Complementary Molecular and Elemental Mass-Spectrometric

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Complementary Molecular and Elemental Mass Spectrometric Imaging of Human Brain Tumors Resected by Fluorescence-Guided Surgery Sabrina Kröger, Ann-Christin Niehoff, Astrid Jeibmann, Michael Sperling, Werner Paulus, Walter Stummer, and Uwe Karst Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03516 • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Analytical Chemistry

Complementary Molecular and Elemental Mass Spectrometric Imaging of Human Brain Tumors Resected by Fluorescence-Guided Surgery Sabrina Kröger1, Ann-Christin Niehoff1, Astrid Jeibmann2, Michael Sperling1,3, Werner Paulus2, Walter Stummer4, Uwe Karst1* 1

Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 30, 48149 Münster, Germany

2

Institute of Neuropathology, University Hospital Münster, Pottkamp 2, 48149 Münster, Germany

3

European Virtual Institute for Speciation Analysis (EVISA), Mendelstraße 11, 48149 Münster, Germany

4

Department of Neurosurgery, University Hospital Münster, Albert-Schweitzer-Campus 1, 48149 Münster, Germany

ABSTRACT: Fluorescence-guided surgery (FGS) has been established as a powerful technique for glioblastoma resection. After oral application of the prodrug 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PpIX) is formed as an intermediate of the heme biosynthesis cascade and accumulates within the tumor. By intraoperative fluorescence microscopy, the specific PpIX fluorescence can be used to differentiate the tumor from healthy brain tissue. To investigate possible chances and limitations of fluorescence diagnosis, the complementary use of molecular and elemental mass spectrometry imaging (MSI) is presented. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is used to examine the distribution of PpIX and heme b in human brain tumors. MALDI-MS/MS imaging is performed to validate MS data and improve the signal-to-noise ratio (S/N). Comparing the imaging results with histological evaluation, an increased PpIX accumulation in areas of high tumor cell density is observed. Heme b and enhanced Fe accumulation is only found in areas of blood vessels and hemorrhage confirming the hampered transformation from PpIX to heme b in glioblastoma tissue. Investigation of non-neoplastic brain tissue and glioblastoma resected without external 5-ALA administration as control samples with true negative fluorescence verified the absence of PpIX accumulation. Analysis of necrotic tumor tissue and gliosarcoma, one rare type of glioma appearing non-fluorescent during FGS, as case examples with false negative fluorescence diagnosis revealed the absence of significant amounts of PpIX, indicating an impairment of PpIX formation. Molecular analysis is complemented by quantitative laser ablation-inductively coupled plasma (LAICP) MSI correlating heme b and Fe distribution. Mathematical pixel-by-pixel correlation of molecular and elemental data revealed a positive correlation with heteroscedasticity for the spatially resolved heme b signal intensities and Fe concentrations.

Glioblastoma, dedicated by the classification of the world health organization (WHO) as grade IV astrocytoma, is the most common and aggressive malignant brain tumor.1 From years 2008-2012, 46.1% of all malignant primary brain and central nervous system tumors were histologically accounted as glioblastoma in the US.2 Due to rapid proliferation, the highly infiltrating growth and the invasive potential, the prognoses for patients with a glioblastoma are poor, with a survival rate of 6.3% after five years.2–4 An extensive resection of malignant glioma cells can significantly improve the

survival rate of the patients.5 Neuronavigational guidance by magnetic resonance imaging (MRI) or positron emission tomography (PET) is routinely used for the localization of tumor tissue. However, the accuracy of resection can be influenced by brain shift and tissue deformation.6,7 The use of intraoperative imaging can help to overcome these limitations, but is expensive and increasing the surgery time.8,9 Therefore, contrast enhancement by a fluorescent dye has evolved to an important technique in brain tumor resection.10,11 1

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In 1998, Stummer et al. introduced fluorescence-guided surgery (FGS) after treatment with the proagent 5aminolevulinic acid (5-ALA).12 Approximately three hours before anesthesia, 5-ALA is orally administered to the patients with a dose of 20 mg/kg body weight.13 This small molecule can cross the abnormal blood-brain barrier into peritumoral tissue and accumulate there.14,15 5-ALA is a natural biochemical precursor of heme b, which can be found in oxygen transport proteins such as hemoglobin.16 An intermediate step in heme-porphyrin biosynthesis pathway is the formation of protoporphyrin IX, which is red fluorescent at 635 nm upon blue light excitation.17 High grade gliomas (HGG), characterized by increased cell proliferation, tumor cell density and microvessel density, usually accumulate high cellular PpIX concentrations resulting in greater fluorescence intensities.18–20 According to Colditz et al., the PpIX accumulation may be caused by a lower heme requirement as tumor cells preferentially gain energy by glycolysis rather than the heme regulated cytochrome oxidative phosphorylation pathway.21 Compared to normal brain tissue, the activity of the enzyme ferrochelatase, incorporating Fe into the porphyrin structure, was found to be downregulated in tumor tissue resulting in a hampered transformation from PpIX to heme b.22 Although FGS using 5-ALA has proven to be a powerful technique in brain tumor resection, false-positive and falsenegative fluorescence cannot be excluded.15 False positive diagnosis can be caused by inflammatory cells, reactive astrocytes or auto-fluorescence of normal brain tissue.11,15,23 The correlation of intense fluorescence to high grade tumor regions is indeed quite reliable. No or weak fluorescence, however, does not indicate the absence of tumor tissue.19,24,25 False negative classification can be attributed to infiltrative regions with low tumor cell density, necrotic portions of tumor tissue, photobleaching, or inadequate timing of 5-ALA administration.12,15 Furthermore, the accuracy of FGS can be limited by the heterogeneity of fluorescence quality observed for different tumor entities.26 To examine these possible limitations and extend the knowledge about biochemical processes related to FGS, the development of adequate analysis methods is of great interest. However, conventional analysis by fluorescence microscopy is limited to the examination of PpIX distribution and photobleaching cannot be excluded. In this study, the complementary use of molecular and elemental mass spectrometry imaging (MSI) is presented as a powerful tool for the investigation of human brain tumors resected by 5ALA based FGS. In the field of molecular MSI, matrixassisted laser desorption/ionization (MALDI)-MS has established as the most frequently used technique allowing the analysis of intact molecules by soft ionization. Recently, Brokinkel et al. presented the complementary use of

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fluorescence microscopy and MALDI-MSI to investigate the applicability of FGS for meningioma.27 For elemental mapping with low limits of detection and the possibility to obtain quantitative data, coupling laser ablation with inductively coupled plasma (LA-ICP)-MS has evolved to an important imaging technique. In a previous in vitro and in vivo mice study by Cho et al., quantitative bioimaging revealed an increasing intracellular Fe concentration 24 h and 48 h after 5ALA administration due to the promotion of heme b biosynthesis.28 A main disadvantage of this destructive technique, however, is the loss of species-specific information. Although the combination of complementary imaging techniques holds a high potential to better address complex questions in biological, pharmaceutical and medical sciences, surprisingly few studies with a multimodal imaging approach have been published yet. Bianga et al. presented the complementary use of MALDI-MSI and LA-ICP-MSI to investigate the penetration and distribution of the Pt-based cytostatics cisplatin and oxaliplatin in human tumor samples.29 However, only for oxaliplatin a drug specific fragment could be detected by MALDI-MS. In a study by Niehoff et al., LAICP-MS and MALDI-MS were combined to monitor and quantify the uptake of arsenic-containing hydrocarbons in the model organism Drosophila melanogaster.30 The use of complementary imaging methods could reveal the quantitative accumulation and identification of the intact arsenolipid in the brain. Recently, Gonzalez de Vega et al. published a multimodal imaging study on the examination of matrix metalloproteinases and the cofactor zinc in breast cancer.31 Here, complementary bioimaging of human brain tumors is performed using MALDI-MSI to investigate the distribution of the fluorescent drug PpIX and the biosynthesis end product heme b, and LA-ICP-MS for quantitative Fe analysis. In addition, MALDI-MS/MS imaging is applied to validate MS data for PpIX and heme b and to improve signal-to-noise ratio (S/N).

MATERIAL AND METHODS Fluorescence-guided surgery of brain tumors. Three to four hours before induction of anesthesia, patients were given oral doses (20 mg/kg body weight) of 5-ALA dissolved in water. Tumor tissue was resected and instantaneously frozen in liquid nitrogen. Data of three glioblastoma after and two glioblastoma without 5-ALA administration, one gliosarcoma, one low grade glioma and two reactive brains are presented. Preparation of tumor sections. Tumor/brain tissue was banked in accordance with research ethics board approval (1IIIPau). Informed consent was obtained from all subjects. For preparation of 10 µm cryosections, tumor tissue was embedded in Tissue TekTM O.T.C.. Consecutive sections 2


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were prepared for complementary bioimaging (for LA-ICPMS on normal glass slides, for MALDI-MS on indium tin oxide (ITO) coated glass slides (70-100 Ω/sq, Sigma Aldrich, Steinheim, Germany)) and histological evaluation (hematoxylin and eosin staining, H&E; glia fibrillary acidic protein, GFAP; cluser of differentiation 31, CD31). Tissue sections for MSI analysis were stored at -80 °C until use. Matrix preparation. For imaging of the tumor sections by MALDI-MS, α-cyano-4-hydroxycinnamic acid (SigmaAldrich, Steinheim, Germany) was used as matrix and prepared by a sublimation/recrystallization protocol as follows. Matrix was sublimed for 20 min at 250 °C and 5∙10-2 Pa using a matrix deposition instrument (iMLayer, Shimadzu, Duisburg, Germany). Subsequently, the matrix was allowed to recrystallize as adopted from Yang et al.32 at 75 °C for 2.5 min in a saturated atmosphere of H2O:methanol (1000:5, v/v). Preparation of HEC standards. For matrix-matched standards, 150 mg 2-hydroxyethyl cellulose (HEC, viscosity average molecular weight Mv ~ 90,000 Da, Sigma-Aldrich, Steinheim, Germany) was spiked with 850 µL Fe solution (ICP standard in 2% HNO3, Sigma-Aldrich, Steinheim, Germany) of different concentrations varying between 0.5-200 mg/L and homogenized at 80 °C. Cryosections of 10 µm thickness were prepared. Determination of the total Fe concentration of the respective standards was performed by dissolving 50 mg of HEC standard in 2% nitric acid (Suprapur, Merck, Darmstadt, Germany). Samples were diluted to final concentrations ranging from 0.1-2.0 µg/L. As internal standard, Rh solution with a final concentration of 1 µg/L was added. As calibration solution, six standards were prepared with Fe concentrations in the range of 0.01-5 µg/L and with 1 µg/L Rh. The Fe concentration of the digested HEC standards was determined using a quadrupole-based iCAP Qc ICP-MS (Thermo Fisher Scientific, Bremen, Germany) equipped with a PFA MicroFlow nebulizer (Elemental Scientific, Omaha, NE), a cyclonic spray chamber (Thermo Fisher Scientific) and a SC-4-S autosampler (Elemental Scientific). A quartz injector pipe with an inner diameter of 3.5 mm was used. To minimize interferences, analysis was performed in kinetic energy discrimination mode (KED) with 4.2 mL/min He as collision gas. The ICP-MS interface was equipped with Pt sampler and Pt skimmer. The analyses were carried out using the following conditions: rf power, 1550 W; cool gas flow, 14 L/min; auxiliary gas flow, 0.8 L/min; and nebulizer gas flow, 1.1 L/min. The isotopes 54Fe, 56Fe and 103 Rh were monitored with a dwell time of 0.1 s each. Analysis by MALDI-MS. For dried droplet experiments, standard solutions of 5-ALA (Acros Organics, Geel, Belgium), PpIX (ABCR GmbH, Karlruhe, Germany) and hemoglobin

(Sigma Aldrich Chemie GmbH, Steinheim, Germany) were used. MALDI-MS analysis was performed on an instrument with ion trap-time of flight mass analyzer (iMScope Trio, Shimadzu, Duisburg, Germany) providing a mass resolution of 10,000 and mass accuracy better than 5 ppm (calculated with external calibration). The MALDI source was working at atmospheric pressure and was equipped with a 1000 Hz frequently-tripled 355 nm Nd:YAG laser. For imaging analysis, a focal laser spot of 20-25 µm with a laser energy of 0.7 µJ, 300 laser shots per pixel (accumulation of 3*100 shots) and a step size (pixel to pixel distance) of 50 µm were used. Four successive positive ion mode measurements were performed with following MS parameters: (1) MS mode with a mass range of m/z 120-170, (2) MS mode with a mass range of m/z 550-620, (3) MS/MS mode with a mass selection window of m/z 563.265 ± 2.5 and a mass range of m/z 400-620 and (4) MS/MS mode with a mass selection window of m/z 616.177 ± 1.5 and a mass range of m/z 400-620. For MS/MS experiments, isolation time and collision-induced dissociation time were set to 0.02 s and 0.03 s, respectively, using Ar as collision gas. Data processing and image generation were performed using Imaging MS Solution (Ver. 1.12, Shimadzu). Analysis by LA-ICP-MS. For the analysis by laser ablation-inductively coupled plasma-mass spectrometry (LAICP-MS), a laser ablation system (LSX 213, CETAC Technologies, Omaha, USA) with a 213 nm Nd:YAG laser was used. The LA was equipped with a low volume custombuilt cell (V ~ 7.5 cm3) providing a laminar gas flow.33 A quadrupole-based mass spectrometer (iCAP Qc, Thermo Fisher Scientific) equipped with a PFA MicroFlow nebulizer (Elemental Scientific) and a cyclonic spray chamber (Thermo Fisher Scientific) was used. The laser ablation parameters were optimized regarding spot size, laser energy, scanning speed and carrier gas flow based on best signal to noise ratio in combination with high scan speed. Cryosections were ablated line by line (0 µm gap) with laser energy of 10 J/cm2, 20 Hz laser shot frequency, 50 µm spot diameter and 100 µm/s scan speed. The generated aerosol was transported to the ICPMS system using a gas mixture of 0.8 L/min helium passing the ablation cell and 0.4 L/min argon added after the ablation cell. A Rh solution (0.3 µg/L) was introduced simultaneously to monitor the sensitivity of the instrumental setup. In order to minimize possible interferences, LA-ICP-MS analysis was performed in KED mode using 4.2 mL/min He as collision gas. All LA-ICP-MS measurements were carried out using the following conditions: nickel sampler, nickel skimmer with insert, rf power, 1550 W; cool gas flow, 14 L/min; auxiliary gas flow, 0.8 L/min; and nebulizer gas flow, 0.5 L/min. The isotopes 54Fe (dwell time: 0.2 s), 56Fe (0.2 s) and 103Rh (0.1 s) were monitored. 56Fe was used for data visualization, whereas 54 Fe served for validation puposes and resulted in identical distribution maps and concentrations. Using this method, 3



limits of detection and limits of quantification (LOD, LOQ) according to the 3- and 10-σ criteria of 1.1 and 3.5 µg/g Fe were achieved, respectively. Data evaluation was performed using a homemade imaging software (MassImager written by R. Schmid). Analysis by complementary imaging. For the mathematical correlation of the Fe and heme b distribution, LA-ICP-MS and MALDI-MS were performed on the same tissue section. The cryosection of the glioblastoma, mounted on an ITO slide, was ablated linewise by the laser ablation system using the same conditions as described above, except the gap between the lines which was set to 25 µm. Additionally, the dwell times were adapted to 0.4 s for both Fe isotopes and 0.2 s for 103Rh. After LA-ICP-MS analysis, CHCA sublimation and recrystallization were conducted analogous to the previously described sample preparation procedure. For MALDI-MSI on the remaining 25 µm wide tissue lines, MS and MS/MS mode experiments were pixelwise alternately conducted analogous to area 2 and 4 of the four pixel arrangement. For the mathematical correlation, the Fe concentrations and heme b signal intensities of nine 50 x 50 µm pixels were averaged to 150 x 150 µm pixels. Data processing and visualization were carried out by Microsoft Office Excel (Microsoft Corporation, Redmond, WA, USA), Origin (Originlab Corporation, Northampton, MA, USA) and the homemade imaging software MassImager (written by R. Schmid).

RESULTS AND DISCUSSION Method development for MALDI-MSI and LA-ICPMSI. To obtain complementary molecular and elemental information on the brain tumors, a MALDI-MSI method including tandem MS and a LA-ICP-MSI method with quantification based on matrix-matched standards were developed. To optimize sensitivity for MALDI-MSI analysis, dried droplet experiments with different matrices (α-cyano-4hydroxycinnamic acid, CHCA; dihydroxybenzoic acid, DHB; sinapinic acid, SA; 2,4,6-trihydroxyacetophenone, THAP) were performed for 5-ALA, PpIX and heme b resulting in best signal intensities for CHCA. 5-ALA was detected as [M+H]+ at m/z 132.066. Fragmentation for this small molecule was, however, not efficient and resulted only in unselective fragment ions (loss of water, data not shown). PpIX was mainly detected as [M+H]+ at m/z 563.265, as well as M+. In MS/MS experiments, fragment ions with a loss of -59 Da, 73 Da and -15 Da were obtained, as presented in Fig. S-1a. Fragmentation conditions were optimized to high signal intensities of the fragment ion with m/z 504.252, which was used for image generation in the following. In case of heme b,

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generation of fragment ions with a loss of 59 Da from m/z 616.177 to m/z 557.163 was observed in MS mode (Fig. S-1b). Both, precursor and fragment ion, were detected as M+. Fragment ion generation in MS mode could not completely be prevented using optimized laser energy. MS/MS experiments also resulted in highest fragment ion signal for m/z 557.163. For the MALDI imaging experiments, a hybridization of an ion trap and a TOF analyzer was used offering the possibility of fragmentation experiments while providing MS detection with a high mass resolution. The capacity and thus the linear range of an ion trap is, however, limited and therefore the ion loading of the trap has to be regulated to reduce space charge effects. In order to limit the number of ions entering the ion trap and thus improve the linear range and the limit of detection, small mass ranges were selected for MALDI-MSI analysis. Therefore, MS mode analysis of 5-ALA and PpIX/heme b was performed in two measurements with a mass range of m/z 120-170 (5-ALA) and m/z 550-620 (PpIX/heme b). Furthermore, MS/MS experiments of PpIX and heme b were carried out following MS experiments. In order to accomplish these four different measurement modes on the same tissue section, a focal laser spot size of 25 µm and a step size of 50 µm were used. A scheme of the four subsequent measurements performed per pixel is shown in Fig. S-1c. This pattern comprising four measurements in one pixel reduces the lateral resolution of the single m/z images from the focal laser spot size of 25 µm to the pattern size of 50 µm, but is still providing sufficient spatial information to distinguish between the relevant morphological structures of the tumor samples. As further extraction-based experiments revealed the presence of 4-hydroxyproline (m/z 132.066) and creatine (m/z 132.077) within the biological tissue (data not shown), interfering with the MS detection of 5-ALA, distribution maps of m/z 132.066 do not exclusively represent the allocation of 5-ALA and are therefore not presented in the following. While quantification possibilities in MALDI-MSI are restricted by strong matrix effects, the complete sample atomization in the plasma allows for reliable quantitative data in LA-ICP-MSI. As the transport and ionization efficiency of the ablation aerosol are, however, dependent on aerosol characteristics like particle size and distribution, suitable standard materials have to be used for quantification by external calibration.33 Standard preparation based on homogenized tumor tissue, to obtain best matrix-matching, provides high naïve Fe concentration and is thus not an appropriate matrix for external calibration. Here, standards based on HEC, which provide a naïve Fe concentration of ~1 µg/g were used. Therefore, extrapolation to lower concentrations is not necessary. In order to obtain quantitative 4


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data, which are not impaired by the material loss during MALDI-MS analysis, consecutive sections were used for LAICP-MSI. Imaging of glioblastomas with true positive fluorescence. To test the capabilities of the method, tissue, which showed fluorescence during surgery, was selected for imaging analysis. Histological staining by H&E and GFAP as established tumor marker revealed glioma tissue with classification as grade IV (glioblastoma). The complementary imaging results for two case examples with true positive fluorescence diagnosis are shown in Fig. 1 and Fig. S-2. Fig. 1 presents the imaging analysis of a glioblastoma with a bigger hemorrhage and a large number of blood vessels. Areas of high blood content are clearly visible in the microscopic images, either by the red-brown color in the bright field images of the analyzed sections (Fig. 1a and 1f) or in the H&E stained tissue section (Fig. 1h). PpIX showed a heterogeneous distribution within the tumor tissue with low signal intensities in areas of the hemorrhage (m/z 563.265; Fig. 1b). Especially

in the right part of the tumor tissue, heterogeneous PpIX allocation with highest signal intensities at the outer part of the section was found. Comparing the PpIX distribution with the GFAP staining, a good correlation can be observed indicating an enhanced transformation of 5-ALA to PpIX in areas of higher tumor density. MS/MS mode (m/z 563.265 m/z 504.252; Fig. 1d) measurements resulted in comparable PpIX distribution validating MS data and giving enhanced evidence on the structural identification of the analyte of interest. The allocation of heme b obtained in MS (m/z 616.177; Fig. 1c) and MS/MS mode (m/z 616.177 m/z 557.163; Fig. 1e) was in good accordance with the location of the hemorrhage and the bigger blood vessels. These imaging results fit well to the expectations as heme b is part of hemoglobin which can be found in the blood. In addition, CD31 staining reflecting the vessel density was performed (see Fig. 1j). In the tumor tissue itself, no heme b was detected. These observations are in line with literature, as the transformation of PpIX to heme b is hampered in glioblastoma tissue due to a downregulated

5



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Figure 1. Imaging of glioblastoma with true positive fluorescence during FGS. Microscopic image (a) with the corresponding MALDI-MS and MALDI-MS/MS images of PpIX (b,d) and heme b (c,e). Microscopic image (f) with the corresponding LA-ICP-MS image of the 56Fe distribution (g). The area for averaging is marked in yellos. Histological evaluation by H&E (h), GFAP (i) and CD31 (j) staining.

activity of the involved enzyme ferrochelatase.22 Fe hotspots correlated well with heme b accumulation and areas of high blood content (Fig. 1g). Mean Fe concentration of 34 µg/g was found within the tumor excluding the hemorrhage area as marked in yellow. As another example for true positive fluorescence during FGS, imaging analysis of fluorescent glioblastoma tissue is shown in Fig. S-2. In case of PpIX distribution (m/z 563.265; Fig. S-2b), high signal intensities were found in the major part of the resected glioblastoma tissue. In the area with lower tumor cell density (upper left),

PpIX was not detected. MS/MS experiments on PpIX resulted in a comparable distribution pattern (m/z 563.265 m/z 504.252; Fig. S-2d). Images of heme b obtained in MS mode (m/z 616.177; Fig. S-2c) and MS/MS mode (m/z 616.177 m/z 557.163; Fig. S-2e) were again in line with the location of blood vessels, as well as with Fe hot spots in the LA-ICP-MS image (Fig. S-2g). Imaging of control samples with true negative fluorescence. As control sample, non-tumorous tissue from a glioblastoma patient who orally received 5-ALA was 6


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analyzed. As expected, the reactive brain tissue appeared nonfluorescent during FGS. MALDI-MS/MS analysis did not reveal the accumulation of PpIX within the non-tumorous tissue (m/z 563.265 m/z 504.252; Fig. S-3b) verifying the principle of FGS based on the selective PpIX accumulation within tumor tissue. The results are in good accordance with a study by Johansson et al. reporting undetectable PpIX concentrations within non-neoplastic tissue.34 Additionally, the absence of endogenous PpIX accumulation within nonneoplastic tissue was verified by the investigation of reactive brain tissue from an epilepsy patient (see Fig. S-3h). As another example, for which no fluorescence was expected, glioblastomas resected without FGS and thus without external administration of 5-ALA to the patient were investigated. The respective MALDI-MS(/MS) and LA-ICPMS images are presented in Fig. S-4. PpIX was not detected within the tumor tissue, neither in MS nor in MS/MS mode (Fig. S-4b and S-4h). The absent PpIX detection validates the necessity of an external 5-ALA administration in excess. Quantitative LA-ICP-MSI analysis resulted in mean Fe concentrations in the range of 30 µg/g (28 µg/g in Fig. S-4f and 32 µg/g in Fig. S-4l) within the tumor tissue. This concentration range is comparable to glioblastomas resected after oral 5-ALA administration (34 µg/g (Fig. 1g) and 30 µg/g (Fig. S-2g), respectively). With a xenograft mouse model, Cho et al. could demonstrate increased intracellular concentrations of heme b and Fe 24 hours after 5-ALA administration.28 The time frame from 5-ALA treatment of the patient to resection, however, seems to be too short to observe this enhanced formation of heme b within the investigated human glioblastomas. These results go along with the consideration that the resection is timed with maximum PpIX accumulation within the tumor tissue, which was found to be 6 hours after 5-ALA exposure in animal studies.18 Imaging of tumor tissue with false negative fluorescence. The reliability of FGS can be limited by the occurrence of false fluorescence diagnosis. The attribution of fluorescence to tumorous tissue is indeed comparably reliable. In contrast to that, false negative fluorescence occurs more often during surgery. Necrotic parts of glioblastoma tissue are known to result in the absence of PpIX fluorescence.12,34 Therefore, a glioblastoma with major areas of necrotic tissue was analyzed. Necrotic areas in the tumor tissue are clearly visible, as they appear darker in H&E stained (Fig. S-5d) and bright field microscopic images (Fig. S-5a and g). Low intensities were obtained in the case of heme b and PpIX. Both analytes were only detected in MS/MS mode with improved S/N. The PpIX image (m/z 563.265 m/z 504.252; Fig. S-5b) shows an inhomogeneous distribution with higher signal intensities in the infiltrating area between necrotic and active tissue. Higher Fe concentrations can be observed in the active tissue

(Fig. S-5h) and distribution correlates (m/z 616.177 m/z 557.163; Fig. S-5c).

with

heme

b

In addition to the analysis of glioblastoma, a gliosarcoma, one rare type of glioma, which is also dedicated as grade IV by the WHO, was analyzed by complementary bioimaging (Fig. S-5, bottom part). This tumor entity was reported to show variable fluorescence using FGS. Hebeda et al. investigated 5-ALA-induced fluorescence quality in two rat brain tumor models resulting in high fluorescence levels with homogeneous distribution versus heterogeneous fluorescence with non-fluorescent parts for different gliosarcoma cell lines.35 MALDI-MS analysis of a human gliosarcoma appearing non-fluorescent during FGS showed no PpIX detection in the whole tumor tissue, neither in MS nor in MS/MS mode (m/z 563.265 m/z 504.252; Fig. S-5j), which is in accordance with the absent intraoperative fluorescence. Higher Fe concentrations (Fig. S-5p) and heme b accumulation (m/z 616.177 m/z 557.163, Fig. S-5k) were detected in areas of blood vessels. The results indicate that the formation of PpIX is impaired in gliosarcoma tissue. An explanation for this observation based on biochemical processes of gliosarcoma in comparison to glioblastoma can presently not be given. Moreover, the imaging method was used for the investigation of low grade glioma (LGG), for which the application of 5-ALA induced PpIX fluorescence has shown a poor diagnostic accuracy. For the vast majority of LGGs, PpIX fluorescence is invisible to intraoperative fluorescence microscopes.36,37 MALDI-MS(/MS) analysis of the LGG tissue investigated in this study indicates comparable observations. The PpIX amount in the tissue is below the detection threshold of the used imaging method (see Fig. S-6). Correlation of molecular and elemental MSI data. For the optical correlation of MALDI-MSI and LA-ICP-MSI data, the use of parallel sections can be advantageous as in this way, the quantification and the lateral resolution of the individual analysis is not affected. However, mathematical correlation requires the data acquisition on one tissue section to prevent restrictions by morphological tissue differences of parallel sections. When MALDI-MSI was conducted first followed by LA-ICP-MSI, the application and removal of the matrix as well as the tissue consumption by MALDI-MSI affected the quantification by LA-ICP-MS. Therefore, in this study quantitative LA-ICP-MS with a laser spot diameter of 25 µm was performed first using a gap of the same size between the ablated lines. Subsequent MALDI-MSI experiments were conducted on the remaining tissue lines with alternating measurments in MS and MS/MS mode. The applied acquisition pattern, schematically presented in Fig. 2d, results in a lateral resolution of 50 µm for the individual molecular and elemental images. The quantitative Fe distribution 7



(Fig. 2b) and the heme b distribution (Fig. 2c) were compared in a pixel-by-pixel overlay visualized in Fig. 2e and 2f. For mathematical correlation, the spatially resolved information of 9 pixels (3 x 3 pixels) was averaged in a bigger pixel with dimensions of 150 x 150 µm to account for errors due to the

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different location of LA-ICP-MS and MALDI-MS acquisition. The scatter plot (see Fig. 2g) clearly depicts the postitive correlation of the signal intensitities of heme b and the Fe concentrations. Obviously, the variance is not constant, but shows an increasing

Figure 2. Complementary imaging of a glioblastoma by LA-ICP-MS and MALDI-MS(/MS) on one tissue section. Microscopic image (a) with the corresponding Fe (b) and the heme b (c) distribution. Scheme of the acquisition pattern comprising laser ablation and subsequent MALDI analysis with a focal laser spot size of 25 µm for both experiments. Overlay of the Fe and heme b distribution for the whole sample (e) and an enlagerd area (f). Scatter plot (g) for the spatially resolved correlation of the signal intensities of heme b and the Fe concentrations.

trend which is mathematically denoted as heteroscedastic. The presence of heteroscedasticity was verified by a residual plot in which the residuals (error terms) are plotted againt the independent variable. The residual plot in Fig. S-7a clearly visualizes the increasing variance in dependency of the Fe concentration. Additionally, the histogram of the residuals is presented in Fig. S-7b, showing a bell-shaped distribution of the deviation around zero. The variance is not completely normally distributed and shows a slight shift towards negative residuals. In conclusion, the used acquisition method comprising quantitative LA-ICP-MSI and subsequent MALDI-MSI on the same tissue section allows for the spatially resolved mathematical correlation of Fe and heme b distribution revealing a proportional relationship with heteroscedasticity. This good correlation is quite striking taking into account that the acquisition is not performed on the exactly same position, that other Fe containing species are also present within the tissue and that the sensitivity of the used techniques is different.

CONCLUSIONS In this study, the complementary use of molecular and elemental imaging has been presented for the spatially resolved analysis of human brain tumors resected by FGS. MALDI-MSI was used to investigate the distribution of the fluorescent metabolic intermediate PpIX and the Fe containing porphyrin heme b. Complementing molecular imaging by quantitative LA-ICP-MS analysis, the correlation of heme b and Fe distribution was examined. In conclusion, MALDIMS(/MS) imaging enabled the detection of PpIX distribution in glioblastomas which showed fluorescence during surgery. Areas of intense PpIX signal could be correlated to high tumor density. Heme b and Fe accumulation were in good accordance with the location of blood vessels and areas of hemorrhage indicating no significant transformation of PpIX to heme b within the tumor tissue on the time scale from administration to resection. In necrotic tissue as an example for the occurrence of false negative fluorescence, no PpIX was detected, correlating well with the absence of fluorescence during FGS. Analysis of a gliosarcoma, which is a HGG but 8


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appearing non-fluorescent during FGS, revealed the absence of PpIX accumulation. Additionally, the spatially resolved signal intensities of heme b and Fe concentrations were mathematically correlated. By conducting quantitative LAICP-MSI and subsequent MALDI-MSI on the same tissue section, the proportional relationship of Fe and heme b could be demonstrated. Finally, the correlation of postoperative MSI and intraoperative fluorescence microscopy can allow for additional conclusions about possibilities and limitations of FGS. This can help to better understand the opportunities of FGS for different tumor entities and evaluate the capabilities of advanced fluorescence microscopes during surgery. MALDI-MSI analysis of PpIX can indicate if the application of more sensitive intraoperative instrumentation for FGS, as already presented in literature38–41, is promising to overcome false negative fluorescence diagnosis with conventional fluorescence microscopes. Indeed, as MSI analysis is invasive and can be quite time-consuming, its application is a great opportunity for research purposes on FGS rather than an intraoperative tool to gain additional information during brain tumor resection.

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. MALDI-MS(/MS) spectra of PpIX and heme b. Scheme of the acquisition pattern for MALDI-MS(/MS) experiments. Imaging results for true positive, true negative and false negative fluorescence during FGS. Residuals plot and histogram for the correlation of Fe and heme b distribution.

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AUTHOR INFORMATION

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Corresponding Author

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*E-mail: [email protected]. Phone: +49 251 83-33141. (23)

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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ACKNOWLEDGMENT Parts of this study were supported by the Cells in Motion Cluster of Excellence (CiM - EXC 1003), Münster, Germany.

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Complementary Molecular and Elemental Mass-Spectrometric

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