells are often related to drug resistance (Yan & Li, 2018). According to the mitochondrial respiration profile (Figure. 3C ), the oxygen consumption in 3D MTSs was much higher than that of 2D monolayer cells, which was mainly due to the significant increase of non-mitochondrial respiration. The results showed that the non-mitochondrial respiration of MTSs was about 4.60 and 3.19 times that of 2D cultures under the control and 5-FU treatment conditions, respectively. The possible reason for the increased non-mitochondrial respiration was that the main energy source shifted from mitochondrial oxidative phosphorylation to glycolysis. Contrary to this, the spare respiratory capacity of MTSs was reduced about 74.98 % and 63.31 relative to the 2D culture, which was associated with mitochondrial dysfunction. Furthermore, the mitochondrial basal respiration and maximum respiration capacity were reduced by 68.56 % and 71.81 % in 3D MTSs relative to the 2D culture under the control conditions, while the ATP synthesis capacity was almost constant. After the 5-FU treatment, the mitochondrial basal respiration and maximum respiration capacity was almost constant, while the ATP synthesis capacity were about 62.40 % reduced in 3D MTSs as compared with the 2D cultures. The further inhibition of 5-FU on the mitochondrial ATP production capacity of 3D MTS aggravated the dependence of MTS on the glycolytic pathway. As the major hub of cellular energy generation, mitochondrion, is also the main source of reactive oxygen species (ROS) and it has been reported that the ROS level can be reduced if glycolysis as the main energy source (Herst, Tan, Scarlett, & Berridge, 2004). However, we found that the ROS production capacity in 3D MTSs was significantly higher than 2D monolayer cells (Figure. 3D ).
As can be seen from Figure. 3E, 3F , in the presence of glucose and glutamine, tumor cells preferred to use glutamine. Under control conditions, cells cultured in 2D monolayer cultures and 3D MTSs mainly used glutamine, and began to consume a small amount of glucose after 60 h of culture. Under the 5-FU treatment, glutamine was still the main energy source in 2D monolayer culture, while cells consumed glutamine and glucose at the same time, in 3D MTSs. In addition, with 5-FU treatment reabsorption phenomenon was found in the 3D MTSs, while HeLa cells continued to secrete ammonia under 2D monolayer culture conditions (Figure. 3G ). It has been reported that the reabsorption and reuse of ammonia was beneficial to the growth of tumor cells (Spinelli et al., 2017). The secretion rate of lactate in 3D MTSs was slightly higher than that in 2D culture under both control and 5-FU treatment conditions (Figure. 3H ,Figure. S4) .
Identification of transcriptional alterations for 5-FU resistance
The biological characteristics of 3D MTSs were more representative than that of 2D monolayer culture, such as hypoxic regions and pH gradients caused by mass transfer limitations, enhanced extracellular matrix (ECM) secretion, drug permeation barriers caused by closely cells contact, increased ECM deposition and the improvement of tumor cell stemness (Dittmer & Leyh, 2015). Therefore, we measured the transcriptional levels of drug resistance-related genes in Hela cells cultured under both 2D cell cultures and 3D MTSs (Figure. 4 ).
In the 3D MTS model, there is often an oxygen diffusion limit of 150~200 μm (Oldham, Clish, Yang, & Loscalzo, 2015). Hence, exceeding this radius would likely form an area of hypoxia in the MTSs, which would lead to genetic and metabolic reprogramming regulated by hypoxic induction factor (HIF1A ) (Denko & Nicholas, 2008). Compared with 2D monolayer cells, after the 5-FU treatment, the transcript level ofHIF1A in the 3D MTSs were significantly increased by 1.60 times (Figure. 4 ). It has been reported that tumor hypoxia microenvironment would cause abnormal activation of the oncogene MET , and consistent with this, the transcription level of MET was 2.54 and 1.8 times higher up-regulated in the 3D MTSs than in the 2D cultures under the control and 5-FU treatment conditions, respectively, which would promote angiogenesis and maintain tumor aggressiveness (Stella, Benvenuti, & Comoglio, 2010). HIF1A could also induce the expression of vascular endothelial growth factor (VEGFA ), which is central to the growth and metastasis of tumors, thereby promoting the malignant progression of tumors (Mohamed, Khalil, & Toni, 2020). As evidenced, we observed that the transcription level of VEGFA was about 5.04 times and 14.87 times higher up-regulated in more 5-FU resistant 3D MTSs than 2D monolayer cells under the control condition and the 5-FU treatment conditions, respectively.
Compared with normal cells, tumor cells display upregulated glycolysis for the provision of intermediates for rapid proliferation, which is mainly manifested by enhanced glucose uptake and lactate excretion (Yong, Stewart, & Frezza, 2019). Similarly, we observed an increase in glucose utilization and lactate excretion in 3D MTSs, especially following the 5-FU treatment, compared with 2D monolayer culture (Figure. 3E, 3H ). Therefore, the transcript level of the genes encoding glucose transporter (GLUT ), lactate dehydrogenase (LDH ) and phosphofructokinase (PFK1 ) were measured. As compared with the 2D monolayer cultures, the transcript levels of GLUT1 , LDHA ,PFK1 were 4.20, 1.60 and 1.21 times higher upregulated in the 3D MTSs under the control conditions (Figure. 4 ). As expected, the transcript levels of these genes were more pronouncedly increased after the 5-FU treatment, showing that 9.72, 2.45 and 1.88 times higher upregulated in the 3D MTSs than in the 2D cultures. This result indicated that 3D MTSs featured enhanced aerobic glycolysis, i.e., the well-known Warburg effect.
The rapid proliferation of tumor cells required a large number of nucleic acids. The only source of thymine in cells is the de novo synthesis pathway, and the high expression of thymidylate synthetase (TYMS ) is often associated with poor prognosis of tumors (Donner et al., 2019). 5-FU blocks DNA synthesis to induce cell death by inhibiting TYMS, while dihydropyrimidine dehydrogenase (DPYD) could decompose and deactivate 5-FU before it was converted into active metabolites (Negarandeh et al., 2020). Compared with the 2D cultures, the transcription level of TYMSwere 2.15 and 3.48 times higher up-regulated, meanwhile the transcript level of DPYD were 3.66 and 1.26 times higher up-regulated in 3D MTSs before and after the 5-FU treatment conditions, respectively (Figure. 4 ). Therefore, the up-regulation of the expression ofTYMS and DPYD were also the reasons for the enhanced resistance of MTSs to 5-FU.
Increasing evidence has ever shown that conventional cancer chemotherapy is seriously limited by the multidrug resistance (MDR) commonly exhibited by tumor cells (Perez-Tomas, 2006). In drug-resistant tumor cells, the main mechanism was the drug accumulation and efflux in which the ATP binding cassette (ABC) transporters played an important role (Orlando & Liao, 2020; Ye et al., 2016). However, we did not observe the up-regulation of ABCB1 and ABCG2 transcription levels in 3D MTSs as expected, but there may be differences in protein or metabolite levels. Lysosome-associated transmembrane protein 4B (LAPTM4B ), a multidrug resistance gene, could stimulate drug resistance and promote cell growth and proliferation by regulating drug efflux mechanism and activating PI3K/Akt signal transduction (Gu et al., 2020). Consistent with this, compared with the 2D monolayer cultures, the expression level ofLAPTM4B was 1.57 and 1.32 times higher up-regulated in 3D MTSs before and after the 5-FU treatment (Figure. 4 ).
Apoptosis defects were also one of the reasons for drug resistance, which were usually regulated by the BCL-2 protein family (Warren, Wong-Brown, & Bowden, 2019). Compared with 2D monolayer cells, the transcription level of the BCL2 encoding anti-apoptotic protein in 3D MTSs was 1.55 times and 3.09 times higher up-regulated before and after the 5-FU treatment, respectively, which contributed to the progression of tumor drug resistance (Figure. 4 ). However, the transcription level of the BAX encoding apoptotic protein was also up-regulated in 3D MTSs, which might be related to the decrease of cell proliferation activity.
Then, the expression of cytokines related to tumor cell proliferation and progression was tested. Transforming growth factor (TGFB1 ) was generally up-regulated in tumor cells, and could induce epithelial-mesenchymal transition and promote tumor cell growth, proliferation and invasion (Fuxe & Karlsson, 2012). The mTOR pathway was a classical signal transduction pathway regulating cell growth and metabolism, and was dysregulated in many cancers (Gao et al., 2009). It has been reported that the glycolytic pathway was affected by the mTOR pathway through two key transcription factors, HIF1A and MYC (Renner et al., 2017). As compared with the 2D monolayer cultures, the transcript levels of TGFB1 ,MTOR , MYC were 1.92, 2.08 and 1.59 times higher upregulated in the 3D MTSs, respectively (Figure. 4 ). As expected, the transcript levels of these genes were more pronouncedly increased after the 5-FU treatment, showing that 1.82, 3.21 and 3.51 times higher upregulated in 3D MTSs than in the 2D monolayer cultures, respectively (Figure. 4 ).
ECM, such as laminin, fibronectin, vimentin, mediated interactions between cells and participated in signal transduction in processes such as cell adhesion, migration, invasion, proliferation and EMT to promote the development of drug resistance, referred to as cell adhesion mediated drug resistance (CAM-DR) (Valkenburg, de Groot, & Pienta, 2018; Wantoch von Rekowski et al., 2019). Although the transcript level of laminin β1 (LAMB1 ) did not change significantly, compared with the 2D cultures, we found 3.68 times higher up-regulation in the expression of fibronectin (FN1 ) in 3D MTSs under the control condition. Meanwhile, the transcript levels of vimentin (VIM ) were 1.70 and 3.18 times higher upregulated in 3D MTSs under the control and 5-FU treatment conditions, respectively (Figure. 4 ). It was found that ECM participated in CAM-DR by stimulating integrin mediated PI3K activation to protect tumor cells from damages caused by radiotherapy and chemotherapy (Hodkinson, Mackinnon, & Sethi, 2007). Compared with the 2D monolayer cultures, the expression level of integrin β1 (ITGB1 ) was 1.76 and 3.59 times higher up-regulated in 3D MTSs before and after the 5-FU treatment, respectively (Figure. 4 ). ITGB1 (also known asCD29 ) and CD44 are reported as tumor stem cell markers, and the presence of tumor stem cells could affect the drug treatment and subsequent tumor recurrence (Tomasetti, Li, & Vogelstein, 2017). It has been shown that CD44 can mediate the stemness of tumor cells and participate in metastasis by binding to hyaluronic acid (Gomez et al., 2020). Compared with 2D monolayer cultures, the expression level of andCD44 was 1.79 and 2.21 times higher upregulated in 3D MTSs than in 2D monolayer culture under the control and 5-FU treatment conditions, respectively (Figure. 4 ).