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

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-017-2474-0) contains supplementary material, which is available to authorized users.

Quantitative Modulation of PpIX Fluorescence and Improved Glioma Visualization

“…These findings will however need to be correlated with effective tumor cell infiltration in brain tumor. We have previously demonstrated that different GBM cell lines may have different PpIX signal intensity when treated with the same parameters (7, 16), a phenomenon also described in vivo (21). This phenomenon can be also seen in our mice GBM tumor model, as demonstrated in Figure 7, where tumor not visible to the naked eye with the conventional blue light of the operation microscope, can be made visible with color coded matrix under the Cp9 microscope.…”

“…The amount of PpIX in tumor cells is favored by both the activity of cytosolic porphobilinogen deaminase (PBGD) during the replication phase, and by the down-regulation of ferrochelatase (FECH) (6). However, other factors such as protein transporters, tyrosine kinase activity and its downstream effect on hemoxygenase-1 (HO-1), availability of free Fe 2+ -ions and its effect on FECH, are related with PpIX fluorescence (7, 8). Because of the above mentioned factors, the final enhancing results are highly variable and difficult to predict both in clinical and laboratory setting, and might also depend on EGFR expression status in glioma cell lines (7).…”

“…However, other factors such as protein transporters, tyrosine kinase activity and its downstream effect on hemoxygenase-1 (HO-1), availability of free Fe 2+ -ions and its effect on FECH, are related with PpIX fluorescence (7, 8). Because of the above mentioned factors, the final enhancing results are highly variable and difficult to predict both in clinical and laboratory setting, and might also depend on EGFR expression status in glioma cell lines (7). Proteins transporters such as ATP-binding cassette subfamily G member 2 (ABCG2) regulate intracellular concentration of PpIX.…”

Molecular and Metabolic Mechanisms Underlying Selective 5-Aminolevulinic Acid-Induced Fluorescence in Gliomas

“…Overexpression of another ATP-binding cassette transporter known to regulate porphyrin synthesis, ABCB6, has also been shown to increase PpIX accumulation in glioma cell lines and subsequently increase PDT effects [ 63 ]. Epidermal growth factor receptor (EGFR) and EGFRvIII co-expression has been shown to influence 5-ALA fluorescence in GBM [ 64 ]. Additionally, PpIX efflux out of the cell was reported in the presence of fetal bovine serum [ 65 ].…”

Role of molecular biomarkers in glioma resection: a systematic review

“…In the past few years, 5-ALA has been established as an effective intraoperative tool to increase EOR in high-grade gliomas [ 47 ]. Fontana et al found that EGFRvIII activated heme oxygenase-1, thereby reducing the 5-ALA-induced fluorescence staining effect in GBMs [ 19 ]. This study explained the low or absent degree of 5-ALA-induced fluorescence staining in some GBMs.…”