Abstract
ABSTRACTGlioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of in vivo responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an in vivo imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.
Highlights
The incidence of primary malignant brain tumors is about five to six cases per 100,000 people per year, and glioblastoma (GBM) accounts for more than half of the cases, being the most common primary malignant brain tumor (Alifieris and Trafalis, 2015; Lapointe et al, 2018)
Injected embryos tolerated the presence of human GBM cells for up to 5 days post injection, which was the last day of observation in our experiments
We successfully micro-injected labeled GBM cells in zebrafish larvae and followed micro-tumor progression using a combination of stereomicroscopy, light sheet fluorescence microscopy (LSFM) and flow cytometry
Summary
The incidence of primary malignant brain tumors is about five to six cases per 100,000 people per year, and glioblastoma (GBM) accounts for more than half of the cases, being the most common primary malignant brain tumor (Alifieris and Trafalis, 2015; Lapointe et al, 2018). New approaches for treatment discovery include the development of targeted drugs and personalized medicine (Holland and Ene, 2015), such as treatments based on evaluating in vitro sensitivity to drugs tested in primary GBM cell cultures from patients (Iwadate et al, 2003). There is a need to utilize new models for drug discovery and testing, especially those that allow reliable determination of in vivo responses to treatment, visualization of tumor microenvironment disruption and better correlation with clinical efficacy
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