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mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Monitoring therapeutic monoclonal antibodies in brain tumor a
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Rima Ait-Belkacem , Caroline Berenguer , Claude Villard , L’Houcine Ouafik , Dominique ac
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Figarella-Branger , Alain Beck , Olivier Chinot
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& Daniel Lafitte
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Aix-Marseille Université Inserm; CRO2 UMR S-911; Marseille, France
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APHM; CHU Nord; Service de Transfert d’Oncologie Biologique; Marseille, France
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APHM; Timone Hospital; Department of Neurosurgery; Marseille, France
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Centre d’Immunologie Pierre Fabre (CIPF); St Julien-en-Genevois, France Accepted author version posted online: 01 Nov 2014.Published online: 15 Dec 2014.
Click for updates To cite this article: Rima Ait-Belkacem, Caroline Berenguer, Claude Villard, L’Houcine Ouafik, Dominique Figarella-Branger, Alain Beck, Olivier Chinot & Daniel Lafitte (2014) Monitoring therapeutic monoclonal antibodies in brain tumor, mAbs, 6:6, 1385-1393, DOI: 10.4161/mabs.34405 To link to this article: http://dx.doi.org/10.4161/mabs.34405
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SHORT COMMUNICATION mAbs 6:6, 1385--1393; November/December 2014; © 2014 Taylor & Francis Group, LLC
Monitoring therapeutic monoclonal antibodies in brain tumor Rima Ait-Belkacem1, Caroline Berenguer1, Claude Villard1, L’Houcine Ouafik2, Dominique Figarella-Branger1,3, Alain Beck4, Olivier Chinot1,3, and Daniel Lafitte1,* 1
Aix-Marseille Universit e Inserm; CRO2 UMR S-911; Marseille, France; 2APHM; CHU Nord; Service de Transfert d’Oncologie Biologique; Marseille, France; 3APHM; Timone Hospital; Department of Neurosurgery; Marseille, France; 4Centre d’Immunologie Pierre Fabre (CIPF); St Julien-en-Genevois, France
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Keywords: bevacizumab, glioblastoma multiforme, MALDI imaging mass spectrometry, monoclonal antibodies, palivizumab, top down in source decay Abbreviations: BBB, blood-brain barrier; CRC, metastatic colorectal cancer; CSF, cerebrospinal fluid; 1; 5 DAN, 1; 5-diaminonaphtalene; EMA, European Medicines Agency; FDA, Food and Drug Administration; GBM, glioblastoma multiforme; IMS, imaging mass spectrometry; ISD, in-source decay; ITO, indium tin oxide; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; mAbs, monoclonal antibodies; MALDI, matrix-assisted laser desorption/ionization; NSCLC, non-small cell lung cancer; pE, pyroglutamate; RMS, root mean square; RP-HPLC, reversed phase high-performance liquid chromatography; TOF, time of flight; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VH, variable domain of the heavy chain; VL, variable domain of the light chain; WHO, world health organization
Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.
Targeted therapies rely on the use of monoclonal antibodies (mAbs) or anti-angiogenesis drugs. These therapies have been designed to overcome some conventional chemotherapy side effects or pitfalls, such as toxicity, resistance, local variations in blood flow or non-effective delivery to the target.1Hybridomas were first described in 1975, and more than 15 y later mAbs obtained clinical validation in the mid-1990s.2 There are currently more than 50 mAbs and related products approved by the Food and Drug Administration (FDA) or the European Medicines Agency (EMA) for therapeutic use.3 In addition, over 30 more are being investigated in late stage clinical trials.4 Two main categories of therapeutic antibodies are in development: antibodies targeting cell markers and antibodies targeting *Correspondence to: Daniel Lafitte; Email: daniel.lafi[email protected] Submitted: 06/18/2014; Revised: 08/04/2014; Accepted: 08/09/2014 http://dx.doi.org/10.4161/mabs.34405
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cytokines. One of the most targeted cytokine is the vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis in normal and disease states. It can be directly secreted by cancer, endothelial, stromal, or blood cells and can be a component of the extracellular matrix surrounding these cellular components.5 VEGF and its receptors, VEGFRs, are the target of many anti-angiogenic therapies that try to prevent angiogenesis and vascular leakage, cell proliferation, survival and migration.6 Bevacizumab (AvastinÒ ) was the first anti-VEGF therapy approved, in combination with cytotoxic chemotherapy, for the first line treatment of metastatic colorectal cancer (CRC),7,8 recurrent or metastatic non-small cell lung cancer (NSCLC),9 metastatic breast cancer10 and renal cancer.11According to the World Health Organization (WHO) classification 2007, glioblastoma multiforme (GBM) or grade IV astrocytoma is the most invasive type of gliomas associated with high mortality and morbidity. GBM is characterized by pronounced hypercellularity, pleomorphism, numerous mitoses, foci of central necrosis, and excessive vascularization.12 It is widely known that tumor blood vessels are leaky, tortuous and dilated, especially in malignancies such as GBM. Assuming that the anti-angiogenic therapy induces normalization of these abnormal blood vessels, making them less leaky,13bevacizumab is used as a single agent for the treatment of recurrent GBM.14-17 An increasing knowledge of the pharmacokinetics properties (absorption, distribution, metabolism and elimination parameters) is necessary to estimate how effectively the therapeutic agents reach their targets, and to tailored the next generation of optimized antibodies (OptimAbs).18 Up to now, in situ concentration measurements have been almost impossible; plasma levels are easier to obtain, offering a proportional relationship with the unbound drug concentration in tissues.19 In the brain, concentrations in micro dialysates and abscesses are not frequently available for humans,20 so the concentration of therapeutic agents is often
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followed by measuring levels in cerebrospinal fluid (CSF). This approach, however, does not take into account tumor anatomic and metabolic heterogeneity, or other impediments to drugs access. Principally in GBM, the interpretation of antibody levels is hampered by the difficulty of taking local measurements, which depend on awareness of the blood-brain barrier (BBB).21 The molecular distribution of large molecules (antibodies) has primarily been investigated using widespread approaches of radio- or fluorescence-labeled probes like electron microscopy,22 intravital fluorescence videomicroscopy,23 autoradiography,24 micellarelectrokinetic capillary25 and positron emission tomography.26 Imaging mass spectrometry (IMS) provides complementary features that overcome many labeling imaging pitfalls, such as the unspecific reactions, the change of affinity due to labeled probes and the inability to distinguish between the parent compound and metabolites. IMS detects label-free analytes,27-29 measuring the distribution of administered compounds and their metabolites in the same experiment.30-32 However, a mass range limitation prevents the detection of large biomolecules, and, therefore, of therapeutic antibodies. To overcome this problem, two solutions can be envisioned.33,34 The first one is the usual bottom-up approach, where the proteins are extracted and digested by proteases into peptides and then analyzed by LCMS/MS. This procedure can, however, induce protein modifications and delocalization.35 Second one is the top down approach that consists of a direct in situ fragmentation of the protein without digestion. This “top-down” approach has been previously used for extensive characterization of post-translational modifications.36,37 In-source decay (ISD) is the top-down approach occurring during the matrix-assisted laser desorption/ionization (MALDI) steps.38,39 This pseudo MS/MS technique, MALDI ISD, uses hydrogen radical transfer from the matrix to analyte molecules to provide fragmentation and sequencing of protein N and C termini.40 Using the appropriate matrix, MALDI ISD can be associated with MALDI IMS, and therefore provides identification and location of exogenous or endogenous molecules at the same time.41,42 In this work, we developed a novel method coupling top-down in source decay fragmentation with matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI ISD IMS). This new strategy was performed to obtain in situ distribution of bevacizumab on treated glioblastoma-bearing mouse brain tissue sections. The main result expected of bevacizumab treatment is to block the angiogenesis. As noted below, angiogenesis and its major regulator, VEGF, represent one of the most important therapeutic targets in GBM treatment. VEGF receptors (VEGFR1/2) are expressed on endothelial, but few other, cells wherein it stimulates several cell responses, including proliferation, migration, survival, and secretion of matrix-degrading enzymes. Through the VEGFR2 and having the stronger kinase activity, it represents the predominant mediator of VEGFinduced angiogenic signaling.14 This signaling pathway can be inhibited by bevacizumab, which binds to and neutralizes all human VEGF-A isoforms (Fig. 1A). Top-down in source decay, using 1, 5-diaminonaphtalene (DAN) as a matrix, is a radical pathway allowing the identification of the immunoglobulin
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amine and carboxyl terminal parts. ISD generally leads to generation of a long series of c- and zC2-ions after cleavage of N-Ca bonds on the peptide backbone (Fig. 1B). In order to verify the correct NH2- and -COOH terminal portions and to identify the set of virtual precursors that are specific to each mAb, we assessed the fragmentation of bevacizumab and palivizumab, two humanized IgG1 therapeutic mAbs. Palivizumab has a totally different epitope and mechanism of action than bevacizumab, and it was therefore chosen as a negative control. It blocks both cell to cell and virus to cell fusion, and targets viral proteins,43 not angiogenesis cytokines. ISD mass spectra were obtained from a mixture of 1, 5 DAN matrix with one of the two purified antibodies (bevacizumab or palivizumab) directly spotted on MALDI target. The best fragmentation range was obtained between m/z 1000–8000 for palivizumab and m/z 2000–8000 for bevacizumab. A sequence of 30 residues corresponding to the variable domain of the heavy (VH) and light (VL) chains of bevacizumab (Fig. 2A) and palivizumab (Fig. 2B) was obtained. Noteworthy, the VH domain of palivizumab carried a pyroglutamate (pE) modification occurring through the rearrangement of the originally synthesized glutamine residue. It is known that both glutamine and glutamate at the N termini of recombinant mAbs can cyclize spontaneously to pyroglutamate (pE) in vitro, making the antibodies more acidic.44 Also, in vivo cyclization can occur as a stabilization mechanism for proteins not impacting their turnover. For therapeutic mAbs, like palivizumab, pE can be one of many posttranslational modifications observed during production and storage.45 Apparently, this cyclized residue is resistant to amino peptidases.46 RP-HPLC is a chromatographic method that was able to show and to quantify post-translational modifications which were also identified by peptide mapping, mass spectrometry and microsequencing.47 The same in source decay fragmentation studies were performed on healthy brain tissue section to obtain the most representative fragments of the studied mAbs in these particular conditions. Wherever the antibody was spotted on the tissue slice, fragmentation occurred and fragment ions were detected showing that ion suppression effect has no impact on our study. Ion suppression usually occurs when an ion suppresses the signal of another species in the sample.48 Antibody fragmentation was measured on tissue using a lowest quantity of 41.75 pmol. In this case, three ions (c20, c21 and c22) corresponding to N termini fragments of bevacizumab were measured (Fig. 2C). To confirm the identity of these ions, a second sequencing step called T3-sequencing was performed directly on tissue sections (Fig. 2D). The generated precursor ions, including the N- or C-terminal sequence generated by ISD fragmentation, were selected in the timed ion gate of a MALDI TOF/TOF mass spectrometer for MS/MS analysis. This new fragmentation (T3-sequencing) generates principally b- and yions, allowing the proper sequencing of both N- and -C termini, respectively, and confirmation of suspected terminal modifications.39,49 We then achieved analysis on mouse xenografts brains after stereotactic cortical tumor U87 cells injection, which was
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performed as previously described.41 To provide protein identification in addition to their localization directly on brain tissue sections, we performed ISD of the entire tissue slice at 80 mm spatial resolution. Most of the obtained signals were protein fragments. Some of them bear the exact mass as bevacizumab c-ions previously measured using the purified antibody (Fig. 3). T3-sequencing confirmed that they were (c20, c21 and c22) ions (results not shown). Figure 1. Bevacizumab mode of action and fragmentation using top down MALDI. (A) Bevacizumab action on VEGF/ The intensities of these VEGFR signaling pathway. (B) In source decay fragmentation of the immunoglobulin amine and carboxyl terminal three ions were summed parts generates a long series of c- and zC2-ions after cleavage of N-Ca bonds on the peptide backbone. to provide the image of antibodies distribution within the brain slices. Bevacizumab is mainly detected on the tumor area, whereas pali- extravasation, antibody binding and demonstrated a detrivizumab is distributed in all the tissue (Fig. 4). It is very difficult mental effect of antibody internalization.51 Micro distribution to compare palivizumab and bevacizumab quantities as we are studies described the phenomena where antibodies are immonot sure that they fragment exactly with the same efficiency. We bilized close to their site of entry and the factors that influwill have to develop absolute quantitation methods using in ence the location of the moving front, such as diffusivity, source decay. antigen density, permeability, and dose.52 Micro distribution Notably, the image obtained with three ion is quite similar to also analyzed the influence of cellular pharmacology, like the one gathered with the most abundant fragment only (c21bev- binding, internalization, and recycling on the distribution acizumab D m/z 2081), but it is more relevant on a statistical around blood vessels.54 basis. It could be interesting to use both c- and z- ions from proGlioblastoma is one of the most vascularized human tumors.5 tein termini for the identification and imaging, however DAN This abnormal tumor vasculature is upregulated by VEGF, fragmentation, via the radical pathway, rather produce c- frag- which serves as a major angiogenic factor in normal vascular ments from the N- terminus of big proteins than z- fragments development.5,55 Angiogenesis is a critical step during glioblasfrom the C- terminus.50 toma development and growth in which tumor requires more The pharmacokinetic properties of bevacizumab are cur- oxygen and nutrients levels for its survival and proliferation. rently measured in plasma. This drug is associated with a low Anti-VEGF treatment can reduce vascular permeability, but this clearance (CL), a limited volume of distribution (Vc), and a may not necessarily reflect tumor cell death. Thus, the macro dislong elimination half-life (21 d) that maintains target thera- tribution and penetration of the bevacizumab into the tumor peutic bevacizumab plasma levels with a range of administra- mass is in agreement with the tumor characteristics and treatment tion schedules (such as 5–10 mg/kg once every 2 or 3 wk). It knowledge. The purpose of coupling MALDI IMS and top-down in is clearly important to obtain direct in situ measurement for a direct pharmacokinetic view closer to the site of action. source decay analysis is to get localization in addition to the proSuch a measurement within the tumor will account for blood tein identification, at the same time and directly on the same tisflow through the tumor, extravasation, convection and diffu- sue section. In situ molecular identification was performed by sion, binding, and internalization. Qualitative and quantita- MALDI ISD, a top-down approach that allows identification of tive mathematical models were constructed previously to the proteins by their N- and C-termini39,41 fragmentation. predict the antibody diffusion through tumors taking into Although limited to the most abundant species, MALDI ISD consideration some of these parameters, such as the effect of IMS was previously shown to be useful for glioma marker characantibody internalization.51-54 Macro distribution models terization41 and post-translational modifications identification.56 (1 cm diameter tumor) predict the role of elevated interstitial We now strengthen its impact with the close investigation of fluid pressure, tumor convection, spatial variation in therapies in situ.
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Figure 2. MALDI ISD characterization of monoclonal antibodies. MALDI mass spectra obtained between m/z 2000–8000 of bevacizumab fragments (A) and m/z 1000–8000 palivizumab fragments (B). (C) In order to identify the precursor ions of bevacizumab, different quantities of antibody were spotted on tissue (167 pmol lower panel, 125.25 middle panel and 41.75 pmol higher panel). Stars correspond to the three precursor ions c20, c21, c22 that will be used in the course of the study. T3-sequencing spectrum of 2081. 13 c-ion precursor showing generated b-ions mainly (D).
Further improvement of antibody quantitation directly on tissue is a possibility under development, in combination with quantitation with triple quadrupole mass spectrometry. New coming sprayer systems will improve spatial resolution of the MALDI imaging combined with in source decay analysis. Going down to 10 mm spatial resolution or less could be important to distinguish antibody location within the tumor cells and their microenvironment. MALDI imaging mass spectrometry could therefore provide both the macro and the micro distribution view described by the mathematical models, as described above. MALDI imaging mass spectrometry may in this way compensate for many conventional imaging modalities limitations by combining a number of real advantages: sensitivity, label free, speed, and reproducibility. Thus, it could play an important role for cancer imaging combined with other modalities in preclinical and clinical practice.
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Materials and Methods Reagents 1, 5-diaminonaphtalene (DAN) was purchased from SigmaAldrich, Saint-Quentin Fallavier, France. Bevacizumab (AvastinÒ , Roche, Grenzach-Wyhlen, Germany) and palivizumab (SynagisÒ , Abbott, Berkshire, UK) were provided by the Centre d’Immunologie Pierre Fabre. Tissues Harvesting of tissues The U87 cells were obtained from a malignant glioma of a female patient by explant technique. Five-weeks-old nude FOXN1 male mice (25–30 g athymic mice) were purchased from Harlan Laboratories, Gannat, France. Glioblastoma cells were injected stereotaxically using: Leica MS5-MZ6 stereomicroscopic device (Leica Microsystems SAS, France), WPI Stereotaxic
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Figure 3. MALDI ISD IMS workflow. After a stereotactic cortical U87 glioblastoma cells injection, tumor growth and immunotherapy, brains are extracted, flash frozen and tissue sections made. Following histological observations and matrix deposition on the entire brain tissue section, MALDI imaging acquisition of the overall average spectrum and image reconstruction (x and y coordinates), an ion-density map was obtained for each chosen m/z signal present on the average mass spectrum. Most of the obtained signals were different protein fragments and some of them bear the exact mass than bevacizumab and palivizumab c-ions previously measured using the purified antibodies. Three c-ions were found to correspond to c-ions of either bevacizumab or palivizumab antibody. The intensities of these three ions are summed to provide the image of antibodies distribution within the brain
Frame 18 deg with Ear Bars (World Precision Instruments, Germany) and Hamilton Syringe 10 ml, Model 701 SN OnColumn Injection SYR, Cemented NDL, 32 ga, 3.35 in, point style 45 mounted on microinjection unit (Hamilton Bonaduz AG,GR, Switzerland). This system enabled inoculation of 105 glioblastoma cells suspended in 2 ml solution using the manual control of the injection system. The striatum on the right frontal lobe at interaural, lateral and depth coordinates of 0.0, 3.0, and 3.0 mm to the bregma was targeted. Eight days after cells injection, the mice were separated to 4 groups: control mice, bevacizumab treated mice, palivizumab treated mice and bevacizumab added to palivizumab treated mice. The intraperitoneal drug injection was done at 200 mg/mouse three times per week during 1 (for bevacizumab and palivizumab) or 3 wk (only for bevacizumab). Mice were anesthetized by the intraperitoneal injection of 6.5 ml/g of anesthetic solution, composed of 50 ml of 2% Rompun with 200 ml of Imalgene 1000 in a final volume of 1000 ml
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of 0.9% NaCl. Mice were then dissected and the total brain blocks extracted and snap-frozen in liquid nitrogen for 15 s. The brains were kept at ¡80 C until use. The drop weight of mice was monitored and the sacrifice was done one week after the latest treatment. Three mice per group were used for MALDI imaging experiment. The animal manipulations have been performed in accordance with international guidelines. Experimental protocols were reviewed and approved by the Institutional Animal Care Committee of the School of Medicine and performed in accordance with INSERM and Aix Marseille Universite School of Medicine polices regarding the use of laboratory animals. Tissues preparation and staining Mouse brain was placed at ¡18 C for 15 min before use. Tissue sections were cut using a Leica CM 1900 UV Mycrosystems cryostat (Leica Microsystems SAS, France) with a microtome chamber and a specimen holder chilled at ¡18 C. The 12 mm
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Figure 4. Monoclonal antibodies localization on brain tissue sections bearing glioblastoma. The first vertical panel represents the scan of Hematoxylin and Eosin histological images of coronal or horizontal tissue sections from control and bevacizumab and/or palivizumab treated mice brains. The tumor region, of a dark pink color, is surrounded by a black-dashed line. After bevacizumab treatment, we note that tumor region is well-defined and localized, even after 30 d of growth. The ion images of in source decay fragments obtained at 80 mm lateral resolution are represented on the other panels. Thus, the anatomical localization of different protein fragments is shown: myelin basic protein is the most abundant protein principally localized in the hippocampus. Bevacizumab and palivizumab fragments localization is found distributed on the entire tissue section. Bevacizumab is more concentrated on tumor region (15 or 30 d). Scale bar D 2 and 1 mm.
thick coronal and horizontal sections were thaw mounted onto Indium Tin Oxide ITO-coated microscopic slides (Bruker Daltonics, Wissembourg, France) adapted for MALDI imaging mass spectrometry. These MALDI target slides were placed in a desiccator for 1 h. ITO slides were washed with isopropanol (twice at 70% for 2 min and once at 96% for 2 min) for MALDI in source decay imaging MS analyses. Following washing, ITO slides were dried in a desiccator for 30 min before matrix deposition. After MALDI imaging analysis, the used slides were washed with pure ethanol and acetone to remove the matrix before histological staining.57 For Hematoxylin and Eosin staining, the tissue sections were immersed in a filtered Harris Hematoxylin solution for 1 min and rinsed with tap water. Then, the sections were immersed in Eosin solution for 30 s and rinsed with tap water.
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The slices were dehydrated in various alcohol solutions (50%, 70%, 80%, 95%, 100%) and cleared with xylene (2x). They were mounted onto a labeled glass slide with Permount. The pictures of H&E stained histological sections were obtained at specific regions with Olympus BH-2microscope at magnification, £ 40 objective. The nuclei and other basophilic structures were colored in blue while cytoplasm and acidophilic structures were colored light to dark red. Matrix deposition for imaging mass spectrometry analyses Brain tissue sections were always scanned before matrix deposition with a histology slide scanner (Opticlab H850 scanner, Plustek). The scanned histological image was used for the teaching in order to obtain a perfect superposition between the histological and the ion images. For MALDI in source decay imaging
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1, 5-diaminonaphtalene (DAN), 10 mg/ml in 50% Acetonitrile 0.2% TFA, matrix deposition was performed with the automatic matrix sprayer device (ImagePrep, BrukerDaltonics) equipped with the new spray head. The spray method was optimized adding more nebulization and drying cycles to obtain a thick matrix deposit compatible with in source decay analysis.
30–50 mm laser spot diameters). Sequences were created on FlexImaging software, version 3.0 (BrukerDaltonics) after the downloading of scanned images. Polygon measurement regions were manually defined using these histological images. A minimum of two images was acquired to ensure reproducibility. Data Analysis
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Data Acquisition MALDI in source decay MS MALDI mass spectra were obtained on an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). ISD analyses were acquired in a reflectron positive mode with an accelerating potential of 20 keV; laser power was increased up to 20% above the ionization threshold to increase fragmentation without losing window (laser power at 50%). The spectra were internally calibrated by using parent and c10-serie ion fragment of bovine serum albumin (BSA) masses. MALDI in source decay imaging MS MALDI in source decay imaging was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). The MALDI ISD IMS experiments were performed at a spatial resolution of 80 mm. Spectra were acquired in positive reflectron ion mode with 1500 laser shots accumulated at each spot and the laser power was optimized at the start of each run and then fixed for the overall MALDI ISD IMS experiment with a frequency of 500 Hz. The mass spectrometer parameters were set according manufacturer’s settings for optimal acquisition performance. Laser spot size was medium (corresponding approximately to References 1. Marin JJG, Romero MR, Blazquez AG, Herraez E, Keck E, Briz O. Importance and limitations of chemotherapy among the available treatments for gastrointestinal tumours. Anti-cancer agents in medicinal chemistry [Internet] 2009 [cited 2014 May 9]; 9:16284. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/19199863 2. DeVita VT, Chu E. A history of cancer chemotherapy. Cancer research [Internet] 2008 [cited 2014 May 1]; 68:8643-53. Available from: http://www.ncbi.nlm.nih. gov/pubmed/18974103 3. Reichert JM, Beck A, Lugovskoy AA, Wurch T, Coats S, Brezski RJ. 9th annual European Antibody Congress, November 11-13, 2013, Geneva, Switzerland. mAbs [Internet] [cited 2014 Jun 18]; 6:309-26. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24492298 4. Reichert JM. Antibodies to watch in 2014: Mid-year update. mAbs [Internet] 2014 [cited 2014 Jun 18]; 6:799-802. Available from: http://www.ncbi.nlm.nih. gov/pubmed/24846335 5. Cea V, Sala C, Verpelli C. Antiangiogenic therapy for glioma. Journal of signal transduction [Internet] 2012 [cited 2014 May 6]; 2012:483040. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid=3399341&tool=pmcentrez&rendertype=abstract 6. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nature medicine [Internet] 2003 [cited 2014 Apr 29]; 9:669-76. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12778165 7. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S,
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MALDI in source decay imaging for monoclonal antibodies distribution IMS data were acquired and normalized with root mean square (RMS) (vector norm) algorithm that provides a very uniform distribution of intense signals. Raw spectra were analyzed with FlexImaging software version 3.0 after baseline subtraction. Masses were selected with a mass precision of § 0.1 Da on the overall average spectrum. Ion density map was created for each signal present on the whole sample average mass spectrum. For signals corresponding to c-ions fragments of mAbs, the spectra lists of interest were exported in .xml format and then, the intensities of all the fragment ions were summed. Finally, we reimported the new spectra list data in FlexImaging software to obtain the image corresponding to the ion sum. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
We acknowledge canceropole PACA, region PACA, Aix Marseille Universite, INSERM, IBISA, and the SIRIC (Grant INCaDGOS-Inserm 6038) for financial support.
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2014 May 6]; Available from: http://www.ncbi.nlm. nih.gov/pubmed/24376047 Debois D, Bertrand V, Quinton L, De Pauw-Gillet MC, De Pauw E. MALDI-in source decay applied to mass spectrometry imaging: a new tool for protein identification. Analytical chemistry [Internet] 2010 [cited 2013 Nov 20]; 82:4036-45. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20397712 Huang K, Incognito L, Cheng X, Ulbrandt ND, Wu H. Respiratory syncytial virus-neutralizing monoclonal antibodies motavizumab and palivizumab inhibit fusion. Journal of virology [Internet] 2010 [cited 2013 Nov 20]; 84:8132-40. Available from: http://www. pubmedcentral.nih.gov/articlerender.fcgi? artid=2916538&tool=pmcentrez&rendertype=abstract Beck A, Wagner-Rousset E, Ayoub D, Van Dorsselaer A, Sanglier-Cianferani S. Characterization of therapeutic antibodies and related products. Analytical chemistry [Internet] 2013 [cited 2014 Jun 18]; 85:715-36. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 23134362 Liu YD, Goetze AM, Bass RB, Flynn GC. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies. The Journal of biological chemistry [Internet] 2011 [cited 2014 Jun 12]; 286:11211-7. Available from: http://www. pubmedcentral.nih.gov/articlerender.fcgi? artid=3064176&tool=pmcentrez&rendertype=abstract Rink R, Arkema-Meter A, Baudoin I, Post E, Kuipers A, Nelemans SA, Akanbi MHJ, Moll GN. To protect peptide pharmaceuticals against peptidases. Journal of pharmacological and toxicological methods [Internet] [cited 2014 Jun 12]; 61:210-8. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/20176117 Beck A, Bussat M-C, Zorn N, Robillard V, KlinguerHamour C, Chenu S, Goetsch L, Corva€ıa N, Van Dorsselaer A, Haeuw J-F. Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. Journal of chromatography B, Analytical technologies in the biomedical and life sciences [Internet] 2005 [cited 2014 Jun 12]; 819:203-18. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 15833284 Amado FML, Domingues P, Gra¸c a Santana-Marques M, Ferrer-Correia AJ, Tomer KB. Discrimination effects and sensitivity variations in matrix-assisted laser desorption/ionization. Rapid Commun Mass Spectrom 1997; 11:1347-52; http://doi.wiley.com/10.1002/(SICI) 1097-0231(199708)11:123.0. CO;2-8 Suckau D, Resemann A. T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry. Analytical chemistry [Internet] 2003 [cited 2014 Jun 12]; 75:5817-24. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ 14588022 Calligaris D, Villard C, Lafitte D. Advances in topdown proteomics for disease biomarker discovery. Journal of proteomics [Internet] 2011 [cited 2014 Aug 4]; 74:920-34. Available from: http://www.ncbi.nlm.nih. gov/pubmed/21477672 Baxter LT, Jain RK. Transport of fluid and macromolecules in tumors. I. Role of interstitial pressure and convection. Microvascular research [Internet] 1989 [cited 2014 Jun 12]; 37:77-104. Available from: http://www. ncbi.nlm.nih.gov/pubmed/2646512 Fujimori K, Covell DG, Fletcher JE, Weinstein JN. A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. Journal of nuclear medicine : official publication, Society of Nuclear Medicine [Internet] 1990 [cited 2014 Jun 12]; 31:1191-8. Available from: http://www.ncbi.nlm.nih. gov/pubmed/2362198 Graff CP, Wittrup KD. Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention. Cancer research [Internet] 2003 [cited 2014 May 29];
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Insights Oncology [Internet] 2013 [cited 2014 Apr 30]; 7:123-35. Available from: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=36827 34&tool=pmcentrez&rendertype=abstract 56. Soltwisch J, Dreisewerd K. Discrimination of isobaric leucine and isoleucine residues and analysis of posttranslational modifications in peptides by MALDI insource decay mass spectrometry combined with collisional cooling. Analytical chemistry [Internet] 2010
[cited 2014 Jun 12]; 82:5628-35. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20524643 57. Yang J, Caprioli RM. Matrix sublimation/recrystallization for imaging proteins by mass spectrometry at high spatial resolution. Analytical chemistry [Internet] 2011 [cited 2013 Jul 23]; 83:5728-34. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid=3136623&tool=pmcentrez&rendertype=abstract
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63:1288-96. Available from: http://www.ncbi.nlm.nih. gov/pubmed/12649189 54. Rippley RK, Stokes CL. Effects of cellular pharmacology on drug distribution in tissues. Biophysical journal [Internet] 1995 [cited 2014 Jun 12]; 69:825-39. Available from: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=1236312&tool=pmcentrez& rendertype=abstract 55. Gil-Gil MJ, Mesia C, Rey M, Bruna J. Bevacizumab for the treatment of glioblastoma. Clinical Medicine
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mAbs Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kmab20
Monitoring therapeutic monoclonal antibodies in brain tumor a
a
a
b
Rima Ait-Belkacem , Caroline Berenguer , Claude Villard , L’Houcine Ouafik , Dominique ac
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Figarella-Branger , Alain Beck , Olivier Chinot
ac
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& Daniel Lafitte
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Aix-Marseille Université Inserm; CRO2 UMR S-911; Marseille, France
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APHM; CHU Nord; Service de Transfert d’Oncologie Biologique; Marseille, France
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APHM; Timone Hospital; Department of Neurosurgery; Marseille, France
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Centre d’Immunologie Pierre Fabre (CIPF); St Julien-en-Genevois, France Accepted author version posted online: 01 Nov 2014.Published online: 15 Dec 2014.
Click for updates To cite this article: Rima Ait-Belkacem, Caroline Berenguer, Claude Villard, L’Houcine Ouafik, Dominique Figarella-Branger, Alain Beck, Olivier Chinot & Daniel Lafitte (2014) Monitoring therapeutic monoclonal antibodies in brain tumor, mAbs, 6:6, 1385-1393, DOI: 10.4161/mabs.34405 To link to this article: http://dx.doi.org/10.4161/mabs.34405
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SHORT COMMUNICATION mAbs 6:6, 1385--1393; November/December 2014; © 2014 Taylor & Francis Group, LLC
Monitoring therapeutic monoclonal antibodies in brain tumor Rima Ait-Belkacem1, Caroline Berenguer1, Claude Villard1, L’Houcine Ouafik2, Dominique Figarella-Branger1,3, Alain Beck4, Olivier Chinot1,3, and Daniel Lafitte1,* 1
Aix-Marseille Universit e Inserm; CRO2 UMR S-911; Marseille, France; 2APHM; CHU Nord; Service de Transfert d’Oncologie Biologique; Marseille, France; 3APHM; Timone Hospital; Department of Neurosurgery; Marseille, France; 4Centre d’Immunologie Pierre Fabre (CIPF); St Julien-en-Genevois, France
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Keywords: bevacizumab, glioblastoma multiforme, MALDI imaging mass spectrometry, monoclonal antibodies, palivizumab, top down in source decay Abbreviations: BBB, blood-brain barrier; CRC, metastatic colorectal cancer; CSF, cerebrospinal fluid; 1; 5 DAN, 1; 5-diaminonaphtalene; EMA, European Medicines Agency; FDA, Food and Drug Administration; GBM, glioblastoma multiforme; IMS, imaging mass spectrometry; ISD, in-source decay; ITO, indium tin oxide; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; mAbs, monoclonal antibodies; MALDI, matrix-assisted laser desorption/ionization; NSCLC, non-small cell lung cancer; pE, pyroglutamate; RMS, root mean square; RP-HPLC, reversed phase high-performance liquid chromatography; TOF, time of flight; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VH, variable domain of the heavy chain; VL, variable domain of the light chain; WHO, world health organization
Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.
Targeted therapies rely on the use of monoclonal antibodies (mAbs) or anti-angiogenesis drugs. These therapies have been designed to overcome some conventional chemotherapy side effects or pitfalls, such as toxicity, resistance, local variations in blood flow or non-effective delivery to the target.1Hybridomas were first described in 1975, and more than 15 y later mAbs obtained clinical validation in the mid-1990s.2 There are currently more than 50 mAbs and related products approved by the Food and Drug Administration (FDA) or the European Medicines Agency (EMA) for therapeutic use.3 In addition, over 30 more are being investigated in late stage clinical trials.4 Two main categories of therapeutic antibodies are in development: antibodies targeting cell markers and antibodies targeting *Correspondence to: Daniel Lafitte; Email: daniel.lafi[email protected] Submitted: 06/18/2014; Revised: 08/04/2014; Accepted: 08/09/2014 http://dx.doi.org/10.4161/mabs.34405
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cytokines. One of the most targeted cytokine is the vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis in normal and disease states. It can be directly secreted by cancer, endothelial, stromal, or blood cells and can be a component of the extracellular matrix surrounding these cellular components.5 VEGF and its receptors, VEGFRs, are the target of many anti-angiogenic therapies that try to prevent angiogenesis and vascular leakage, cell proliferation, survival and migration.6 Bevacizumab (AvastinÒ ) was the first anti-VEGF therapy approved, in combination with cytotoxic chemotherapy, for the first line treatment of metastatic colorectal cancer (CRC),7,8 recurrent or metastatic non-small cell lung cancer (NSCLC),9 metastatic breast cancer10 and renal cancer.11According to the World Health Organization (WHO) classification 2007, glioblastoma multiforme (GBM) or grade IV astrocytoma is the most invasive type of gliomas associated with high mortality and morbidity. GBM is characterized by pronounced hypercellularity, pleomorphism, numerous mitoses, foci of central necrosis, and excessive vascularization.12 It is widely known that tumor blood vessels are leaky, tortuous and dilated, especially in malignancies such as GBM. Assuming that the anti-angiogenic therapy induces normalization of these abnormal blood vessels, making them less leaky,13bevacizumab is used as a single agent for the treatment of recurrent GBM.14-17 An increasing knowledge of the pharmacokinetics properties (absorption, distribution, metabolism and elimination parameters) is necessary to estimate how effectively the therapeutic agents reach their targets, and to tailored the next generation of optimized antibodies (OptimAbs).18 Up to now, in situ concentration measurements have been almost impossible; plasma levels are easier to obtain, offering a proportional relationship with the unbound drug concentration in tissues.19 In the brain, concentrations in micro dialysates and abscesses are not frequently available for humans,20 so the concentration of therapeutic agents is often
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followed by measuring levels in cerebrospinal fluid (CSF). This approach, however, does not take into account tumor anatomic and metabolic heterogeneity, or other impediments to drugs access. Principally in GBM, the interpretation of antibody levels is hampered by the difficulty of taking local measurements, which depend on awareness of the blood-brain barrier (BBB).21 The molecular distribution of large molecules (antibodies) has primarily been investigated using widespread approaches of radio- or fluorescence-labeled probes like electron microscopy,22 intravital fluorescence videomicroscopy,23 autoradiography,24 micellarelectrokinetic capillary25 and positron emission tomography.26 Imaging mass spectrometry (IMS) provides complementary features that overcome many labeling imaging pitfalls, such as the unspecific reactions, the change of affinity due to labeled probes and the inability to distinguish between the parent compound and metabolites. IMS detects label-free analytes,27-29 measuring the distribution of administered compounds and their metabolites in the same experiment.30-32 However, a mass range limitation prevents the detection of large biomolecules, and, therefore, of therapeutic antibodies. To overcome this problem, two solutions can be envisioned.33,34 The first one is the usual bottom-up approach, where the proteins are extracted and digested by proteases into peptides and then analyzed by LCMS/MS. This procedure can, however, induce protein modifications and delocalization.35 Second one is the top down approach that consists of a direct in situ fragmentation of the protein without digestion. This “top-down” approach has been previously used for extensive characterization of post-translational modifications.36,37 In-source decay (ISD) is the top-down approach occurring during the matrix-assisted laser desorption/ionization (MALDI) steps.38,39 This pseudo MS/MS technique, MALDI ISD, uses hydrogen radical transfer from the matrix to analyte molecules to provide fragmentation and sequencing of protein N and C termini.40 Using the appropriate matrix, MALDI ISD can be associated with MALDI IMS, and therefore provides identification and location of exogenous or endogenous molecules at the same time.41,42 In this work, we developed a novel method coupling top-down in source decay fragmentation with matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI ISD IMS). This new strategy was performed to obtain in situ distribution of bevacizumab on treated glioblastoma-bearing mouse brain tissue sections. The main result expected of bevacizumab treatment is to block the angiogenesis. As noted below, angiogenesis and its major regulator, VEGF, represent one of the most important therapeutic targets in GBM treatment. VEGF receptors (VEGFR1/2) are expressed on endothelial, but few other, cells wherein it stimulates several cell responses, including proliferation, migration, survival, and secretion of matrix-degrading enzymes. Through the VEGFR2 and having the stronger kinase activity, it represents the predominant mediator of VEGFinduced angiogenic signaling.14 This signaling pathway can be inhibited by bevacizumab, which binds to and neutralizes all human VEGF-A isoforms (Fig. 1A). Top-down in source decay, using 1, 5-diaminonaphtalene (DAN) as a matrix, is a radical pathway allowing the identification of the immunoglobulin
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amine and carboxyl terminal parts. ISD generally leads to generation of a long series of c- and zC2-ions after cleavage of N-Ca bonds on the peptide backbone (Fig. 1B). In order to verify the correct NH2- and -COOH terminal portions and to identify the set of virtual precursors that are specific to each mAb, we assessed the fragmentation of bevacizumab and palivizumab, two humanized IgG1 therapeutic mAbs. Palivizumab has a totally different epitope and mechanism of action than bevacizumab, and it was therefore chosen as a negative control. It blocks both cell to cell and virus to cell fusion, and targets viral proteins,43 not angiogenesis cytokines. ISD mass spectra were obtained from a mixture of 1, 5 DAN matrix with one of the two purified antibodies (bevacizumab or palivizumab) directly spotted on MALDI target. The best fragmentation range was obtained between m/z 1000–8000 for palivizumab and m/z 2000–8000 for bevacizumab. A sequence of 30 residues corresponding to the variable domain of the heavy (VH) and light (VL) chains of bevacizumab (Fig. 2A) and palivizumab (Fig. 2B) was obtained. Noteworthy, the VH domain of palivizumab carried a pyroglutamate (pE) modification occurring through the rearrangement of the originally synthesized glutamine residue. It is known that both glutamine and glutamate at the N termini of recombinant mAbs can cyclize spontaneously to pyroglutamate (pE) in vitro, making the antibodies more acidic.44 Also, in vivo cyclization can occur as a stabilization mechanism for proteins not impacting their turnover. For therapeutic mAbs, like palivizumab, pE can be one of many posttranslational modifications observed during production and storage.45 Apparently, this cyclized residue is resistant to amino peptidases.46 RP-HPLC is a chromatographic method that was able to show and to quantify post-translational modifications which were also identified by peptide mapping, mass spectrometry and microsequencing.47 The same in source decay fragmentation studies were performed on healthy brain tissue section to obtain the most representative fragments of the studied mAbs in these particular conditions. Wherever the antibody was spotted on the tissue slice, fragmentation occurred and fragment ions were detected showing that ion suppression effect has no impact on our study. Ion suppression usually occurs when an ion suppresses the signal of another species in the sample.48 Antibody fragmentation was measured on tissue using a lowest quantity of 41.75 pmol. In this case, three ions (c20, c21 and c22) corresponding to N termini fragments of bevacizumab were measured (Fig. 2C). To confirm the identity of these ions, a second sequencing step called T3-sequencing was performed directly on tissue sections (Fig. 2D). The generated precursor ions, including the N- or C-terminal sequence generated by ISD fragmentation, were selected in the timed ion gate of a MALDI TOF/TOF mass spectrometer for MS/MS analysis. This new fragmentation (T3-sequencing) generates principally b- and yions, allowing the proper sequencing of both N- and -C termini, respectively, and confirmation of suspected terminal modifications.39,49 We then achieved analysis on mouse xenografts brains after stereotactic cortical tumor U87 cells injection, which was
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performed as previously described.41 To provide protein identification in addition to their localization directly on brain tissue sections, we performed ISD of the entire tissue slice at 80 mm spatial resolution. Most of the obtained signals were protein fragments. Some of them bear the exact mass as bevacizumab c-ions previously measured using the purified antibody (Fig. 3). T3-sequencing confirmed that they were (c20, c21 and c22) ions (results not shown). Figure 1. Bevacizumab mode of action and fragmentation using top down MALDI. (A) Bevacizumab action on VEGF/ The intensities of these VEGFR signaling pathway. (B) In source decay fragmentation of the immunoglobulin amine and carboxyl terminal three ions were summed parts generates a long series of c- and zC2-ions after cleavage of N-Ca bonds on the peptide backbone. to provide the image of antibodies distribution within the brain slices. Bevacizumab is mainly detected on the tumor area, whereas pali- extravasation, antibody binding and demonstrated a detrivizumab is distributed in all the tissue (Fig. 4). It is very difficult mental effect of antibody internalization.51 Micro distribution to compare palivizumab and bevacizumab quantities as we are studies described the phenomena where antibodies are immonot sure that they fragment exactly with the same efficiency. We bilized close to their site of entry and the factors that influwill have to develop absolute quantitation methods using in ence the location of the moving front, such as diffusivity, source decay. antigen density, permeability, and dose.52 Micro distribution Notably, the image obtained with three ion is quite similar to also analyzed the influence of cellular pharmacology, like the one gathered with the most abundant fragment only (c21bev- binding, internalization, and recycling on the distribution acizumab D m/z 2081), but it is more relevant on a statistical around blood vessels.54 basis. It could be interesting to use both c- and z- ions from proGlioblastoma is one of the most vascularized human tumors.5 tein termini for the identification and imaging, however DAN This abnormal tumor vasculature is upregulated by VEGF, fragmentation, via the radical pathway, rather produce c- frag- which serves as a major angiogenic factor in normal vascular ments from the N- terminus of big proteins than z- fragments development.5,55 Angiogenesis is a critical step during glioblasfrom the C- terminus.50 toma development and growth in which tumor requires more The pharmacokinetic properties of bevacizumab are cur- oxygen and nutrients levels for its survival and proliferation. rently measured in plasma. This drug is associated with a low Anti-VEGF treatment can reduce vascular permeability, but this clearance (CL), a limited volume of distribution (Vc), and a may not necessarily reflect tumor cell death. Thus, the macro dislong elimination half-life (21 d) that maintains target thera- tribution and penetration of the bevacizumab into the tumor peutic bevacizumab plasma levels with a range of administra- mass is in agreement with the tumor characteristics and treatment tion schedules (such as 5–10 mg/kg once every 2 or 3 wk). It knowledge. The purpose of coupling MALDI IMS and top-down in is clearly important to obtain direct in situ measurement for a direct pharmacokinetic view closer to the site of action. source decay analysis is to get localization in addition to the proSuch a measurement within the tumor will account for blood tein identification, at the same time and directly on the same tisflow through the tumor, extravasation, convection and diffu- sue section. In situ molecular identification was performed by sion, binding, and internalization. Qualitative and quantita- MALDI ISD, a top-down approach that allows identification of tive mathematical models were constructed previously to the proteins by their N- and C-termini39,41 fragmentation. predict the antibody diffusion through tumors taking into Although limited to the most abundant species, MALDI ISD consideration some of these parameters, such as the effect of IMS was previously shown to be useful for glioma marker characantibody internalization.51-54 Macro distribution models terization41 and post-translational modifications identification.56 (1 cm diameter tumor) predict the role of elevated interstitial We now strengthen its impact with the close investigation of fluid pressure, tumor convection, spatial variation in therapies in situ.
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Figure 2. MALDI ISD characterization of monoclonal antibodies. MALDI mass spectra obtained between m/z 2000–8000 of bevacizumab fragments (A) and m/z 1000–8000 palivizumab fragments (B). (C) In order to identify the precursor ions of bevacizumab, different quantities of antibody were spotted on tissue (167 pmol lower panel, 125.25 middle panel and 41.75 pmol higher panel). Stars correspond to the three precursor ions c20, c21, c22 that will be used in the course of the study. T3-sequencing spectrum of 2081. 13 c-ion precursor showing generated b-ions mainly (D).
Further improvement of antibody quantitation directly on tissue is a possibility under development, in combination with quantitation with triple quadrupole mass spectrometry. New coming sprayer systems will improve spatial resolution of the MALDI imaging combined with in source decay analysis. Going down to 10 mm spatial resolution or less could be important to distinguish antibody location within the tumor cells and their microenvironment. MALDI imaging mass spectrometry could therefore provide both the macro and the micro distribution view described by the mathematical models, as described above. MALDI imaging mass spectrometry may in this way compensate for many conventional imaging modalities limitations by combining a number of real advantages: sensitivity, label free, speed, and reproducibility. Thus, it could play an important role for cancer imaging combined with other modalities in preclinical and clinical practice.
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Materials and Methods Reagents 1, 5-diaminonaphtalene (DAN) was purchased from SigmaAldrich, Saint-Quentin Fallavier, France. Bevacizumab (AvastinÒ , Roche, Grenzach-Wyhlen, Germany) and palivizumab (SynagisÒ , Abbott, Berkshire, UK) were provided by the Centre d’Immunologie Pierre Fabre. Tissues Harvesting of tissues The U87 cells were obtained from a malignant glioma of a female patient by explant technique. Five-weeks-old nude FOXN1 male mice (25–30 g athymic mice) were purchased from Harlan Laboratories, Gannat, France. Glioblastoma cells were injected stereotaxically using: Leica MS5-MZ6 stereomicroscopic device (Leica Microsystems SAS, France), WPI Stereotaxic
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Figure 3. MALDI ISD IMS workflow. After a stereotactic cortical U87 glioblastoma cells injection, tumor growth and immunotherapy, brains are extracted, flash frozen and tissue sections made. Following histological observations and matrix deposition on the entire brain tissue section, MALDI imaging acquisition of the overall average spectrum and image reconstruction (x and y coordinates), an ion-density map was obtained for each chosen m/z signal present on the average mass spectrum. Most of the obtained signals were different protein fragments and some of them bear the exact mass than bevacizumab and palivizumab c-ions previously measured using the purified antibodies. Three c-ions were found to correspond to c-ions of either bevacizumab or palivizumab antibody. The intensities of these three ions are summed to provide the image of antibodies distribution within the brain
Frame 18 deg with Ear Bars (World Precision Instruments, Germany) and Hamilton Syringe 10 ml, Model 701 SN OnColumn Injection SYR, Cemented NDL, 32 ga, 3.35 in, point style 45 mounted on microinjection unit (Hamilton Bonaduz AG,GR, Switzerland). This system enabled inoculation of 105 glioblastoma cells suspended in 2 ml solution using the manual control of the injection system. The striatum on the right frontal lobe at interaural, lateral and depth coordinates of 0.0, 3.0, and 3.0 mm to the bregma was targeted. Eight days after cells injection, the mice were separated to 4 groups: control mice, bevacizumab treated mice, palivizumab treated mice and bevacizumab added to palivizumab treated mice. The intraperitoneal drug injection was done at 200 mg/mouse three times per week during 1 (for bevacizumab and palivizumab) or 3 wk (only for bevacizumab). Mice were anesthetized by the intraperitoneal injection of 6.5 ml/g of anesthetic solution, composed of 50 ml of 2% Rompun with 200 ml of Imalgene 1000 in a final volume of 1000 ml
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of 0.9% NaCl. Mice were then dissected and the total brain blocks extracted and snap-frozen in liquid nitrogen for 15 s. The brains were kept at ¡80 C until use. The drop weight of mice was monitored and the sacrifice was done one week after the latest treatment. Three mice per group were used for MALDI imaging experiment. The animal manipulations have been performed in accordance with international guidelines. Experimental protocols were reviewed and approved by the Institutional Animal Care Committee of the School of Medicine and performed in accordance with INSERM and Aix Marseille Universite School of Medicine polices regarding the use of laboratory animals. Tissues preparation and staining Mouse brain was placed at ¡18 C for 15 min before use. Tissue sections were cut using a Leica CM 1900 UV Mycrosystems cryostat (Leica Microsystems SAS, France) with a microtome chamber and a specimen holder chilled at ¡18 C. The 12 mm
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Figure 4. Monoclonal antibodies localization on brain tissue sections bearing glioblastoma. The first vertical panel represents the scan of Hematoxylin and Eosin histological images of coronal or horizontal tissue sections from control and bevacizumab and/or palivizumab treated mice brains. The tumor region, of a dark pink color, is surrounded by a black-dashed line. After bevacizumab treatment, we note that tumor region is well-defined and localized, even after 30 d of growth. The ion images of in source decay fragments obtained at 80 mm lateral resolution are represented on the other panels. Thus, the anatomical localization of different protein fragments is shown: myelin basic protein is the most abundant protein principally localized in the hippocampus. Bevacizumab and palivizumab fragments localization is found distributed on the entire tissue section. Bevacizumab is more concentrated on tumor region (15 or 30 d). Scale bar D 2 and 1 mm.
thick coronal and horizontal sections were thaw mounted onto Indium Tin Oxide ITO-coated microscopic slides (Bruker Daltonics, Wissembourg, France) adapted for MALDI imaging mass spectrometry. These MALDI target slides were placed in a desiccator for 1 h. ITO slides were washed with isopropanol (twice at 70% for 2 min and once at 96% for 2 min) for MALDI in source decay imaging MS analyses. Following washing, ITO slides were dried in a desiccator for 30 min before matrix deposition. After MALDI imaging analysis, the used slides were washed with pure ethanol and acetone to remove the matrix before histological staining.57 For Hematoxylin and Eosin staining, the tissue sections were immersed in a filtered Harris Hematoxylin solution for 1 min and rinsed with tap water. Then, the sections were immersed in Eosin solution for 30 s and rinsed with tap water.
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The slices were dehydrated in various alcohol solutions (50%, 70%, 80%, 95%, 100%) and cleared with xylene (2x). They were mounted onto a labeled glass slide with Permount. The pictures of H&E stained histological sections were obtained at specific regions with Olympus BH-2microscope at magnification, £ 40 objective. The nuclei and other basophilic structures were colored in blue while cytoplasm and acidophilic structures were colored light to dark red. Matrix deposition for imaging mass spectrometry analyses Brain tissue sections were always scanned before matrix deposition with a histology slide scanner (Opticlab H850 scanner, Plustek). The scanned histological image was used for the teaching in order to obtain a perfect superposition between the histological and the ion images. For MALDI in source decay imaging
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1, 5-diaminonaphtalene (DAN), 10 mg/ml in 50% Acetonitrile 0.2% TFA, matrix deposition was performed with the automatic matrix sprayer device (ImagePrep, BrukerDaltonics) equipped with the new spray head. The spray method was optimized adding more nebulization and drying cycles to obtain a thick matrix deposit compatible with in source decay analysis.
30–50 mm laser spot diameters). Sequences were created on FlexImaging software, version 3.0 (BrukerDaltonics) after the downloading of scanned images. Polygon measurement regions were manually defined using these histological images. A minimum of two images was acquired to ensure reproducibility. Data Analysis
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Data Acquisition MALDI in source decay MS MALDI mass spectra were obtained on an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). ISD analyses were acquired in a reflectron positive mode with an accelerating potential of 20 keV; laser power was increased up to 20% above the ionization threshold to increase fragmentation without losing window (laser power at 50%). The spectra were internally calibrated by using parent and c10-serie ion fragment of bovine serum albumin (BSA) masses. MALDI in source decay imaging MS MALDI in source decay imaging was performed using an Ultraflextreme TOF/TOF mass spectrometer controlled by the FlexControl 3.3 software (BrukerDaltonics). The MALDI ISD IMS experiments were performed at a spatial resolution of 80 mm. Spectra were acquired in positive reflectron ion mode with 1500 laser shots accumulated at each spot and the laser power was optimized at the start of each run and then fixed for the overall MALDI ISD IMS experiment with a frequency of 500 Hz. The mass spectrometer parameters were set according manufacturer’s settings for optimal acquisition performance. Laser spot size was medium (corresponding approximately to References 1. Marin JJG, Romero MR, Blazquez AG, Herraez E, Keck E, Briz O. Importance and limitations of chemotherapy among the available treatments for gastrointestinal tumours. Anti-cancer agents in medicinal chemistry [Internet] 2009 [cited 2014 May 9]; 9:16284. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/19199863 2. DeVita VT, Chu E. A history of cancer chemotherapy. Cancer research [Internet] 2008 [cited 2014 May 1]; 68:8643-53. Available from: http://www.ncbi.nlm.nih. gov/pubmed/18974103 3. Reichert JM, Beck A, Lugovskoy AA, Wurch T, Coats S, Brezski RJ. 9th annual European Antibody Congress, November 11-13, 2013, Geneva, Switzerland. mAbs [Internet] [cited 2014 Jun 18]; 6:309-26. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24492298 4. Reichert JM. Antibodies to watch in 2014: Mid-year update. mAbs [Internet] 2014 [cited 2014 Jun 18]; 6:799-802. Available from: http://www.ncbi.nlm.nih. gov/pubmed/24846335 5. Cea V, Sala C, Verpelli C. Antiangiogenic therapy for glioma. Journal of signal transduction [Internet] 2012 [cited 2014 May 6]; 2012:483040. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid=3399341&tool=pmcentrez&rendertype=abstract 6. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nature medicine [Internet] 2003 [cited 2014 Apr 29]; 9:669-76. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12778165 7. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S,
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MALDI in source decay imaging for monoclonal antibodies distribution IMS data were acquired and normalized with root mean square (RMS) (vector norm) algorithm that provides a very uniform distribution of intense signals. Raw spectra were analyzed with FlexImaging software version 3.0 after baseline subtraction. Masses were selected with a mass precision of § 0.1 Da on the overall average spectrum. Ion density map was created for each signal present on the whole sample average mass spectrum. For signals corresponding to c-ions fragments of mAbs, the spectra lists of interest were exported in .xml format and then, the intensities of all the fragment ions were summed. Finally, we reimported the new spectra list data in FlexImaging software to obtain the image corresponding to the ion sum. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
We acknowledge canceropole PACA, region PACA, Aix Marseille Universite, INSERM, IBISA, and the SIRIC (Grant INCaDGOS-Inserm 6038) for financial support.
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