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.