Deferoxamine may enhance 5-aminolevulinic acid-based fluorescence in glioma surgery

Glioblastoma multiforme (GBM) is one of the most malignant brain tumors, because of its proliferative and invasive characteristics. The median survival is 12−16 months, despite multidisciplinary therapies, including surgery, radiation, and chemotherapy (1,2). GBM tumor cells migrate into the brain parenchyma far from the tumor mass and recurrence is common along the periphery of the tumor removal cavity, even in cases where the enhanced lesion has completely disappeared postoperatively (2). However, it is difficult to evaluate tumor spread of GBM on magnetic resonance imaging (MRI). We have previously reported a positron emission tomography (PET) study in which we compared the methionine (MET) uptake area to the area of gadolinium (Gd) enhancement on MRI in patients with GBM (3). We showed that the MET uptake area completely enveloped the Gd-enhanced area. In most cases, the MET uptake area enclosed the outer region of the Gd-enhanced area, with an additional 30 mm expansion. In contrast, the Gd-enhanced area coincided with only 58.6% of the MET-uptake area on average. Based on these results, we identified three cases of possible GBM recurrences after complete resection; they presented as new Gd-enhanced lesions in the MET uptake area at the edge of the surgical removal cavity (3). Hence, MET-PET may be useful for predicting local recurrence, given the metabolic abnormalities in residual tumor cells even before local tumor recurrence could be discerned on MRI (4).

The resection rate is one of the prognostic factors in GBM. It has been reported that resection of >78% of GBM lesions could improve survival; moreover, aggressive resection of 95–100% of the tumor increases survival in a stepwise fashion (5). Accordingly, it seems necessary to remove the infiltrating tumor along with the surrounding normal brain tissue, in order to achieve gross total resection.

It is considered that 5-aminolevulinic acid (5-ALA) may be useful for discriminating high-grade glioma from normal brain tissues during surgery. A randomized controlled trial showed that 5-ALA fluorescence-guided resection of glioblastoma improved patients’ 6-month progression-free survival as compared to those undergoing microsurgical resection under conventional white light (6). Subsequently, analysis of data from a randomized controlled trial provided level 2b evidence supporting the benefit of resection under 5-ALA fluorescence guidance on overall survival (7).

We have previously reported the sensitivity, specificity, positive-predictive value, and negative-predictive value of 5-ALA for identifying more than 50% of olig2-positive cells remaining in the wall of the removal cavity, as examined on immunohistochemistry of the postoperative surgical specimens, as 90.9%, 44.4%, 66.7%, and 80%, respectively (8). In other words, it is not possible to identify all the tumor cells with conventional usage of 5-ALA.

Glioblastoma stem cells (GSCs) have been reported to play a crucial role in the initiation, progression, resistance to therapy, and recurrence of GBM (9,10). In our recurrent cases, it appears that GSCs must have remained around the removal cavity, acting as the major source of the recurrence, after escaping the effect of multimodal therapies (2). GBM is considered to be composed of heterogeneous populations of tumor cells and to include specific subpopulations, i.e., GSCs and non-GSCs. Wang et al. showed that GSCs are less able to accumulate protoporphyrin IX (PpIX) than non-GSCs, and a subset of side population (SP)-defined C6 glioma cells, which are GSCs, most likely escapes from detection and resection under 5-ALA-based photodynamic diagnosis (PDD) (11). Additionally, they demonstrated that a subpopulation of C6 SP-defined GSCs possessing low PpIX fluorescence exhibit markedly higher tumorigenic activity and much more rapid progression of tumors than that of SP-derived cells possessing high PpIX fluorescence. In contrast, main population (MP)-derived cells, i.e., non-GSCs showing low PpIX fluorescence, did not present rapid progressions. This phenomenon indicates that more aggressive tumor cells can elude 5-ALA-based resection, and lead to tumor recurrence. This leads to the question of how SP-derived cells with low PpIX fluorescence can be removed. To achieve this, Wang et al. propounded deferoxamine (DFO) treatment. The agent has a specific affinity for free iron in the blood stream, resulting in the suppression of the metabolism of PpIX to heme by an iron-chelation effect. Valdés et al. have shown that DFO-mediated iron chelation increases 5-ALA-mediated PpIX accumulation in U251 malignant glioma cells in vivo (12). Wang et al. hypothesized that DFO may suppress this PpIX metabolism process, resulting in the restoration of PpIX accumulation in GSCs (11). They then demonstrated that DFO treatment significantly increased the percentage of PpIX fluorescence-positive cells among C6 SP-derived cells, as compared to that in MP-derived cells (11). They certainly showed a distinctive decrease in the frequency of cells with poor PpIX accumulation using this DFO treatment. However, the DFO-induced enhancement effect was not observed in MP-derived cells. They first reported that DFO-mediated iron chelation could enhance PpIX accumulation in C6 SP-defined GSCs. Further clinical studies are required to test the applicability of DFO in the enhancement of PpIX accumulation in GSCs in GBM (11). Because DFO has been clinically used as treatment for hemochromatosis, to reduce liver iron concentration without significant side effects (13,14), this agent could be applied in glioma surgery in future.

At the molecular level, what causes the low accumulation of PpIX in C6 SP-defined GSCs? In a search for iron metabolism-associated genes, HO-1, which encodes a rate-limiting enzyme for heme degradation and which functions as an inducible protective gene against cellular stress and oxidative injury, was identified (15). Semi-quantitative RT-PCR analysis of HO-1 revealed significantly enhanced expression in C6 SP-derived cells treated with 5-ALA, as comparing to that without 5-ALA treatment. This difference was not observed in C6 MP-derived cells. Accordingly, C6 SP-derived cells, namely GSCs, in the GBM may strongly express HO-1 under 5-ALA treatment, which could accelerate PpIX/heme metabolism, leading to poor accumulation of 5-ALA. Elevated expression of HO-1 was seen in human GBMs as compared to non-tumorous tissue or low grade gliomas, based on publicly available datasets, including the TCGA_GBM HG-U133A platform, Rembrandt, and Gravendeel datasets. In addition, HO-1 was verified as a prognostic factor in GBMs based on Kaplan-Meier survival analysis. In fact, the lower expression group showed significantly longer survival than did the high expression group in all datasets. Taken together, administration of 5-ALA could activate HO-1, leading to reduced accumulation of PpIX through accelerated metabolism of PpIX to heme in GSCs.

In conclusion, Wang et al. showed that an iron chelator could be used to improve 5-ALA-based PDD of SP-defined GSCs in glioma. Clinical usage of a combination of 5-ALA and DFO may be valuable in glioma surgery.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Ning Huang (Department of Neurosurgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China).

Conflicts of Interest: Dr. T Iwama accepted payment for research expenses from Otsuka Pharmaceutical Co. Ltd. and Ogaki Tokushukai Hospital in 2013. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Ishida J, Kurozumi K, Ichikawa T, et al. Evaluation of extracellular matrix protein CCN1 as a prognostic factor for glioblastoma. Brain Tumor Pathol 2015;32:245-52. [Crossref] [PubMed]
2. Hide T, Makino K, Nakamura H, et al. New treatment strategies to eradicate cancer stem cells and niches in glioblastoma. Neurol Med Chir (Tokyo) 2013;53:764-72. [Crossref] [PubMed]
3. Miwa K, Shinoda J, Yano H, et al. Discrepancy between lesion distributions on methionine PET and MR images in patients with glioblastoma multiforme: insight from a PET and MR fusion image study. J Neurol Neurosurg Psychiatry 2004;75:1457-62. [Crossref] [PubMed]
4. Yano H, Shinoda J, Iwama T. Clinical utility of positron emission tomography in patients with malignant glioma. Neurol Med Chir (Tokyo) 2017;57:312-20. [Crossref] [PubMed]
5. Sanai N, Polley MY, McDermott MW, et al. An extent of resection threshold for newly diagnosed glioblastomas. J Neurosurg 2011;115:3-8. [Crossref] [PubMed]
6. Stummer W, Pichlmeier U, Meinel T, et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol 2006;7:392-401. [Crossref] [PubMed]
7. Stummer W, Reulen HJ, Meinel T, et al. Extent of resection and survival in glioblastoma multiforme: identification of and adjustment for bias. Neurosurgery 2008;62:564-76. [Crossref] [PubMed]
8. Yano H, Nakayama N, Ohe N, et al. Pathological analysis of the surgical margins of resected glioblastomas excised using photodynamic visualization with both 5-aminolevulinic acid and fluorescein sodium. J Neurooncol 2017;133:389-97. [Crossref] [PubMed]
9. Baumann M, Krause M, Hill R. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 2008;8:545-54. [Crossref] [PubMed]
10. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005;5:275-84. [Crossref] [PubMed]
11. Wang W, Tabu K, Hagiya Y, et al. Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation. Sci Rep 2017;7:42070. [Crossref] [PubMed]
12. Valdés PA, Samkoe K, O'Hara JA, et al. Deferoxamine iron chelation increases delta-aminolevulinic acid induced protoporphyrin IX in xenograft glioma model. Photochem Photobiol 2010;86:471-5. [Crossref] [PubMed]
13. Aydinok Y, Kattamis A, Cappellini MD, et al. Effects of deferasirox-deferoxamine on myocardial and liver iron in patients with severe transfusional iron overload. Blood 2015;125:3868-77. [Crossref] [PubMed]
14. Bring P, Partovi N, Ford JA, et al. Iron overload disorders: treatment options for patients refractory to or intolerant of phlebotomy. Pharmacotherapy 2008;28:331-42. [Crossref] [PubMed]
15. Otterbein LE, Soares MP, Yamashita K, et al. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol 2003;24:449-55. [Crossref] [PubMed]