Selective uptake of boronophenylalanine by glioma stem/progenitor cells

https://doi.org/10.1016/j.apradiso.2012.04.005Get rights and content

Abstract

The success of boron neutron capture therapy (BNCT) depends on the amount of boron in cells and the tumor/blood and tumor/(normal tissue) boron concentration ratios. For the first time, measurements of boron uptake in both stem/progenitor and differentiated glioma cells were performed along with measurements of boron biodistribution in suitable animal models. In glioma stem/progenitor cells, the selective accumulation of boronophenylalanine (BPA) was lower, and retention of boron after BPA removal was longer than in differentiated glioma cells in vitro. However, boron biodistribution was not statistically significantly different in mice with xenografts.

Highlights

► Uptake of BPA was analyzed in stem/progenitor and differentiated glioma cells. ► Selective accumulation of BPA was lower in glioma stem/progenitor cells. ► Retention of boron after BPA removal was longer in glioma stem/progenitor cells. ► Boron biodistribution was not statistically different in mice with xenografts.

Introduction

Human glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer with poor prognosis (Lamszus and Günther, 2010, Wen and Kesari, 2008). Even with aggressive treatments, including surgery, radiation therapy and chemotherapy, patient survival is typically 12–18 months post-diagnosis (Grossman et al., 2010, Stupp et al., 2009). Despite the progress in research in the molecular aspects of malignant glioma, the prognosis of these brain tumors continues to be dismal partly because mechanisms responsible for gliomagenesis and progression remain elusive. It is traditionally believed that glioma may develop from neural stem cells (NSCs) that undergo abnormal differentiation or from differentiated cell types that acquire malignance by de-differentiating in response to oncogenic mutation.

Recent observations related to isolation and characterization of brain tumor-initiating cells support the concept that transformed NSCs may seed gliomas (Vescovi et al., 2006). Currently, it is a widely accepted hypothesis that a highly tumorigenic subpopulation of cancer cells, called glioma stem cells (GSCs), is responsible for tumor initiation and recurrence. The diffuse growing pattern and cell infiltration are more pronounced in glioblastoma derived from GSC xenografts than in general glioma line xenografts (Brehar et al., 2010), and transplanted model tumor established by GSCs in nude mice is more likely to be malignant than that established by cancer cells (Jin et al., 2011). The characterization of cancer stem cells will likely help to develop novel targeted therapies designed to eradicate the most dangerous tumor cells (Bautch, 2010). More importantly, the characterization of glioma cells with stem-like properties has opened up the development of novel targeted therapies (Hede et al., 2011). Targeting GSCs may effectively reduce tumor recurrence and significantly improve GBM treatment. Recent studies indicate that cancer stem cells have critical distinctions, which provide important clues to useful therapeutic targets (Huang et al., 2010). The extensive infiltration growing patterns of xenografts derived from GSCs make them useful models for studying the mechanisms and therapy strategies in gliomas (Brehar et al., 2010).

Boron neutron capture therapy (BNCT) is a unique high-dose tumor-selective radiotherapy for cancer treatment. In principle, BNCT allows for preferential destruction of boron (10B)-loaded tumor cells while sparing the normal tissue that does not contain 10B (Yamamoto et al., 2008). It is based on the nuclear capture and fission reactions that occur when the stable isotope 10B is irradiated with thermal neutrons to produce high-energy (>2.3 MeV) α-particles and recoiling 7Li nuclei (Barth et al., 2005). These products of fission reaction have high linear energy transfer (LET) and provide a disruptive effect within a spatial range of 5–10 μm. These characteristics ensure a well-localized effect of BNCT, which is virtually confined to the cells containing a critical amount of 10B (Yang et al., 2009a, Yang et al., 2009b). For a successful BNCT, a sufficient number of 10B atoms (∼109 atoms/cell or ∼20 μg of 10B/g) must be delivered to the tumor, and enough thermal neutrons must be absorbed by the 10B atoms to sustain the lethal 10B(n,α)7Li reaction (Barth et al., 2005). Recent clinical studies of BNCT have focused mainly on the treatment of high-grade gliomas and cutaneous melanomas worldwide. Over 350 high-grade gliomas have been treated with a combination of surgery and BNCT (Yamamoto et al., 2008). Two 10B delivery agents are used in the current clinical protocols to treat melanomas and brain tumors, namely, boronophenylalanine (BPA), and sodium borocaptate (BSH; Capala et al., 2003, Hideghéty et al., 2003). However, a large body of literature has demonstrated a higher effectiveness of BPA as compared with BSH in the BNCT of brain malignances (Capala et al., 1996, Capuani et al., 2002). This could be attributed to different micro-distributions and tumor incorporations of the two compounds (Ono et al., 1996, Ono et al., 1999)

BPA (C9H12 10BNO4), a derivative of the neutral amino acid phenylalanine, was first used clinically by Mishima in 1988–1989 to treat patients with cutaneous malignant melanomas (Mishima et al., 1989). It was not until 1994 that the first clinical trial was initiated at the Brookhaven National Laboratory to evaluate BPA–fructose complex for BNCT of patients with GBM (Diaz, 2003). Since then, BPA–fructose has become the most frequently used clinical boron delivery agent for both intra- and extra-cranial tumors (Barth, 2009). At the 13th International Congress on Neutron Capture Therapy, a number of papers were presented on boron delivery agents, but no new boron compound has reached the level of Phase I in clinical biodistribution study (Altieri et al., 2009).

Boron uptake in GSCs is a prerequisite for killing cells by BNCT. Boron concentrations in the tumor, normal brain tissue, and blood (especially the ratios of the concentration in tumor to the concentrations in normal tissue and blood) are very important in planning treatment with BNCT. Until relatively recently, detailed biodistribution and pharmacokinetic data for BPA in GSCs were not available. In this work, we studied absorption, retention and biodistribution of boron administered with BPA in glioma stem/progenitor cells in vitro and in model mice with their xenografts. The goal was to support radiobiological studies for development of BNCT in human brain tumors.

Section snippets

Cell lines

SU2 cells, a gift from Professor Qiang Huang, are a stem/progenitor cell line of Chinese glioma origin. It was isolated from a 52-year-old female patient with recurrent glioma who had undergone two operations in a 6-month interval because of rapid re-growth of a tumor mass in the right temporal lobe; pathology identified it as GBM (WHO Grade IV). The SU2 cells were used in this work as a glioma stem/progenitor cell (GSPC) line. The characteristics of the SU2 cell line demonstrate successful

Xenograft formation in BALB/c nude mice

There was a sharp boundary between tumor mass and surrounding normal brain tissue. Round and densely arranged tumor cells, abundant mitosis and vascularization were observed in the SHG-44 cell xenografts by HE staining (Fig. 1A). More notably, nucleolus mitosis occurred in the SU2 cell subcutaneous xenograft (Fig. 1D). GFAP, one of the important markers for GBM, was highly expressed in cytoplasm of tumor cells (Fig. 1B of SHG-44 cell xenograft, Fig. 1E of SU2 cell xenograft).

Discussion

Although the view of selective uptake of BPA by glioma cells has been remarkably unchanged in the last decade, there has been some doubt if glioma stem cells could absorb it and what the imbibed amount is. In this study, we analyzed BPA uptake in vitro and in vivo by measuring boron concentration with ICP-AES, which is deemed a common and adequate method for cells and tissues in BNCT studies. The results demonstrate that both differentiated glioma cells and glioma stem/progenitor cells

Acknowledgments

The current research was supported by the Young Teacher Natural Science Foundation of Suzhou University (Q312202810) and the Major Issues Foundation of the Health Department of Jiangsu Province (K201106). We would like to express thanks to Professor Weilian Yang, who works in the Department of Pathology of the Ohio State University in the USA, for theoretical support.

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