The clinical significance of cathepsin S expression in human astrocytomas

Abstract

Early local invasion by astrocytoma cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-Leu-HomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma cells and provide evidence for a potential role for CatS in invasion.

Figure 1.

RT-PCR of mRNA extracted from cultures derived from astrocytomas of World Health Organization grade I (1 to 3), World Health Organization grade II (4 to 7), World Health Organization grade III (8 and 9), and World Health Organization grade IV. 10 and 11: RT-PCR of mRNA extracted from a normal brain sample. 12: PCR, without RT step, of mRNA extracted from World Health Organization grade IV cell culture to demonstrate the absence of DNA contamination in the mRNA preparation. 13: Positive control mRNA sample of 500 bp. 14: Water (negative control). 15: Size markers. 16: CatS is represented by the fraction of 996 bp.

Figure 2.

A: CatS immunostaining of World Health Organization grade II astrocytoma. B: CatS immunostaining of World Health Organization grade IV astrocytoma (inset shows area of endothelial hyperplasia (e) with CatS-negative endothelial cells). C: CatS immunostaining of normal brain. n, neurone; a, astrocyte; o, oligodendrocyte; m, macrophage. D: CD68 staining of tumor-associated microglia/macrophages in World Health Organization grade IV astrocytoma (inset shows perivascular macrophages). E: CatS immunostaining of brain with gliosis from a World Health Organization grade II astrocytoma (r, reactive astrocyte). Original magnification, ×400.

Figure 3.

A: Immunostaining of grade IV astrocytoma to show CD68-positive microglia/macrophages (arrows). B: CatS/CD68 dual labeling. Microglia/macrophages are positive for CatS and CD68 (arrows) whereas astrocytoma cells (arrowheads) are only positive for CatS. C: CatS (red)/GFAP (green) dual labeling. The lysosomal localization of CatS immunostaining (I) is distinct from that of GFAP which is predominantly within tumor cell foot processes (p). m, mitotic figure; n, outline of nucleus; bv, blood vessel. D: CatS immunostaining of astrocytoma cells in vitro. Nuclei are counterstained with propidium iodide. Original magnification, ×400.

Figure 4.

Specific activity of CatS in lysates derived from biopsies of astrocytomas I-IV.

Figure 5.

Specific activity of CatS in astrocytoma cultures (grades I to IV). a: Intracellular activity. b: Extracellular activity. c: Plot of extracellular CatS versus intracellular CatS activity for each culture.

Figure 6.

In vitro invasion assay. Photomicrographs (magnification, ×200) show Hoechst-stained nuclei of U251MG glioblastoma cells which have invaded to the lower side of the Matrigel-coated microporous membrane. a: Control assay. b: 10 nmol/L LHVS incorporated in the invasion assay. c: 50 nmol/L LHVS incorporated in the assay. The histogram is a quantitative summary of the data. Each assay was performed in triplicate and four fields were counted in each assay.