Cell surface Nestin is a biomarker for glioma stem cells

Highlights

• Cell surface Nestin expresses in human glioblastoma and glioma stem cells.

• Cell surface Nestin-positive glioma stem cells possess robust tumorsphere-forming ability.

• γ-Secretase regulates the generation of cell surface Nestin in gloma stem cells.

Abstract

Cancer stem cells (CSCs) are the most aggressive cell type in many malignancies. Cell surface proteins are generally used to isolate and characterize CSCs. Therefore, the identification of CSC-specific cell surface markers is very important for the diagnosis and treatment of malignancies. We found that Nestin (a type VI intermediate filament protein), like the glioma stem cell (GSC) markers CD133 and CD15, exhibited different levels of expression in primary human glioblastoma specimens. Similar to our previous finding that cytoplasmic Nestin is expressed as a cell surface form in mouse GSCs, the cell surface form of Nestin was also expressed at different levels in human GSCs. We isolated cell surface Nestin-positive cell populations from human GSCs by fluorescence-activated cell sorting FACS analysis, and observed that these populations exhibited robust CSC properties, such as increased tumorsphere-forming ability and tumorsphere size. Mechanistically, we found that DAPT, a γ-secretase (a multi-subunit protease complex) inhibitor, reduced the proportion of cell surface Nestin-positive cells in human GSCs in a time- and dose-dependent manner, without significant changes in total Nestin expression, implying that a post-translational modification was involved in the generation of cell surface Nestin. Taken together, our data provides the first evidence that cell surface Nestin may serve as a promising GSC marker for the isolation and characterization of heterogeneous GSCs in glioblastomas.

Introduction

Glioblastoma multiforme (GBM, WHO grade IV) is the most aggressive malignancy of the central nervous system with a median survival time of only 12–15 months despite vigorous treatments such as surgical resection, radiotherapy, and chemotherapy [1]. Recently, glioma stem cells (GSCs), a subpopulation of glioma cells [2], were identified as the main cause of tumor propagation or tumor recurrence after anti-cancer therapy [3], [4], [5].

A number of cell surface markers are generally used to isolate and characterize cancer stem cells (CSCs) [6]. In GBM, 2 cell surface markers, CD133 [7] and CD15 [8], are generally used to isolate and characterize GSCs. CD133 is a glycoprotein that specifically localizes to the outer cellular membrane [9], [10], and is expressed in hematopoietic stem cells [11], endothelial progenitor cells [12], neural stem cells, and brain tumors [10], [13]. However, several recent reports have indicated that CD133 may not be a robust marker for GSCs [14], [15], [16], [17], [18]. CD15 is a carbohydrate adhesion molecule that is also known as stage-specific embryonic antigen 1 (SSEA1) [19], and is expressed in embryonic or adult central nervous system stem cells [20], [21], leukemias [22], and GBM [23].

Many cellular factors, including transcriptional factors (e.g., Sox2, Nanog, and Oct3/4) [24], cytoskeletal proteins (e.g., Nestin) [25], post-transcriptional factors (e.g., Musashi 1) [25], and Polycomb transcriptional suppressors (e.g., Bmi1 and Ezh2) [26], [27], are also considered GSC markers. However, in contrast to CD133 and CD15, these cellular factors are not useful for the isolation of live GSCs from tumor tissues given their intracellular localization, such as in the nucleus or cytoplasm.

During the early developmental stage of the central nervous system, Nestin is primarily expressed in neural progenitor/stem cells [28]. The Nestin protein is mainly localized in the cytoplasm and functions as a type VI intermediate filament with a high molecular weight (240 kDa) [29]. We have previously reported that an ∼60-kDa N-terminal isotype of Nestin (hereafter, referred to as cell surface Nestin) is expressed on the outer cellular membrane of Id4-driven murine GSCs [30]. Here, we report the expression of cell surface Nestin in human GBM specimens and human GSCs, the isolation of live cell surface Nestin-positive GSCs, characterization of their self-renewal property, and a plausible mechanism underlying the generation of cell surface Nestin.