Development of a 3D Tumor Spheroid Model from the Patient's Glioblastoma Cells and Its Study by Metabolic Fluorescence Lifetime Imaging.

IF 1.1 Q4 MEDICINE, RESEARCH & EXPERIMENTAL
Sovremennye Tehnologii v Medicine Pub Date : 2023-01-01 Epub Date: 2023-03-29 DOI:10.17691/stm2023.15.2.03
D V Yuzhakova, M M Lukina, D A Sachkova, G M Yusubalieva, V P Baklaushev, A M Mozherov, V V Dudenkova, A I Gavrina, K S Yashin, M V Shirmanova
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引用次数: 0

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.

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利用患者胶质母细胞瘤细胞建立三维肿瘤球状模型并通过代谢荧光寿命成像进行研究
患者特异性体外肿瘤模型是研究肿瘤发生机制和个性化药物选择的一个前景广阔的平台。对于胶质脑肿瘤,开发和使用此类模型尤为重要,因为此类肿瘤的治疗效果仍然极不理想。本研究的目的是根据患者的手术材料建立一个三维肿瘤胶质母细胞瘤球体模型,并通过荧光寿命成像显微镜研究其代谢辅酶的代谢特征:研究使用的肿瘤样本来自确诊为胶质母细胞瘤(IV 级)的患者。从肿瘤组织样本中分离出原代培养物,对培养物进行形态学和免疫细胞化学鉴定,然后将其种植到圆底超低粘附性平板中。种植细胞的数量根据经验选择。将细胞培养物的生长特征与来自 U373 MG 稳定系人类胶质母细胞瘤患者的胶质母细胞瘤球体进行了比较。球形细胞中烟酰胺腺嘌呤二核苷酸(磷酸)NAD(P)H 和黄素腺嘌呤二核苷酸(FAD)代谢辅酶的自发荧光可视化是通过带有 FLIM 模块(Becker & Hickl GmbH,德国)的 LSM 880 激光扫描显微镜进行的。研究了常氧和缺氧条件(3.5% О2)下的自发荧光衰减参数:结果:开发出了一种用于三维胶质母细胞瘤球体培养的原创方案。从患者的手术材料中获得了原始胶质培养物,并对其进行了表征。分离出的胶质母细胞瘤细胞形态呈纺锤形,有许多突起和明显的颗粒状胞质。所有培养物均表达胶质纤维酸性蛋白(GFAP)。最佳播种剂量为每孔 2000 个细胞;应用该方法可形成结构致密的球形细胞,并在 7 天内稳定生长。FLIM 方法有助于确定患者材料中的球形细胞与稳定品系的球形细胞具有大致相似的新陈代谢,但它们表现出更明显的新陈代谢异质性。在缺氧条件下培养球形细胞,发现其新陈代谢过渡到更多的糖酵解类型,表现为游离形式的 NAD(P)H 对荧光衰减的贡献增加:结论:从患者胶质母细胞瘤中提取的肿瘤球体模型与荧光显微成像技术相结合,可作为研究肿瘤代谢特征和开发评估抗肿瘤治疗效果的预测测试工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Sovremennye Tehnologii v Medicine
Sovremennye Tehnologii v Medicine MEDICINE, RESEARCH & EXPERIMENTAL-
CiteScore
1.80
自引率
0.00%
发文量
38
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