Sabrina Kröger (Presenter)
University of Münster
Bio: PhD Student, Research Group of Prof. Dr. Uwe Karst, Institute of Inorganic and Analytical Chemistry, University of Münster
Authorship: Sabrina Kröger (1), Ann-Christin Niehoff (1), Astrid Jeibmann (2), Walter Stummer (3), Uwe Karst (1)
(1) Institute of Inorganic and Analytical Chemistry, University of Münster, Münster; (2) Institute of Neuropathology, University Hospital of Münster, Münster; (3) Department of Neurosurgery, University Hospital of Münster, Münster
Contrast enhancement by fluorescence-guided surgery (FGS) can significantly improve the resection of glioblastoma, the most common malignant brain tumor. The major proagent for FGS is 5-aminolevulinic acid (5-ALA), a natural biochemical precursor of heme b, with fluorescent protoporphyrin IX (PPIX) as intermediate product. To investigate limitations by false negative and false positive fluorescence, mass spectrometry imaging is presented. A complementary molecular and elemental imaging method combining spatially resolved information from matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is developed and applied for the investigation of 5-ALA, PPIX and heme b distribution in human brain tumor tissue.
Glioblastoma is the most common and aggressive malignant brain tumor. It is classified as grade IV astrocytoma by the World Health Organization (WHO) . The survival rate can be significantly enhanced by total surgical resection of malignant glioma cells . However, this can only be achieved in less than 20 % of all cases . Resection can be improved by contrast enhancement using fluorescence-guided surgery (FGS). The major proagent for FGS is 5-aminolevulinic acid (5-ALA). 5-ALA is a natural biochemical precursor of heme b, with fluorescent protoporphyrin IX (PPIX) as intermediate product. After oral administration of 5-ALA to the patients, it is transported through the blood-brain barrier into tumor tissue. Based on the fluorescence of formed PPIX, tumor tissue can be differentiated from healthy brain tissue [4,5]. To extent the knowledge about biochemical processes related to FGS and examine possible limitations by false positive and false negative fluorescence, the development of suitable imaging methods is of great interest. In this study, a complementary molecular and elemental imaging method combining spatially resolved information from matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is developed and applied for the investigation of 5-ALA, PPIX and heme b distribution in human brain tumor tissue.
After resection of the human cerebral tumor, 10 µm cryosections were prepared for hematoxylin and eosin staining, MALDI-MS and LA-ICP-MS analysis. Based on H&E staining, the tumor classification and the tumor grade were evaluated. The distribution of 5-ALA, PPIX and heme b in the tumor tissue was investigated by MALDI-MS imaging. The MALDI source was equipped with a frequently-tripled 355 nm Nd:YAG laser operating at atmospheric pressure. Four positive ion mode experiments in MS and MS/MS mode were performed using a focal laser spot of 20-25 µm and a step size of 50 µm. The matrix α-cyano-4-hydroxycinnamic acid (CHCA) was sublimated and recrystallized before analysis.
The Fe distribution was examined coupling a laser ablation to a quadrupole based ICP-MS operating in kinetic energy discrimination mode to minimize interferences. Cryosections were ablated linewise with a 213 nm Nd:YAG laser using 20 Hz laser shot frequency, 50 µm spot diameter and 100 µm/s scan speed. The LA system was equipped with a low volume custom-built ablation cell providing a laminar gas flow. The generated aerosol was transported to the ICP with helium as carrier gas. An additional wet aerosol of a Rh standard was simultaneously introduced into the plasma to monitor the sensitivity of the instrumental setup. Quantitative data were obtained by external calibration with matrix-matched standards based on 2-hydroxyethyl cellulose (HEC).
Complementary molecular and elemental mass spectrometry imaging is presented to investigate the distribution of 5-ALA, PPIX and heme b in human brain tumor tissues. Therefore, method development for MALDI-MS and quantitative LA-ICP-MS imaging was performed.
Experimental conditions for MALDI-MS measurements of 5-ALA, PPIX and heme b were optimized by dried droplet analysis of the pure substances. To improve the signal-to-noise ratio (S/N), MS/MS experiments proved to be suitable for PPIX and heme b detection. MALDI-MS analysis of the tumor tissue was successively performed in four different modes using a shift in the corresponding pixel arrangement. Due to the system setup with an octapole, an ion trap and a TOF-MS arranged in series, small mass ranges were selected to obtain highest sensitivity. Therefore, MS analysis of 5-ALA and PPIX/heme b was performed in two measurements of different mass ranges. Furthermore, MS/MS imaging experiments of PPIX and heme b improved limit of detection and validated MS data. The developed MALDI-MS and MALDI-MS/MS imaging method was applied to several human brain tumor samples. Next to 5-ALA detection, the correlation between PPIX accumulation and high tumor cell density was shown for glioblastomas. Heme b accumulation correlated with blood vessel distribution. Necrotic tissue showed no detection of 5-ALA or PPIX, which is in accordance with the absence of fluorescence during FGS. Furthermore, a gliosarcoma, one rare type of glioma, which appears non-fluorescent during FGS, was investigated. The imaging results verified the penetration of 5-ALA through the blood-brain barrier. PPIX was not detected in gliosarcoma tissue which correlates with the non-fluorescent appearance of this kind of tumor during FGS. An explanation for this limited fluorescence has not yet been reported.
Next to molecular analysis, elemental bioimaging of Fe, corresponding to heme b, was performed by LA-ICP-MS. For quantification, external calibration with homemade matrix-matched standards was used. Best matrix-matching using homogenized tumor tissue is not appropriate due to high native Fe concentration. Matrix-matched standards based on HEC provided low limits of detection (LOD) and quantification (LOQ) of 1.1 µg/g and 3.5 µg/g, respectively. Fe accumulations of over 200 µg/g could be detected in the tumor tissue correlating well with heme b and blood vessel distribution.
Complementary molecular and elemental imaging by MALDI-MS(/MS) and LA-ICP-MS is presented as a powerful tool to investigate the distribution of 5-ALA, PPIX and heme b in human brain tumor resected by fluorescence-guided surgery.
References & Acknowledgements:
 Ostrom, Q. T.; Gittleman, H.; Liao, P.; Rouse, C.; Chen, Y.; Dowling, J.; Wolinsky, Y.; Kruchko, C.; Barnholtz-Sloan, J. Neuro Oncol. 2014, 16, iv1–iv63.
 Kawano, H.; Hirano, H.; Yonezawa, H.; Yunoue, S.; Yatsushiro, K.; Ogita, M.; Hiraki, Y.; Uchida, H.; Habu, M.; Fujio, S.; Oyoshi, T.; Bakhtiar, Y.; Sugata, S.; Yamahata, H.; Hanaya, R.; Tokimura, H.; Arita, K. Br. J. Neurosurg. 2015, 29, 206–212.
 Willems, P. W. A.; Taphoorn, M. J. B.; Burger, H.; Berkelbach van der Sprenkel, J. W.; Tulleken, C. A. F. J. Neurosurg. 2006, 104, 360–368.
 Ennis, S. R.; Novotny, A.; Xiang, J.; Shakui, P.; Masada, T.; Stummer, W.; Smith, D. E.; Keep, R. F. Brain Res. 2003, 959, 226–234.
 Hadjipanayis, C. G.; Widhalm, G.; Stummer, W. Neurosurgery 2015, 77 (5), 663–673.
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