Morphological and functional responses of three-dimensional-cultured colorectal cancer spheres to anticancer drugs

Figure 1 Schema of the experimental method. Colorectal adenocarcinoma cells were cultured in low-attachment 96-well plates for 3 days to form spheres. After sphere formation, anticancer drugs were added to each well at a concentration of 30 μM. The day of anticancer drug administration was defined as day 0, and the areas of the spheres were measured on days 0, 1, 3, 5, and 7 using an imaging system. 5-FU: 5-fluorouracil; WST-8: Water-soluble tetrazolium 8; qRT-PCR: Quantitative reverse transcriptase PCR; CSC: Cancer stem cell; EMT: Epithelial-mesenchymal transition.

Figure 2 Effects of three anticancer drugs on the growth and morphology of DLD-1 spheres. DLD-1 spheres were imaged using a three-dimensional imaging system on days 0, 1, 3, 5, and 7 after anticancer drug treatment. Day 7 spheres were examined using scanning electron microscopy. Black scale bar = 200 μm; white thick scale bar = 100 μm; white thin scale bar = 50 μm. 5-FU: 5-fluorouracil; SEM: Scanning electron microscopy.

Figure 3 Effects of three anticancer drugs on the growth and morphology of CACO-2 spheres. CACO-2 spheres were imaged using a three-dimensional imaging system on days 0, 1, 3, 5, and 7 after anticancer drug treatment. Day 7 spheres were examined using scanning electron microscopy. Black scale bar = 200 μm; white thick scale bar = 50 μm; white thin scale bar = 25 μm. 5-FU: 5-fluorouracil; SEM: Scanning electron microscopy.

Figure 4 Effects of three anticancer drugs on the growth and morphology of HCT-15 spheres. HCT-15 spheres were imaged using a three-dimensional imaging system on days 0, 1, 3, 5, and 7 after anticancer drug treatment. Day 7 spheres were examined using scanning electron microscopy. Black scale bar = 200 μm; white thick scale bar = 100 μm; white thin scale bar = 50 μm. 5-FU: 5-fluorouracil; SEM: Scanning electron microscopy.

Figure 5 Effects of three anticancer drugs on the growth and morphology of SW480 spheres. SW-480 spheres were imaged using a three-dimensional imaging system on days 0, 1, 3, 5, and 7 after anticancer drug treatment. Day 7 spheres were examined using scanning electron microscopy. Black scale bar = 200 μm; white thick scale bar = 100 μm; white thin scale bar = 50 μm. 5-FU: 5-fluorouracil; SEM: Scanning electron microscopy.

Figure 6 Effects of three anticancer drugs on the growth and morphology of COLO-320 spheres. COLO-320 spheres were imaged using a three-dimensional imaging system on days 0, 1, 3, 5, and 7 after anticancer drug treatment. Day 7 spheres were examined using scanning electron microscopy. Black scale bar = 200 μm; white thick scale bar = 100 μm; white thin scale bar = 50 μm. 5-FU: 5-fluorouracil; SEM: Scanning electron microscopy.

Figure 7 Cell viability and caspase-3/7 activity in colorectal adenocarcinoma spheres after anticancer drug treatment. Colorectal adenocarcinoma cells were cultured in 96-well low-attachment plates, and anticancer drugs (30 μM) were added after 3 days. On day 7 post-treatment, cell viability was assessed using the ATP and water-soluble tetrazolium 8 (WST-8) assays. ATP luminescence and WST-8 absorbance (450 nm) were normalized to those of controls (set at 100%). Apoptosis was evaluated on day 7 using the Caspase-Glo 3/7 3D Assay, with luminescence measured after a 3-hour incubation at 23 °C. Data represent the means ± SD from three independent experiments (each performed in triplicate). ^aP < 0.05 vs control group; ^bP < 0.0001 vs control group. 5-FU: 5-fluorouracil; WST-8: Water-soluble tetrazolium 8.

Figure 8 Effects of three anticancer drugs on the growth of three-dimensional-cultured colorectal cancer spheres. DLD-1, CACO-2, HCT-15, SW480, and COLO-320 spheres were imaged using a three-dimensional (3D) imaging system on days 0, 1, 3, 5, and 7 after treatment with three anticancer drugs (5-fluorouracil, irinotecan, and oxaliplatin). Sphere areas were quantified from the 3D images using an integrated artificial intelligence-based deep learning algorithm. Solid lines indicate anticancer drug-treated groups, and dashed lines indicate the corresponding control groups. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. 5-FU: 5-fluorouracil.

Figure 9 Quantitative reverse transcriptase PCR analysis of cancer stem cell and epithelial-mesenchymal transition markers on colorectal adenocarcinoma spheres. HCT-15 and SW480 cells were cultured in 96-well low-attachment plates, and anticancer drugs (30 μM) were added on day 3. Total RNA was extracted on day 7 post-treatment, and quantitative reverse transcriptase PCR was performed to assess the expressions of cancer stem cell markers (CD44, ALDH1A1, and PROM1) and epithelial-mesenchymal transition markers (CDH1, VIM, and ZEB1). Gene expression levels were normalized to GAPDH and expressed relative to the control group (set to 1.0). Data represent the means ± SD from three independent experiments, each analyzed in duplicate (biological replicates, n = 3). ^aP < 0.001 vs control group; ^bP < 0.0001 vs control group. 5-FU: 5-fluorouracil; CSC: Cancer stem cell; EMT: Epithelial-mesenchymal transition.