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Updated: Jul 25, 2025
A 3D Spheroid Model for Glioblastoma
Published on: April 9, 2020
Development of a 3D Tumor Spheroid Model from the Patient's Glioblastoma Cells and Its Study by Metabolic
D V Yuzhakova1, M M Lukina2, D A Sachkova3
1Researcher, Laboratory of Genomics of Adaptive Antitumor Immunity, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.
Abstract:
Patient-specific in vitro tumor models are a promising platform for studying the mechanisms of oncogenesis and personalized selection of drugs. In case of glial brain tumors, development and use of such models is particularly relevant as the effectiveness of such tumor treatment remains extremely unsatisfactory. The aim of the study was to develop a model of a 3D tumor glioblastoma spheroid based on a patient's surgical material and to study its metabolic characteristics by means of fluorescence lifetime imaging microscopy of metabolic coenzymes.
Materials And Methods:
The study was conducted with tumor samples from patients diagnosed with glioblastoma (Grade IV). To create spheroids, primary cultures were isolated from tumor tissue samples; the said cultures were characterized morphologically and immunocytochemically, and then planted into round-bottom ultra low-adhesion plates. The number of cells for planting was chosen empirically. The characteristics of the growth of cell cultures were compared with spheroids from glioblastomas of patients with U373 MG stable line of human glioblastoma. Visualization of autofluorescence of metabolic coenzymes of nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) in spheroids was performed by means of an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany). The autofluorescence decay parameters were studied under normoxic and hypoxic conditions (3.5% О2).
Results:
An original protocol for 3D glioblastoma spheroids cultivation was developed. Primary glial cultures from surgical material of patients were obtained and characterized. The isolated glioblastoma cells had a spindle-shaped morphology with numerous processes and a pronounced granularity of cytoplasm. All cultures expressed glial fibrillary acidic protein (GFAP). The optimal seeding dose of 2000 cells per well was specified; its application results in formation of spheroids with a dense structure and stable growth during 7 days. The FLIM method helped to establish that spheroid cells from the patient material had a generally similar metabolism to spheroids from the stable line, however, they demonstrated more pronounced metabolic heterogeneity. Cultivation of spheroids under hypoxic conditions revealed a transition to a more glycolytic type of metabolism, which is expressed in an increase in the contribution of the free form of NAD(P)H to fluorescence decay.
Conclusion:
The developed model of tumor spheroids from patients' glioblastomas in combination with the FLIM can serve as a tool to study characteristics of tumor metabolism and develop predictive tests to evaluate the effectiveness of antitumor therapy.
Insights
Researchers developed a 3D glioblastoma spheroid model from patient samples to study tumor metabolism. This model, combined with fluorescence lifetime imaging microscopy (FLIM), reveals metabolic heterogeneity and responses to hypoxia, aiding in personalized cancer therapy development.
Area of Science:
- Oncology
- Biotechnology
- Medical Imaging
Background:
- Patient-specific in vitro tumor models are crucial for understanding oncogenesis and guiding personalized drug selection.
- Glial brain tumors, particularly glioblastoma, have unsatisfactory treatment outcomes, necessitating advanced research models.
- Developing 3D glioblastoma spheroids from patient surgical material offers a relevant platform for studying tumor biology.
Purpose of the Study:
- To establish a 3D glioblastoma spheroid model using patient-derived surgical samples.
- To investigate the metabolic characteristics of these patient-specific spheroids.
- To utilize fluorescence lifetime imaging microscopy (FLIM) for metabolic coenzyme analysis.
Main Methods:
- Isolation and characterization of primary glial cultures from glioblastoma patient tumor samples.
- Cultivation of cells into 3D spheroids using ultra low-adhesion plates with optimized cell seeding (2000 cells/well).
- Metabolic coenzyme analysis (NAD(P)H, FAD) using FLIM under normoxic and hypoxic conditions.
Main Results:
- A reproducible protocol for 3D glioblastoma spheroid cultivation from patient material was developed.
- Patient-derived spheroids exhibited similar metabolism to the U373 MG cell line but showed greater metabolic heterogeneity.
- Hypoxia induced a shift towards glycolytic metabolism, evidenced by increased free NAD(P)H fluorescence.
Conclusions:
- The developed patient-derived glioblastoma spheroid model is a valuable tool for studying tumor metabolism.
- FLIM analysis of these spheroids provides insights into metabolic characteristics and responses to environmental changes.
- This model holds potential for developing predictive tests for antitumor therapy effectiveness.
