Determination of the concentration of extracellular metabolites
During the cell culture process, the concentration of glucose,
glutamine, lactate, and ammonia in the supernatant was determined by a
biochemical analyzer (Roche, Basel, CH). Control: 2000 cells were seeded
in 96-well plates per well without treatment under both 2D monolayer
culture and 3D MTSs. 5-FU treatment: under 2D monolayer culture, 2000
cells were seeded in 96-well plates per well and cultured for 24 h.
After that, the supernatant discarded and then cultured with 16 μM 5-FU;
under 3D MTSs, 2000 cells were seeded in 96-well plates per well and
cultured for 6 days. After that, the supernatant discarded and then
cultured with 16 μM 5-FU.
Transcriptional analysis
of tumor resistance-related genes
According to the manufacturer’s protocol, total RNA was extracted using
TRIeasy™ Total RNA Extraction Reagent (10606ES60, Yeasen, Shanghai, CN),
and RNA concentration and purity were determined by Nano Drop
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA
purity was considered as qualified when
A260/A280 was between 1.8 and 2.0. The
procedures about reverse transcription (11141ES10, Yeasen, Shanghai, CN)
and quantitative polymerase chain reaction (qPCR, 11198ES08, Yeasen,
Shanghai, CN) were detailed in the kit instructions. The qPCR reaction
was performed by real-time fluorescent quantitative PCR system (Thermo
Fisher Scientific, Waltham, MA, USA). The primer sequences involved in
this study can be found in the Table S1 , and the transcription
level of each gene was standardized by the internal control gene,ACTB , which is encoding for β-actin.
Proteome analysis
2D monolayer and 3D MTSs cells
treated with and without 5-FU were collected, with three biological
replicates in each group and about 5 × 106 cells in
each sample. The proteome was determined using Tandem Mass Tag (TMT)
technique. The main processes can be summarized as follows: protein
extraction, enzymatic hydrolysis, peptide labeling, LC-MS detection,
database analysis and processing. For
specific procedures, please refer to the Supplementary
materials . The LC-MS data were imported into the Uniprot database
(http://www.uniprot.org/) for qualitative and quantitative analysis of
proteome data. Firstly, the reliability of the results was determined by
principal component analysis (PCA).
The proteins
with T test p-value <
0.05 and Fold change > 1.5 were determined as differential
proteins. Subsequently, the
differential protein data were imported into the Gene Ontology (GO)
database (http://geneontology.org/) to analyze the biological process,
cellular component and molecular function. Through the KEGG database
(https://www.kegg.jp/kegg/pathway.html), pathway enrichment analysis of
differential proteins was performed. The STRING database
(https://www.string-db.org/) was used to analyze the interactions
between proteins, select the hub proteins, and draw the network diagram
of the hub protein interaction.
Metabolome
analysis
The cells were collected in the same way as the proteome analysis. The
main process of non-targeted metabolomics detection includes metabolite
extraction, GC-MS analysis, database analysis and processing. For
specific procedures, please refer to Supplementary materials .
First, the PCA analysis was used to determine the reliability of the
results. Orthogonal partial least
squares-discriminant analysis (OPLS-DA) was used to screen differential
metabolites. The screening criteria were VIP value of PC1 >
1 in OPLS-DA and T test p-value < 0.05. Qualitative analysis
was performed on metabolome data from the LUG database (Untargeted
database of GC-MS from Lumingbio). As for headspace sampling GC-MS
experiments for studying volatile substances and similar substances, the
NIST database (https://webbook.nist.gov/chemistry/) was used for
qualitative analysis of substances. Next, the bioinformatics analyses
were then performed in the same way as proteomics.