Generation of glioblastoma patient-derived organoids and mouse brain orthotopic xenografts for drug screening

Abstract

Summary Robust patient-derived platforms that recapitulate the cellular and molecular fingerprints of glioblastoma are crucial for developing effective therapies. Here, we describe a chemically defined protocol for 3D culture and propagation of glioblastoma in 3D gliospheres, patient-derived organoids (PDOs), mouse brain orthotopic xenografts (PDOXs), and downstream drug and immunofluorescence assays. This simple-to-follow protocol allows assessing drug sensitivity, on-target activity, and combined drug synergy. Promising therapies can then be validated in PDOXs for translation in precision medicine oncology trials. For complete details on the use and execution of this protocol, please refer to Chadwick et al. (2020) and Patrizii et al. (2018) .

Highly Invasive Fluorescent/Bioluminescent Patient-Derived Orthotopic Model of Glioblastoma in Mice

“…According to the standard techniques, human GBM cells are inoculated in a mouse brain in the amount of up to 5 × 10 5 tumor cells in 2–5 μl of serum-free media (the final concentration of the cell suspension is ~1–1.6 × 10 5 cells/μl) at the rate of 1 μl/min ( 10 , 11 ). In our study, different cell doses and rates of injection were tested to generate tumors from patient-based GBM7-Luc2-mKate2 cells.…”

“…The important property of PDXs is their ability to recapitulate the original tumor features such as heterogeneous histology, molecular profile, malignant phenotypes and genotypes, tumor vasculature, and invasive capacity. Classical PDX is established as a model at low passage numbers, passed in animals, and/or propagated in cell culture conditions ( 8 , 10 , 11 ). The main challenges with PDX are that the availability of fresh tumor tissue is often limited and the primary tumor cells have low viability, which reduces the reproducibility of experimental results and success rate for generating intracranial tumors ( 5 , 6 ).…”

“…They demonstrate a faithful recapitulation of human GBM features such as intratumoral heterogeneity, specific molecular profile, diffuse invasion into the brain, endovascular proliferation, and pseudopalisading necrosis (5)(6)(7)(8). While a plethora of protocols exists in the literature for the establishment of a PDX, they are not always successful, and the engraftment success rate often depends on the experience of the lab, varying from 10% to 60% on average (4,(8)(9)(10)(11). A typical protocol includes the obtaining of single-cell suspension from the tumor, maintaining the cells in a culture for a short time (1-2 weeks), intracranial implantation into immunodeficient mice, and passaging in mice to have a stable growth.…”

“…We show the similarity of the established model to the original patient's tumors in terms of histological and immunohistochemical features and demonstrate the possibility of dual non-invasive bioluminescence and fluorescence imaging of the GBM model in immunodeficient nude mice. The morphological and behavioral properties of the new model were compared with commonly used human GBM model U87 MG, which is one of the most widely studied gliomas used in experimental neuro-oncology (5)(6)(7)(8)(9)(10)(11)(12). Furthermore, the duallabeled PDX model was validated using advanced optical diagnostics methods macro-FLIM and CP OCT.…”

Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays

“…Recovery of organoids was performed as described for other organoid types [ 50 , 51 ] with modifications. In order to use the organoids for analyses, it is necessary to remove them from the Matrigel.…”