Osteopontin expression in intratumoral astrocytes marks tumor progression in gliomas induced by prenatal exposure to N-ethyl-N-nitrosourea

Abstract

To better study early events in glioma genesis, markers that reliably denote landmarks in glioma development are needed. In the present study, we used microarray analysis to compare the gene expression patterns of magnetic resonance imaging (MRI)-localized N-ethyl-N-nitrosourea (ENU)-induced tumors in rat brains with those of uninvolved contralateral side and normal brains. Our analysis identified osteopontin (OPN) as the most up-regulated gene in glioma. Using immunohistochemistry we then confirmed OPN expression in every tumor examined (n = 17), including those with diameters as small as 300 mum. By contrast, no OPN immunostaining was seen in normal brain or in brains removed from ENU-exposed rats before the development of glioma. Further studies confirmed that OPN was co-localized exclusively in intratumoral glial fibrillary acidic protein-expressing cells and was notably absent from nestin-expressing ones. In conjunction with this, we confirmed that both normal neurosphere cells and ENU-im-mortalized subventricular zone/striatal cells produced negligible amounts of OPN compared to the established rat glioma cell line C6. Furthermore, inducing OPN expression in an immortalized cell line increased cell proliferation. Based on these findings, we conclude that OPN overexpression in ENU-induced gliomas occurs within a specific subset of intratumoral glial fibrillary acidic protein-positive cells and becomes evident at the stage of tumor progression.

Figure 1

A–C: H&E sections demonstrating early neoplastic proliferation (ENP) (A), microtumor (B), and macrotumor (C) that were noted in 90-, 115-, and 150-day-old rats, respectively, after ENU exposure in utero. Similar lesions are never observed in non-ENU-exposed rats. Tumor (T) margins are demarcated with dotted lines. D: Graphic depiction of tumor size in serially sectioned brains from ENU-exposed rats that were stained with H&E. Each data point represents an individual brain (n = 38) (there are less than 38 data points visualized because of overlap at 0 and >1 mm sizes). Line and r value represent results of a regression analysis.

Figure 2

Heavy T2-weighted image of an asymptomatic P210 rat exposed to ENU in utero. Tumor that was sampled is located in left cerebral cortex (arrow). Imaging 30 days before this study did not reveal tumor. Right:H&E of tumor sample. Pathology verified astrocytoma.

Figure 3

Representative examples of OPN staining in ENU-induced gliomas. Tumors in A–C correspond to those depicted in Figure 1. Insets in B represent typical OPN-expressing cells visualized within all gliomas examined. In addition, a fine speckled pattern of staining is noted in extracellular spaces. Below each panel (D–F) are the same sections counterstained with the hematoxylin method to demonstrate tumor in more detail.

Figure 4

OPN is not expressed in normal brain, even in areas such as the SVZ, an area containing migratory, proliferating cells. Adjacent coronal sections at the level of the striatum stained with H&E (left), BrdU and nestin (middle), and OPN (right). No OPN staining is noted.

Figure 5

No evidence of co-localization of OPN and nestin in ENU-induced gliomas. Arrows delineate nestin⁺ cells expressing tetramethyl-rhodamine isothiocyanate (A) and arrowheads OPN⁺ cells expressing fluorescein isothiocyanate (B). A merged figure (C) with arrows and arrowheads included reveals no evidence of co-localization.

Figure 6

Co-localization of OPN and intratumoral GFAP-expressing cells. A: Brain-glioma interface (glioma is to the left of the dotted line) illustrating increased size of GFAP⁺ cells within tumor (arrows) compared to normal astrocytes (arrowhead). B and C: Higher magnification photomicrograph of area enclosed within the inset in A illustrating GFAP (green) and OPN (red) co-localization. A similar result was noted when the fluorophores were reversed.

Figure 7

Confocal 2.2-μm optical sections of GFAP and OPN staining in ENU-induced tumor cells of 150-day-old rat. GFAP (A) and OPN (B) co-exist in the same cell (D). Cell nucleus is stained blue with DAPI in C. Arrows in panels designate location of nucleus of OPN- and GFAP-labeled cell. Scale bar, 20 μm. Original magnifications, ×800.

Figure 8

Representative section of reactive astrocytosis produced by a stab wound illustrating no co-expression of GFAP with OPN. Normal rat was sacrificed 14 days after wounding and the section double stained for GFAP (green) and OPN (red). Cell density is higher in the wound area on the left of photoimage. Reactive GFAP cells are aggregated at the edge of the injured brain in center of photograph. Hypertrophic GFAP-expressing cells similar to what was noted within ENU-induced gliomas were not seen. No OPN is visualized in the area; reddish substance within the wound reflects nonspecific staining of blood products. Arrow points to fluorescein isothiocyanate-labeled GFAP⁺ cell within the stab wound.

Figure 9

C6 glioma cells express more OPN mRNA as assessed by real-time PCR and secretes more OPN protein as assessed by ELISA than either immortalized cell lines ENU3.2, ENU4.30, and ENU4.1 (described in Savarese et al15) or normal neurosphere cells (RNS) isolated from P1 rat pups (isolation methods described in Savarese et al15). *P < 0.001, one-way analysis of variance. There is no statistical significance in the levels of OPN mRNA or protein between normal neurosphere cells and immortalized lines.

Figure 10

ENU3.2^OPN expresses intermediate levels of OPN mRNA as assessed by real-time PCR compared to native ENU3.2 and C6 glioma. The differences in expression between each line is significant (*P < 0.05, Student-Newman-Keuls method).