Figure legends
Figure 1 . Schematic illustration of the generation of single tumor spheroid.
Figure 2 . Physiological characterizations of the growth dynamics of 3D MTSs over the culture age. (A) Optical imaging of 3D MTSs. (B) Diameter of 3D MTSs. (C) Circularity of 3D MTSs. (D) Growth curve of 3D MTSs. (E) The morphology and microstructure. (F) Cell apoptosis and cell cycle.
Figure 3 . The effect of 5-FU on 3D MTSs. (A) Cytotoxicity assay. (B) Cell apoptosis and cell cycle. (C) Mitochondrial respiration profile. (D) ROS measurement. The measured concentrations of extracellular (E) glucose, (F) glutamine, (G) ammonium and (H) lactate.
Figure 4 . Transcriptional analysis of putative drug resistance genes associated with HIF1A -induced signal pathways, glucose metabolism, nucleotide metabolism, multidrug resistance-related geneLAPTM4B , ECM secretion, tumor stem cell and signaling pathways related to cell growth and proliferation.
Figure 5 . Proteomic differences between cells in 2D monolayer and 3D MTS. (A) Heatmap of differential proteins between 2D monolayer and 3D MTS. 50 differential proteins in the top KEGG pathways (sorted according to the -log10 p-value) were selected to draw a heat map. (B) The top 20 KEGG pathways of proteome between 2D monolayer and 3D MTS (sorted according to the -log10 p-value). (C) Chord diagram of GO analysis. The top 6 GO terms with the number of differential proteins between 3~50 was selected by sorted according to the -log10 p-value in descending order. The relationship between the selected GO term and the corresponding differential proteins were shown in the chord diagram. (D) Protein-protein interaction network of the top 25 proteins in connectivity degree. The molecular chaperones related to protein folding, assembly, and secretion exerted the most obvious effects, and the heat shock protein family was dominant.
Figure 6 . Metabolomic differences between cells in 2D monolayer and 3D MTS. (A) Heatmap of differential metabolites between 2D monolayer and 3D MTS. 50 differential metabolites in the top KEGG pathways (sorted according to the -log10 p-value) were selected to draw a heat map. (B) The top 20 KEGG pathways of proteome between 2D monolayer and 3D MTS (sorted according to the -log10 p-value).