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. 2009 Aug;175(2):856-66.
doi: 10.2353/ajpath.2009.090019. Epub 2009 Jul 16.

MYCN promotes the expansion of Phox2B-positive neuronal progenitors to drive neuroblastoma development

Goleeta Alam  1 , Hongjuan Cui, Huilin Shi, Liqun Yang, Jane Ding, Ling Mao, William A Maltese, Han-Fei Ding
Affiliations

MYCN promotes the expansion of Phox2B-positive neuronal progenitors to drive neuroblastoma development

Goleeta Alam et al. Am J Pathol. 2009 Aug.

Abstract

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B(+) neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin(+) cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B(+) neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development.

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Figures

Figure 1
Figure 1
Early postnatal sympathetic ganglia of TH-MYCN mice contain multiple clusters of proliferating cells. Sections of SCG from postnatal day 12 (P12) wild-type and TH-MYCN mice were stained with H&E (A), anti-MYCN (B), anti-Ki-67 (C), or anti-pHH3 (E). Wild-type SCG consist of mainly large neurons and small glial satellite cells (A) that express no detectable levels of MYCN (B), and the proliferating cells marked by Ki-67 (C) or pHH3 (E, arrowhead) were evenly distributed. By contrast, TH-MYCN SCG contain multiple clusters of small round blue cells (A) that express MYCN (B) and are in a state of active proliferation (C, E). Scale bars = 100 μm. D, F: Quantitative analysis of ganglionic cells expressing either Ki-67 (D) or pHH3 (F) in wild-type SCG (n = 4) and in non-hyperplastic (Non-Hyper) and hyperplastic (Hyper) regions of TH-MYCN SCG (n = 4). Data are presented as means ± SD.
Figure 2
Figure 2
Hyperplastic lesions are composed predominantly of proliferating Phox2B+ cells. A: Sections of SCG from P14 wild-type and TH-MYCN mice were stained with anti-Ki-67 (red) and anti-BLBP (green), an early marker for glial cells. Most of the Ki-67+ cells in wild-type SCG also express BLBP, whereas Ki-67+ cells in hyperplastic lesions (arrows) are negative for BLBP. B: Immunofluorescent staining of a representative SCG section from a P16 TH-MYCN mouse shows a hyperplastic lesion (arrow) consisting predominantly of cells expressing both Ki-67 (red) and Phox2B (green). Nuclei were stained with DAPI (blue). Scale bars = 100 μm.
Figure 3
Figure 3
Phox2B+ hyperplastic cells are arrested in neuronal differentiation. A: Sections of SCG from P4, P7, and P14 wild-type mice were stained with anti-Phox2B (red) and anti-TH (green), a late marker for sympathetic neurons. Most of the Phox2B+ cells display the morphology of mature neurons and express TH. Only a few Phox2B+ cells in P4 and P7 SCG stained negatively for TH (arrows). B: Sections of SCG from P12 and P16 TH-MYCN mice were stained with anti-Phox2B (red) and/or anti-TH (green). Most of the hyperplastic cells are Phox2B+TH− neuronal progenitors. Nuclei were stained with DAPI (blue). Scale bars = 100 μm.
Figure 4
Figure 4
Phox2B+ progenitor cells are the major cellular component of mouse primary neuroblastoma tumors and xenograft tumors derived from human neuroblastoma cell lines. A: Sections of neuroblastoma tumors from TH-MYCN mice were stained with H&E, anti-Ki-67, or anti-Phox2B (red) and anti-TH (green). Nuclei were stained with DAPI (blue). The small round blue tumor cells are organized in nests surrounded by thin fibrovascular septa (H&E), and most of them express Ki-67 and Phox2B, but are negative for TH. B: Immunofluorescent staining of a representative mouse neuroblastoma section for Ki-67 (green) and Phox2B (red). Nuclei were stained with DAPI (blue). C: Sections of xenograft tumors derived from human neuroblastoma cell lines were stained with anti-Phox2B (red) and anti-TH (green). Nuclei were stained with DAPI (blue). Most of the tumor cells express Phox2B. SK-N-DZ and BE(2)-C xenografts also contain significant numbers of TH+ cells. The cells stained yellow in SK-N-DZ xenograft section are probably necrotic cells. Scale bars = 100 μm (A-C). D, E: Quantification of Phox2B+TH− progenitor cells (D) in xenograft tumors of different human neuroblastoma cell lines reveals a positive correlation with the tumor growth rates (E). Data are presented as means ± SD and analyzed with two-tailed Student’s t-test with P values indicated. F: Immunoblot analysis of MYCN protein levels in human neuroblastoma cell lines. α-tubulin levels are shown as loading control.
Figure 5
Figure 5
Hyperplasia and primary neuroblastoma tumors contain a minor population of nestin+ progenitor cells. A: Sections of hyperplastic lesions were stained with anti-Phox2B (red), anti-nestin (green), and DAPI (blue), and examined with a confocal microscope. Arrows and asterisks indicate nestin+ and nestin+Phox2B+ cells, respectively. B: Sections of wild-type and MYCN sympathetic ganglia at P14 were stained with anti-nestin (green) and DAPI (blue). The arrow indicates a hyperplastic lesion in the MYCN SCG. C: Quantitative analysis showing a fourfold increase in the number of nestin+ cells in TH-MYCN SCG with hyperplastic lesions compared with age-matched wild-type SCG (P14 to P16). Data are presented as means ± SD and analyzed by two-tailed Student’s t-test with the P value indicated. D, E: Sections of representative primary neuroblastoma tumors from TH-MYCN mice were stained with anti-nestin (green) alone (D) or with anti-TH (red), anti-BLBP (red) or anti-Phox2B (red) (E). Nuclei were stained with DAPI (blue). Nestin+ tumor cells are negative for TH and BLBP, and some of them express Phox2B (arrowheads). Scale bars: 10 μm (A); 50 μm (B, D, and E).
Figure 6
Figure 6
A simplified schematic diagram for the cellular pathway in MYCN-induced neuroblastoma development. See text for discussion.
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