Utility of Glioblastoma Patient-Derived Orthotopic Xenografts in Drug Discovery and Personalized Therapy

Abstract

Despite substantial effort and resources dedicated to drug discovery and development, new anticancer agents often fail in clinical trials. Among many reasons, the lack of reliable predictive preclinical cancer models is a fundamental one. For decades, immortalized cancer cell cultures have been used to lay the groundwork for cancer biology and the quest for therapeutic responses. However, cell lines do not usually recapitulate cancer heterogeneity or reveal therapeutic resistance cues. With the rapidly evolving exploration of cancer "omics," the scientific community is increasingly investigating whether the employment of short-term patient-derived tumor cell cultures (two- and three-dimensional) and/or patient-derived xenograft models might provide a more representative delineation of the cancer core and its therapeutic response. Patient-derived cancer models allow the integration of genomic with drug sensitivity data on a personalized basis and currently represent the ultimate approach for preclinical drug development and biomarker discovery. The proper use of these patient-derived cancer models might soon influence clinical outcomes and allow the implementation of tailored personalized therapy. When assessing drug efficacy for the treatment of glioblastoma multiforme (GBM), currently, the most reliable models are generated through direct injection of patient-derived cells or more frequently the isolation of glioblastoma cells endowed with stem-like features and orthotopically injecting these cells into the cerebrum of immunodeficient mice. Herein, we present the key strengths, weaknesses, and potential applications of cell- and animal-based models of GBM, highlighting our experience with the glioblastoma stem-like patient cell-derived xenograft model and its utility in drug discovery.

Keywords: glioblastoma; mouse models; patient-derived xenografts; personalized therapy; precision medicine.

Figure 1

Analysis of neurospheres derived from primary glioblastoma surgical tissues. (A) Model of patient-derived sphere assay. Neurospheres are kept in serum-free growth factor condition. To examine their differentiation potential, they are cultured in polyornithine-coated plates. (B) Neurosphere clonogenic potential of four different patient-derived glioblastoma multiforme (GBM) cells. (C) Immunofluorescence for NESTIN, which is used as a marker of stem cells, being expressed in central nervous system (CNS) stem cells but not in mature CNS cells, and TUBULIN and glial fibrillary acidic protein (GFAP; differentiation markers). TUBULIN-beta-III is a neuron-specific early marker of signal commitment in primitive neurepithelium. GFAP is used as a marker of CNS mature astrocytes and ependymal cells. DAPI is used to label the nuclei, while phalloidin is used to mark the cell architecture within the spheres. (D) GBM spheres transduced with lentiviruses expressing either luciferase (Luc) and EGFP or DsRed. (E) The two types of transduced spheres were used to generate patient-derived orthotopic xenografts (PDOXs) when injected into NSG mouse brains with 2 × 10⁵ labeled cells and monitored with the IVIS system. Luc was used to longitudinally track tumor growth in vivo. Images were from PDOX mice imaged at 8-weeks postinjection. Please note that the image showing Luc and DsRed is zoomed in to demonstrate the brain regions. Color scale was generated with the IVIS software and represents pixel intensities in luminescent or fluorescent images. Color scale units are photons/cm²/second. Scale bars are 200 µM in (D).

Figure 2

Histologic and immunophenotypic parity between original glioblastoma tissue and patient-derived orthotopic xenografts (PDOXs). (A) Strategy to generate spheres and PDOX from primary glioblastoma tissue. The diagram displays the process of microinjecting sphere cells into the cerebrum region of the mouse brain. The location of burr drilling hole (red circle) in NSG mice skull is demonstrated for microinjections using stereotactic infusion pump resulting in effective (90% take) generation of orthotopic glioblastoma multiforme (GBM) PDOXs. (B) Histological H&E analysis of original patient-derived glioblastoma tissue (patient (Pt) #46) and four different PDOX lines generated from the same patient-derived spheres. Note that the cell density is different in these sections as it depends on the number of cells engrafted into the PDOXs. The lower panels are 1,000× higher magnification of the outlined areas in the top panels. (C) Representative sections for comparison of the expression of stem cell proteins (BMI1, NESTIN, and SOX9) and the proliferation marker Ki67 in the original patient GBM tissue and the corresponding PDOX. Red arrows indicate positive cells. (D) Representative sections for comparison of the clustered expression of the GBM stem cell marker (CD15) in vascular niches and with the hypoxia protein carbonic anhydrase IX (CA9) near blood vessels expressing CD31, both in the original patient GBM tissue and the corresponding PDOX. Black and/or red arrows indicate positive cells. Scale bars are 500 µM in the upper panel of (B) and 20 µM in the lower or magnified panel of (B–D).