Cancerous stem cells can arise from pediatric brain tumors

Abstract

Pediatric brain tumors are significant causes of morbidity and mortality. It has been hypothesized that they derive from self-renewing multipotent neural stem cells. Here, we tested whether different pediatric brain tumors, including medulloblastomas and gliomas, contain cells with properties similar to neural stem cells. We find that tumor-derived progenitors form neurospheres that can be passaged at clonal density and are able to self-renew. Under conditions promoting differentiation, individual cells are multipotent, giving rise to both neurons and glia, in proportions that reflect the tumor of origin. Unlike normal neural stem cells, however, tumor-derived progenitors have an unusual capacity to proliferate and sometimes differentiate into abnormal cells with multiple differentiation markers. Gene expression analysis reveals that both whole tumors and tumor-derived neurospheres express many genes characteristic of neural and other stem cells, including CD133, Sox2, musashi-1, bmi-1, maternal embryonic leucine zipper kinase, and phosphoserine phosphatase, with variation from tumor to tumor. After grafting to neonatal rat brains, tumor-derived neurosphere cells migrate, produce neurons and glia, and continue to proliferate for more than 4 weeks. The results show that pediatric brain tumors contain neural stem-like cells with altered characteristics that may contribute to tumorigenesis. This finding may have important implications for treatment by means of specific targeting of stem-like cells within brain tumors.

Fig. 1.

Tumor-derived progenitors form neurospheres in culture that give rise to both neuronal and glial cells. Neurospheres from one tumor, BT1, were cultured at medium (A–D) and clonal (E–H) densities. (A) A typical primary neurosphere. (B and C) Undifferentiated primary neurospheres expressed high levels of nestin protein (B, green) and low levels of β-III-tubulin (C, red) and GFAP (C, green). (D) Expression of β-III-tubulin and GFAP, after 7 days of differentiation on substrate. (E) Nestin expression in undifferentiated clonal neurosphere cells. (F) Musashi-1 (green) expression in undifferentiated clonal neurospheres. (G) β-III-tubulin (red) and GFAP (green) expression in a differentiated clonal neurosphere. Some cells (arrows) expressed both markers. (H) Hu (green) expression in a differentiated neurosphere. Some nuclei were counterstained with DAPI (F and H, blue). (Scale bar in H = 30 μm in A, G, and H, 60 μm in B–F.)

Fig. 2.

Neurospheres derived from multiple types of tumors give rise to cells expressing neuronal and glial markers in various proportions. (Left) Average count of cells expressing nestin, TuJ1 alone, GFAP alone, or both markers in clonal neurospheres (NS) from BT1–5 (A–E) before (white) and after (black) differentiation. (Right) Fates of clonal neurospheres (NS) after differentiation. Markers used are TuJ1 for neurons (N) and GFAP for astrocytes (A).

Fig. 3.

Immunohistochemical characteristics of original tumor samples. Paraffin-embedded sections were labeled with antibodies to nestin (green; A–D) or TuJ1 (red; E–H) to recognize neurons or GFAP (green; E–H) to recognize glia. Area denoted by asterisk in C and F delineates normal brain tissue adjacent to the tumor. (Scale bar in H = 60 μm in A–H.)

Fig. 4.

Tumor-derived neurospheres give rise to neurons that proliferate aberrantly. (A) Clonal neurosphere derived from the BT4 tumor, double-labeled for β-III-tubulin (red) and Ki-67 (green). Nuclei were counterstained with DAPI (blue). (B) Double-label for BrdUrd (green), visualized after a 14-h pulse and TuJ1 (red). (Scale bar in B = 15 μm in A and B.)