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Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated
Reiko Ikeda-Motonakano1,2, Fumika Hirabayashi-Nishimuta2, Naomi Yada3
1Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan.
Abstract:
Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.
Insights
This study developed a microwell device for generating cancer stem cells (CSCs) from oral cancer. The spheroid culture system efficiently produces uniform CSCs, offering a promising tool for cancer research and therapy development.
Area of Science:
- Biomedical Engineering
- Cancer Biology
- Stem Cell Research
Background:
- Cancer stem cells (CSCs) drive tumor recurrence, metastasis, and treatment resistance.
- Targeting CSCs is crucial for developing effective cancer therapies.
- Oral squamous cell carcinoma (OSCC) presents significant therapeutic challenges due to CSCs.
Purpose of the Study:
- To develop a novel spheroid culture system for generating cancer stem cells (CSCs) from oral cancer.
- To evaluate the characteristics and CSC marker expression of spheroids derived from oral cancer cell lines.
- To assess the potential of this system as a high-throughput tool for CSC production.
Main Methods:
- Fabrication of a polyethylene glycol-tagged microwell device for spheroid formation.
- Culture of human oral squamous carcinoma cell lines (HSC-3 and Ca9-22) in the microwell device.
- Analysis of spheroid morphology, cell viability, CSC marker mRNA expression (Cd44, Oct4, Nanog, Sox2), cisplatin resistance, and xenograft tumor growth.
Main Results:
- Uniform oral cancer spheroids were generated within 24-48 hours, maintaining integrity for up to five days.
- Spheroids exhibited significantly higher mRNA expression of CSC markers compared to monolayer cultures.
- Spheroid-cultured cells showed enhanced resistance to cisplatin and CSC marker upregulation upon outgrowth.
- Tumor growth in xenograft models was comparable between spheroid and monolayer implantation groups.
Conclusions:
- The developed microwell spheroid culture system is an effective high-throughput method for generating uniform cancer stem cells from oral cancer.
- This system facilitates the study of CSC properties, including drug resistance and marker expression.
- The findings suggest potential applications in cancer research, drug screening, and therapeutic strategy development targeting CSCs.
