Proteome analysis
Tumor cells synthesize a variety of proteins that interact with each other to perform cellular functions through transcriptional, translational and post-translational modifications (Manzoni et al., 2018). Genetic variation and changes of the tumor microenvironment would affect the expression, structure and interaction of proteins, leading to changes in the activities of cells. The differences in protein expression levels between 2D monolayer cultures and 3D MTSs cultures before and after the 5-FU treatment were determined through Tandem Mass Tag (TMT) technology. The principal component analysis (PCA) result showed that there were significant differences among all sample groups and good repeatability within the sample group (Figure. S5A ), which proved that the obtained experimental data could support the following data analysis. A total of 5262 proteins were determined, and the number of differential proteins was shown in Table 1 . We found that the effect of 16 μM 5-FU on the expression profile of protein in 3D MTSs was less pronounced than that in 3D monolayer cells. Under the 5-FU treatment, there were 133 proteins significantly different in 2D monolayer cells compared with the control condition, while only 27 differential proteins were significantly changed in the 3D MTSs, which indicated that 5-FU induced response in 3D MTS was attenuated. Our results showed that, between the control condition and 5-FU treatment, there were significant differences in cellular processes related to cell growth and death (e.g., cell cycle, apoptosis and senescence) under 2D monolayer culture (Figure. S5B ). However, the differential proteins in the 3D MTSs were not enriched in specific metabolic pathways. While upregulating the negative cell cycle regulatory protein RB1 to block the cell cycle, 2D monolayer cells upregulated cyclin (CCNB1, CCNB2), apoptosis inhibitor BIRC5 to maintain growth activity with the 5-FU treatment (Figure. S5C ). In the 3D MTSs, we also observed the up-regulation of BIRC5 with the 5-FU treatment. Meanwhile, 5-FU treatment reduced the absorption and transport of glucose, folate and amino acids in 2D monolayer cultures, and down-regulated the members of solute carrier family members, including SLC2A1, SLC19A1, SLC38A1 and SLC38A2, which were not significant in the 3D MTSs (Fig S5C ).
Table 1 shows 367 and 288 differential proteins between 2D monolayer cultures and 3D MTS before and after the 5-FU treatment, respectively. Based on this, we selected 50 differential proteins which exerted great influence on the growth, metabolism and drug resistance of tumor cells, and then their expression levels were hierarchically clustered between 2D monolayer cultures and 3D MTSs before and after the 5-FU treatment. The differential proteins between 2D monolayer cultures and 3D MTSs displayed similar profiles (Figure. 5A ). In order to further analyze the metabolic pathway differences, KEGG pathway enrichment analysis was performed with all differential proteins between 2D monolayer cultures and 3D MTSs (Figure. 5B ). Processes related to protein synthesis, processing and transportation, mainly in heat shock protein family, exhibited the most significant differences between 2D monolayer cultures and 3D MTSs. In addition, there were significant differences in the mutual conversion of amino acids, in which mitochondrial glutamate oxaloacetic acid transaminase (GOT2) was significantly down-regulated in 3D MTSs. GOT2 plays an important role in amino acid metabolism and TCA cycle (Yang et al., 2015). Consistent with the transcription level (Figure. 4 ), we found more multidrug resistance-related proteins (ABCC4) and ECM proteins in 3D MTSs, including laminin (e.g., LAMA5) and collagen (e.g., COLO1A1). In the control condition, LAMA5 and COLO1A1 was upregulated in 3D MTSs compared with 2D monolayer cultures (Figure. 5A ). After the 5-FU treatment, 3D MTSs upregulated more kinds of ECM protein, including LAMA1, LAMB1, LAMB2, LAMC1 and COL4A1. Similarly, under the 5-FU treatment, we also found that integrin α5 (ITGA5) was up-regulated in 3D MTSs (Figure. 5A ), which was consistent with the up-regulated transcription level of ITGB1 (Figure. 4 ). Notably, glucose transport related proteins, such as SLC2A1, were significantly up-regulated, which further proved the possibility of up-regulation of glycolytic flux of 3D MTSs. In KEGG pathway enrichment analysis, we found that signaling pathways such as PI3K/Akt and HIF-1 were significantly altered after the 5-FU treatment (Figure. 5B ). In this study, HYOU1 in 3D MTSs was also significantly up-regulated regardless of the 5-FU treatment (Figure. 5A ). Hypoxia up-regulated 1 (HYOU1), a member heat shock protein 70 family, maintains endoplasmic reticulum (ER) homeostasis under hypoxia conditions while promotes the growth, metastasis and invasion of tumor cells by activating the PI3K/Akt signaling pathway (Li et al., 2019).
Through GO analysis, the gene functions of the differential proteins were analyzed (Figure. 5C ,Table S3 andTable S4 ). Under both control and 5-FU treatment conditions, the differential proteins between 2D monolayer cultures and 3D MTSs were similar in enrichment levels. The molecular functions of 3D MTSs were mainly performed by up-regulating proteins related to calcium ion binding, ECM constituent, integrin binding, and down-regulating proteins related to ATP binding, RNA binding, ubiquitin protein ligase binding. While for the biological process, 3D MTSs mainly up-regulated processes related to ECM organization, cellular protein metabolic process tissue development, blood vessel development and protein folding in endoplasmic reticulum, and down-regulated processes related to mitochondrial function, including mitochondrial translation, mitochondrial transcription and mitochondrial electron transport, etc. In addition, with the 5-FU treatment, MTSs also up-regulated proteins related to cell adhesion and down-regulated proteins related to cell division and cell redox homeostasis. On the other hand, for cellular components, the proteins up-regulated in MTSs were mainly distributed in ER or extracellular regions such as exosomes, ECM, basement membrane, while the down-regulated proteins were mainly distributed in the mitochondria. This is reasonable that the increase of the production and deposition of ECM enhanced the physical barrier of drug penetration, as well as promoted the EMT process caused by enhanced mechanical stimulation, thereby promoting the development of tumor resistance (Hirschhaeuser et al., 2010; Joyce et al., 2018). Mitochondrion, an important signal transduction hub, can regulate cell apoptosis and participate in cell communication and tumor formation through ROS, nitric oxide, and calcium ions and proteins involved in the apoptotic cascade (Frezza, 2014). It has been reported that the down-regulation of mitochondria-related genes promoted the up-regulation of EMT pathway and promoted the invasion and metastasis of tumor cells (Gaude, 2018). Therefore, the up-regulation of ECM construction and the down-regulation of mitochondrial function genes might lead to the enhanced resistance of 3D MTSs to 5-FU. The interactions between differential proteins were further analyzed using the STRING database for the protein-protein interaction network (Figure. 5D ), we found that the molecular chaperones related to protein folding, assembly, and secretion exerted the most obvious effects, and the heat shock protein family was dominant (such as, HSPA5, HSP90B1, HSPG2). The increased expression of molecular chaperones was conducive to maintaining the growth of tumor cells in unfavorable environments, and the overexpression of HSP90 is related to the poor prognosis response of tumors and the increase of drug resistance (Jarosz, 2016).