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).