Cell viability
Single cell suspensions from both 2D monolayer culture and 3D MTSs were prepared as described above. About 106 cells were collected for cell cycle and apoptosis analysis by cell cycle analysis kit (C1052, Beyotime, Shanghai, CN) and apoptosis analysis kit (40302ES60, Yeasen, Shanghai, CN), respectively. The operation steps were detailed in the kit instructions. The flow cytometer (Beckman Coulter, Brea, CA, USA) was used for detection, and the obtained data were analyzed by FlowJo 10.5.
Cytotoxicity assay
The cytotoxicity of 5-FU to tumor cells cultured under both 2D monolayer and 3D MTSs was determined. 2D monolayer culture: HeLa cells (10000 cells/well) were seeded in the 96-well plates and incubated for 24 h; 3D MTS: HeLa cells (2000 cells/well) were seeded in the 96-well plates and cultured for 6 days. Then removed the medium and incubated with different concentrations of 5-FU (MS0249, Mkbio, Shanghai, CN) for 48 h. Then removed the medium and added 200 μL CCK-8 staining solution (40203ES92, Yeasen, Shanghai, CN) diluted with 1:10 medium to each well. After incubation at 37 °C for 2 h, the absorbance of each well was determined at λ = 450 nm by fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Cellular viability was calculated according to the following equation. Half inhibitory concentration (IC50) was calculated using GraphPad Prism 8.
\begin{equation} Cellular\ viability=\frac{A_{450,\ \ dose}-A_{450,\ blank}}{A_{450,\ 0\ dose}-A_{450,\ blank}}\times 100\%\nonumber \\ \end{equation}