Epithelial growth factor receptor expression influences 5-ALA induced glioblastoma fluorescence

Andrea O Fontana,Floriana Burgio,Michael Reinert,Uwe Pieles,Deborah Piffaretti,Sandra Pinton,Paolo Paganetti,Ana Bela Faia-Torres,Francesco Marchi

doi:10.1007/s11060-017-2474-0

Abstract

The extent of 5-aminolevulinic acid (5-ALA) guided tumor resection has a determining impact in high-grade glioma and glioblastoma surgery. Yet the intensity of the 5-ALA induced fluorescence may vary within the tumor. We aimed to correlate 5-ALA induced fluorescence with the expression of epithelial growth factor receptor (EGFR) and its constitutively active version EGFRvIII in different glioblastoma (GBM) cell lines. To elucidate the role of EGFR in the metabolism of 5-ALA in GBM cell lines with variable EGFR expression status, we analyzed the activation of EGFR by its primary ligand EGF, and its downstream effect on Heme oxygenase-1 (HO-1), a key enzyme regulating the metabolism of Protoporphyrin IX (PpIX), the fluorescent metabolite of 5-ALA. Effects of direct pharmacological inhibition by Tin(IV)-Protoporphyrin (SnPP) or gene knockdown by small interfering RNA (siRNA) on HO-1 enzyme were analyzed in respect to 5-ALA induced fluorescence. Furthermore, inhibition of EGFR by Gefitinib was tested. A significant difference in 5-ALA induced fluorescence was obtained in U87MG (low EGFR expression) and LN229EGFR cells (EGFR overexpression) compared to BS153 (EGFR overexpression/EGFRvIII+). Treatment of U87MG and LN229EGFR cells with EGF significantly reduced cellular fluorescence, by promoting HO-1 transcription and expression in a concentration-dependent manner. This effect could be reversed by EGFR-specific siRNA treatment, which reduced protein expression of about 80% in U87MG. Remarkably, inhibition of HO-1 activity by SnPP or reduction of HO-1 protein levels by siHO-1 treatment restored fluorescence in all cell lines, independently of EGFR quantitative and qualitative expression. Gefitinib treatment was able to restore fluorescence after EGF stimulation in U87MG cells but not in BS153 cells, overexpressing EGFR/EGFRvIII. In GBM cell lines, 5-ALA induced fluorescence is variable and influenced by EGF-induced downstream activation of HO-1. HO-1 protein expression was identified as a negative regulator of 5-ALA induced fluorescence in GBM cells. We further propose that co-expression of EGFRvIII but not quantitative EGFR expression influence HO-1 activity and therefore cellular fluorescence.

Highlights

High-grade gliomas and especially glioblastoma (GBM) is the most common and progressive tumor of the central nervous system
During fluorescence assisted tumor resection a variance of the fluorescence intensity within GBM can be observed, especially at the infiltrating zone [4, 8, 10]. 5-aminolevulinic acid (5-ALA) induced fluorescence may vary upon many factors such as cell density, proliferation index, mitochondrial mass and index and exogenous factors such as application or fading during surgery [5,6,7,8]
Even though high grade glioma and GBM are considered diffuse and infiltrating diseases, increasing the magnitude of surgical tumor resection can impact overall survival in studies of high-grade gliomas, including GBM. 5-ALAaided surgery has been established as an efficient tool to increase the accuracy and extent of resection of high-grade tumors and is part of the standard procedure during

Summary

Introduction

High-grade gliomas and especially glioblastoma (GBM) is the most common and progressive tumor of the central nervous system. The extent of resection in patients with high-grade. Over the last several years, 5-aminolevulinic acid (5-ALA) has been established as an intra-operative tool to augment the extent of resection in high-grade glioma and GBM surgery [4]. 5-ALA is resorbed through the upper intestine into the blood, where it diffuses through the blood–brain barrier, which has been typically disrupted by infiltrative tumor cells [5,6,7,8,9]. 5-ALA induced fluorescence may vary upon many factors such as cell density, proliferation index, mitochondrial mass and index and exogenous factors such as application or fading during surgery [5,6,7,8]. Schwake et al have shown that in GBM cell lines continuous exposition of 5-ALA continuously increased 5-ALA induced fluorescence up to 24 h [11]

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