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Development of patient-derived ortotopic glioblastoma model in nude mice

D.V. Yuzhakova1; E.B Kiseleva1; M.M. Lukina1; V.V. Dudenkova1; G.M. Yusubalieva2; A.I. Gavrina1; V.P. Baklaushev2; M.V. Shirmanova1

1 Privolzhsky Research Medical University, Nizhny Novgorod, Russia;
2 Federal Research and Clinical Center, Federal Medical and Biological Agency, Moscow, Russia


Glioblastoma is the most common primary malignant brain tumor in adult. Despite the complex treatment approach (neurosurgical resection, radiotherapy, chemotherapy), the outcome is extremely poor, with 100% mortality. Discovering the new effective therapies requires establishing the animal models that accurately mimic human glioblastomas.
The purpose of this study was to develop the new patient-derived ortotopic model of glioblastoma, labeled by fluorescent protein m-Kate, in nude mice and investigate its functional features.
Glioblastoma primary cell culture was obtained by enzymatic disaggregation of human glioblastoma multiforme Grade IV tissue and cultivation of cells in DMEM F12 with 10% of fetal bovine serum. Patient-derived glioblastoma cells, stably expressing m-Kate, were obtained by lentiviral transduction. Intracranial injection of the glioblastoma m-Kate-labelled cells in mouse brain was performed with a stereotactic system (RWD Life Science, China). Verification of tumor formation and non-invasive monitoring of tumor growth was carried out by in vivo fluorescence imaging using an IVIS-Spectrum imaging system (Caliper Life Sciences, USA). Probing of the metabolic status of excised brain with glioblastoma was performed ex vivo by measuring endogenous fluorescence lifetime using a confocal macro-FLIM system (Becker&Hickl GmbH, Germany). Structure of tumor and peritumoral tissues were studied ex vivo with a multimodal optical coherence tomography (OCT) system (IAP RAS, Russia).
Using new primary fluorescent-protein labelled cell culture, we demonstrated the 100% formation of patient-derived glioblastoma in nude mice. 10 days after intracranial inoculation of tumor cells, histological analysis confirmed the presence of glioblastoma in the mouse brains.
We detected in vivo the tumor in a mouse brain and evaluated the growth rate non-invasively by measuring the fluorescent signal of m-Kate. Using macro-FLIM of endogenous fluorescence, we showed the ability to detect differences between the tumor and normal tissues, associated with biochemical alterations. The results of OCT demonstrated that in color-coded maps the patient-derived glioblastoma can be perfectly differentiated from the surrounding white matter.
In summary, we develop, for the first time, the novel patient-derived ortotopic glioblastoma in nude mice and reported easy and efficient ways to screen its functional features using optical imaging techniques.
This work was supported by the Russian Foundation for Basic Research (grant # 18-29-01022, tumor model, FLIM; grant # 18-29-01049, OCT study).

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Diana Yuzhakova
Privolzhsky Research Medical University


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