A clinically relevant orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary tumor and preserves cancer stem cells in vivo

Abstract

Limited availability of in vitro and in vivo model systems has hampered efforts to understand tumor biology and test novel therapies for ependymoma, the third most common malignant brain tumor that occurs in children. To develop clinically relevant animal models of ependymoma, we directly injected a fresh surgical specimen from a 9-year-old patient into the right cerebrum of RAG2/severe complex immune deficiency (SCID) mice. All five mice receiving the initial transplantation of the patient tumor developed intracerebral xenografts, which have since been serially subtransplanted in vivo in mouse brains for 4 generations and can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors shared nearly identical histopathological features with the original tumors, harbored 8 structural chromosomal abnormalities as detected with spectral karyotyping, maintained gene expression profiles resembling that of the original patient tumor with the preservation of multiple key genetic abnormalities commonly found in human ependymomas, and contained a small population (<2.2%) of CD133(+) stem cells that can form neurospheres and display multipotent capabilities in vitro. The permanent cell line (BXD-1425EPN), which was derived from a passage II xenograft tumor and has been passaged in vitro more than 70 times, expressed similar differentiation markers of the xenograft tumors, maintained identical chromosomal abnormalities, and formed tumors in the brains of SCID mice. In conclusion, direct injection of primary ependymoma tumor cells played an important role in the generation of a clinically relevant mouse model IC-1425EPN and a novel cell line, BXD-1425EPN. This cell line and model will facilitate the biological studies and preclinical drug screenings for pediatric ependymomas.

Fig. 1.

Ependymoma xenograft in the mouse brain. (A) Gross appearance of H&E-stained mouse brain with huge xenograft tumors (left panel), and changes of animal survival times during serial subtransplantation from passage I (P-I) to passage III (P-III) and from cryopreserved xenograft tumor cells at passage III (P-rIII) (middle panel) as well as reduced cell numbers (right panel). P < .01 between passages I and IV, and P < .001 between mice injected with 50 000 and 25 000 cells. (B) H&E staining showing the replication of pseudorosettes in xenograft tumors (×10). (C) Representative images of IHC showing the positive staining of human-specific mitochondrial (MT) antigen, cell proliferation marker (Ki-67), neuronal marker (TuJ), neural progenitor marker (Nestin), VEGFR, as well as microvessel density (vWF) (×40). (D) Detection of invasive tumor cells using human-specific antibodies against VIM (×40).

Fig. 2.

Cellular and molecular genetic features of IC-1425EPN xenograft tumors. (A) Identification of structural chromosomal abnormalities with SKY (left panel) and inverted-DAPI banding (right panel). (B) The heat map of the correlation coefficients among samples. The scale bar at the right illustrated the r² value. Clustering of the samples was illustrated at the dendrogram at the top. (C) The hierarchical clustering of samples including the normal samples. Green rectangles represented down-expression, whereas red rectangles signified up-regulation. (D) List of gene numbers that are differentially expressed using normal brain tissue and patient tumor as references. (E) The hierarchical clustering of samples excluding the normal samples. The dendrogram at the right identified the grouping of samples. Green rectangles represented down-expression, whereas red rectangles signified up-regulation.

Fig. 3.

CD133⁺ cells in IC-1425EPN xenograft tumors. (A) Representative graphs showing the isolation of CD133⁺ cells from an IC-1425EPN xenograft tumor (passage II) with FACS using PE-conjugated monoclonal antibodies against human CD133. (B) In vitro formation of neurosphere from isolated CD133⁺ cells in the presence of EGF and bFGF (upper panel), and induced differentiation of preformed neurosphere with FBS (lower panel). (C and D) Representative IMF images showing the expression of human-specific mitochondrial (MT) antigen, markers of neuroprogenitor (Nestin), early (TuJ-1), and mature (MAP2 and SYP) neurons, glial precursor (A2B5), astrocyte (GFAP), as well as intermediate filament VIM (×60).

Fig. 4.

Characteristics of the ependymoma cell line BXD-1425EPN. (A) Morphology of cells with 50% (left panel) and 100% (right panel) confluence. (B) Representative images of IMF staining. Cells were processed for immunofluorescent labeling to detect the presence of MT, Notch1, Nestin, GFAP, and MAP2. Nuclei were counterstained with DAPI. (C) Log-rank analysis of animal survival times of the mice injected with different amount of cells, ranging from 1 × 10⁴ (10K) to 1 × 10⁵(100K), from passage 10 (p10) and passage 68 (p68) into right cereberum. (D) H&E staining of orthotopic xenograft tumors generated from passage 10 (p10) and passage 68 cells (a and c, ×20; b and d, ×40).