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Updated: Apr 18, 2026
Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs
Published on: December 16, 2022
Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer
Yoshinori Imamura1, Toru Mukohara1, Yohei Shimono1
1Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
Abstract:
It is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs; therefore three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The purpose of this study was to reveal the differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474 and T-47D) developed dense multicellular spheroids (MCSs) in 3D-culture, and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB‑231) developed only loose MCSs in 3D, and showed drug sensitivities similar to those found in the 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved-PARP expression in the 2D-culture compared with the 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected the cells from paclitaxel-induced apoptosis. Hypoxia was observed only in the dense 3D-MCSs. BT-549 had fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0-dormant subpopulation was responsible for its drug resistance in the 3D-culture. BT-474 had a lower level of caspase-3 in the 3D- than in the 2D-culture, suggesting that the 3D-environment was anti-apoptotic. Finally, we compared staining for Ki-67 and caspases in the 2D- and 3D-primary‑cultured cells originating from a patient-derived xenograft (PDX), fresh PDX tumor, and the patient's original tumor; 2D-cultured cells showed greater proportions of Ki-67-positive and caspase-3-positive cells, in agreement with the view that 3D-primary culture better represents characteristics of tumors in vivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.
Insights
Three-dimensional (3D) cell cultures better mimic the in vivo tumor microenvironment than 2D cultures, showing increased drug resistance due to hypoxia and dormancy. This highlights 3D models
Area of Science:
- Oncology
- Cell Biology
- Drug Discovery
Background:
- Two-dimensional (2D) cell cultures are limited in accurately predicting oncology drug efficacy.
- Three-dimensional (3D) culture systems offer a more relevant model of the in vivo tumor microenvironment.
- Understanding differential drug effects in 2D vs. 3D cultures is crucial for improving cancer therapy selection.
Purpose of the Study:
- To investigate the distinct responses of breast cancer cells to chemotherapeutic drugs in 2D and 3D culture systems.
- To elucidate the underlying mechanisms contributing to observed differences in drug sensitivity and resistance.
- To compare 3D-cultured cells with patient-derived xenografts and original tumors for in vivo relevance.
Main Methods:
- Cultured three breast cancer cell lines (BT-549, BT-474, T-47D) in both 2D and 3D formats.
- Assessed drug sensitivity to paclitaxel and doxorubicin, alongside oxygen status, Ki-67, and caspase expression.
- Compared 2D and 3D primary cultures from patient-derived xenografts (PDX) and patient tumors.
Main Results:
- Dense 3D multicellular spheroids (MCSs) exhibited greater resistance to paclitaxel and doxorubicin than 2D cultures.
- Hypoxia, increased G0-dormancy (Ki-67), and anti-apoptotic features (caspase-3) were observed in dense 3D MCSs.
- 3D primary cultures showed characteristics more aligned with in vivo tumors than 2D cultures.
Conclusions:
- Dense 3D-cultured cells, particularly those forming MCSs, provide a more accurate simulation of in vivo tumor characteristics.
- 3D culture models reveal mechanisms of drug resistance, including hypoxia, dormancy, and anti-apoptotic effects.
- The findings support the use of 3D culture systems for more precise oncology drug screening and development.
