A mouse model of the most aggressive subgroup of human medulloblastoma

Abstract

Medulloblastomas that display a large cell/anaplastic morphology and overexpress the cellular c-MYC gene are highly aggressive and carry a very poor prognosis. This so-called MYC-subgroup differs in its histopathology, gene expression profile, and clinical behavior from other forms of medulloblastoma. We generated a mouse model of MYC-subgroup medulloblastoma by transducing Trp53-null cerebellar progenitor cells with Myc. The cardinal features of these mouse medulloblastomas closely mimic those of human MYC-subgroup tumors and significantly differ from mouse models of the Sonic-Hedgehog- and WNT-disease subgroups. This mouse model should significantly accelerate understanding and treatment of the most aggressive form of medulloblastoma and infers distinct roles for MYC and MYCN in tumorigenesis.

Figure 1. MBs derived from orthotopic transplants in the cortices of naïve recipient mice of cerebellar progenitors overexpressing Myc and Mycn

(A) Kaplan-Meier survival curves of mice transplanted with cerebellar cells purified from [Cdkn2c^−/−, Trp53^−/−, Atoh1-GFP] mice infected with Myc-RFP (red line), Mycn-RFP (blue line) or RFP empty vector (orange line). (B) Pathology of human and mouse MBs (H&E): (a) a human tumor with MYCN amplification and classic morphological features, and (b) a human anaplastic tumor with MYC amplification. Note the difference in nuclear pleomorphism between classic and anaplastic tumors. The anaplastic tumor also shows a paving stone-like pattern of cell molding and contains abundant apoptotic cells, a hallmark of this variant. Mycn-tumors with a classic morphology of round cells (c, e) contrast with Myc-tumors that show an anaplastic morphology with cell molding and abundant mitotic figures and apoptotic bodies (d, f). Scale bar = 50μm. (C) Myc- tumor cells are significantly larger, and are more likely to undergo apoptosis, than their SHH- and WNT-subgroups counterparts. Error bars indicate standard deviation. *, p<0.05 for SHH- and WNT-subgroup tumors compared to the MYC-subgroup. See also Figure S1.

Figure 2. Comparative molecular analysis of engineered mouse MBs and GNPs

(A) Affymetrix gene chip analysis of mouse Myc- and Mycn-tumors, GNPs purified from the cerebellum of P6 [Cdkn2c^−/−, Trp53^−/−, Atoh1-GFP] mice, from spontaneous MBs of the SHH-subgroup from [Cdkn2c^−/−, Trp53^−/−, Nestin-cre] and [Cdkn2c^−/−, Ptch1^+/−] mice and from the mouse WNT-subgroup from [Ctnnb1^{+/lox (ex3)}; Blbp-Cre; Trp53^−/−] mice. Arrowheads indicate Myc-tumors from FACS-sorted GFP-positive (GFP⁺) (green) and GFP-negative (GFP⁻) (red) cerebellar cells. (B) Heatmap of differentially regulated genes between Myc- and Mycn- mouse MBs and the SHH-subgroup MBs from the Ptch1^+/− and Trp53-null mice, the WNT-subgroup of tumors and GNPs. (C–D) Quantitative RT-PCR (QRT-PCR) analysis of Atoh1, Gli1, Boc and Sfrp1 (C) and Myc and Mycn (D) in Myc-engineered MBs (Myc-1,2,3) compared to spontaneous Trp53 and Ptch MBs, Mycn-engineered (Mycn-1,2,3) MBs, and GNPs. See also Figure S2.

Figure 3. Comparison of the gene expression signature of human MYC-, SHH-, and WNT-subgroup MB with that of mouse MBs reveals three representative murine models of the human disease

Signature genes that specifically distinguish the human WNT-, SHH-, and MYC-subgroup tumors by their broad subgroup over-expression relative to the others were compiled from three recent publications on subgroups of MB (Cho et al., 2011; Northcott et al., 2011; Thompson et al., 2006). 52 signature genes relatively over-expressed by the human MYC-subgroup, 54 by the human WNT-subgroup, and 53 by the human SHH-subgroup were found to have corresponding mouse orthologs on the Affymetrix 430v2 chip. Shown here are representative heat maps columns which compare mean average expression of the orthologs in the three mouse medulloblastoma models (Myc, Shh, Wnt). Red indicates a log scale relative increase of expression while green signifies a log scale relative decrease of expression. The ortholog name and the corresponding first author of the study from which the signature gene was described are listed in the columns immediately to the right of the heat map. (A) 31 of 52 orthologs (60%) with increased expression in the human MYC-medulloblastoma subgroup and in the mouse Myc-tumors. (B) 40 of 54 orthologs (74%) with increased expression in the human WNT-subgroup and in the mouse WNT-tumors. (C) 31 of 53 orthologs (58%) with increased expression in the human SHH-subgroup and mouse Ptch-tumors. See also Figure S3.

Figure 4. Mouse MYC-subgroup MBs before and after orthotopic transplants

(A) Kaplan-Meier survival curve of mice subjected to orthotopic cranial injection of decreasing number of Myc- primary tumor cells. (B–K) Sections of tumors were immunostained with H&E (B, G), Ki67, a marker of proliferating cells (C, H), cleaved Caspase-3, a marker of apoptosis (D, I), GFAP, that marks glial and neural progenitor cells (E, J), and synaptophysin that characterizes mature neurons (F, K). Scale bar = 50 μm. See also Figure S5.

Figure 5. Myc-induced tumor cells grow as neurospheres in vitro

(A) Tumor cells from mouse MYC-subgroup MBs form red spheres by day 6 after plating. Scale bar = 200 μm. (B) Spheres can be passaged continuously, here shown up to passage 10 with the cell number increasing up to 25 fold. (C) Immunostaining of tumor spheres with antibodies against Ki67, Npr3, Nestin, Sox2, Oct4, Nanog, Lgr5 and GFAP. Scale bar = 50 μm. (D) Spheres share a similar molecular signature (lower levels of Shh signature genes and higher levels of Lgr5 and Npr3) than that of GNPs after several passages up to passage 10. Data are represented as the mean ± SD. See also Figure S6.

Figure 6. Tumor sphere cells contain tumor-propagating cells

(A) Myc-engineered MBs form after transplant of purified tumor cells or tumor sphere cells at passage 2 (2×10⁵ cells) into the cortices of recipient mice. (B) Comparison of the expression of RFP, GFP, Ki67, Npr3, Nestin, Sox2, Oct4, Nanog and Lgr5 by IHC between primary MB (Primary MB) and those induced after transplant of spheres (Secondary MB). Scale bar = 50 μm. (C) Comparison of gene signature among Trp53-tumors, Myc-primary and Myc-secondary tumors.

Figure 7. Myc- tumor cells are resistant to SHH signaling inhibitors

(A) GNPs purified from the cerebellum from 7 days old pups and purified tumor cells were grown in serum-free medium in the absence (Control, black square) or the presence of cyclopamine (light grey square) or BMP4 (dark grey square). Proliferation was assessed by FACS by calculating the percent of BrdU positive cells. Proliferation index was defined as the ratio of the percentage of BrdU-incorporated cells with Shh signaling antagonists to that of control. (B) GNPs and tumor cells were analyzed by qRT-PCR for specific gene expression to validate the expression of genes in the SHH signaling pathway. (C) Treatment of MYC-tumor sphere cells with or without SHH signaling antagonists under neurosphere culture conditions. Proliferation index represents the ratio of cell number of cultured neurosphere cells with SHH signaling antagonists to that of control, 6 and 12 days after plating. Data in graphs A and C represent the mean ± SD.