Xenotransplantation of Human glioblastoma in Zebrafish larvae: in vivo imaging and proliferation assessment

Abstract

Glioblastoma (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.

Modeling neoplastic disease with spheroids and organoids

“…The zebrafish model overcomes such limitations: the small size coupled with the optical transparency of the embryos allows the visualization of growing tumors with single-cell resolution [198,199]. Zebrafish embryos can be grown in multiwell plates and this, coupled with their low cost, highlight their potential as models for high-throughput screening of new anticancer drugs to increase the power of statistical analysis [200,201]. This leads to more robust and reliable data before moving to mammalian models.…”

Patient-Derived Glioma Models: From Patients to Dish to Animals

“…Compared with standard confocal systems, this method has low phototoxicity, allowing imaging of zebrafish for a longer period of time, and has been used successfully to follow GBM growth and therapeutic response [102]. Although simultaneous use of these modalities has been successful for GBMs [104], several factors need to be considered; zebrafish embryos usually grow at 28 °C, a much lower temperature than mammalian cells. Various studies using GBM xenografts reported working with temperatures between 25 and 36 °C, with a survival rate between 87.5 and 95% [105].…”

Fishing for contact: Modeling perivascular glioma invasion in the zebrafish brain

“…While traditional glioma models utilize rodents studies have now demonstrated zebrafish are a suitable model to elucidate CNS cancer biology 16, 17, 20 . As a means to ask whether glioma-vessel interactions may occur in the zebrafish brain, we opted to inject fewer cells than previously published.…”