Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumour Model

Transcription

1 Supporting Information Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumour Model Nadine E. Mascini 1, Menglin Cheng 2, Lu Jiang 2, Asif Rizwan 2, Helen Podmore 3, Dhaka R. Bhandari 4, Andreas Römpp 5, Kristine Glunde 2,6,*, Ron M.A. Heeren 1,7,* 1 FOM Institute AMOLF, 1098 XG Amsterdam, The Netherlands. 2 The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 3 Thermo Fisher Scientific, Stafford House, Boundary Way, Hemel Hempstead, Herts, United Kingdom. 4 TransMIT GmbH TransMIT Center for Mass Spectrometric Developments, Schubertstrasse 60, Giessen, Germany. 5 Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, Giessen, Germany. 6 Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. 7 The Maastricht Multimodal Molecular Imaging institute (M4I), 6229 ER Maastricht, The Netherlands. *Corresponding authors. r.heeren@maastrichtuniversity.nl, kglunde@mri.jhu.edu. Table of contents Page S-2 and S-3. Supplementary Materials and Methods. Page S-4. Putative chemical structures of the identified pimonidazole-derived metabolites, Figure S-1. Page S-5. Co-registration data for all tumors, Figure S-2. Page S-6. Fragmentation spectra of endogenous molecules, Figure S-3. S-1

2 Supplementary Materials and Methods Chemicals and reagents We obtained alpha-((2-nitroimidazol-1-yl)methyl)-1-piperidineethanol (pimonidazole), α-cyano- 4-hydroxycinnamic acid (CHCA) and trifluoroacetic acid (TFA) from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) and methanol (MeOH) were purchased from Biosolve (Valkenswaard, The Netherlands). Cresyl Violet was obtained from Thermo Scientific (cat# 40576, PA, USA). Ponceau S (cat# P3504), Mayer s hematoxylin and eosin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Paraformaldehyde was purchased from Santa Cruz Biotechnology (Dallas, TX, USA.). Other reagents used for immunostaining were purchased from EMD Millipore (MA, USA), unless stated otherwise. Hematoxylin and eosin (H&E) staining One frozen slide from each set was thawed and fixed with 4% paraformaldehyde for 30 min, then washed with water. Fresh hematoxylin was applied on top of the tissue sections for 1 min. After washing with distilled water, tissue sections were stained with eosin for 1 min. The slides were then washed with water until there was minimal colouring visible on the gelatin area of the sections. The slides were then mounted with a cover glass using mounting medium (DAKO Faramount aqueous mounting medium, cat# S3025, Carpinteria, CA, USA) and photomicrographs were taken on a Nikon microscope equipped with a CCD camera. Immunohistochemical (IHC) staining for pimonidazole One tissue section from each set was thawed, fixed in 4% paraformaldehyde for 30 min and washed three times with TBS/0.1% Tween-20. Peroxidase activity was quenched with 3% H 2 O 2 for 10 min, and tissue sections were washed again three times with TBS/0.1% Tween-20. Non- S-2

3 specific binding was blocked with protein blocking reagent (cat# 20773, Millipore, Billerica, MA, USA) and rabbit serum, each for 10 min. Tissue sections were rinsed three times with TBS/0.1% Tween-20. For staining, tissue sections were incubated with primary antibody (primary mouse anti-pimonidazole antibody conjugated with FITC, HP2-100 Kit, Hypoxyprobe, Burlington, MA, USA) diluted to 1:75 with antibody dilution buffer (TBS/0.1% Tween-20) for 30 min. Samples were washed five times with TBS/0.1% Tween-20, followed by incubation with secondary antibody (secondary rabbit anti-fitc antibody, HP2-100 Kit, Hypoxyprobe, Burlington, MA, USA) diluted to 1:75 with antibody dilution buffer for 30 min. Samples were washed five times with TBS/0.1% Tween-20. 3,3'-Diaminobenzidine (DAB) staining was performed for 10 min (DAB Quanta, cat #TA-060-QHDX, Thermo Scientific). The reaction was stopped by washing with water. Samples were washed once with TBS/0.1% Tween-20. Counterstaining was performed with hematoxylin for 1 min. Finally, mounting medium was applied and a cover glass attached. Images were acquired using a Nikon microscope equipped with a CCD camera. S-3

4 Figure S-1. Putative chemical structures of the identified pimonidazole-derived metabolites. The indicated pathway is based on existing literature (see Results and Discussion ). For each species the detected m/z value is given. R = side chain. G = glutathione. S-4

5 Figure S-2. Co-registration of anti-pimonidazole stained tissue sections and normalized and thresholded MS images of pimonidazole-derived ions (green) from adjacent tissue sections. Data are shown for all three tumors. H&E stained adjacent tissue sections are added for comparison. Necrotic regions are indicated by a dashed line. Scale bar 2 mm. S-5

6 Figure S-3. Fragmentation spectra of endogenous molecules detected from MDA-MB-231 breast tumor xenograft tissue. (a) C 8 H 18 NO 2, (b) acetylcarnitine, (c) 1-methylnicotinamide, and (d) stearoylcarnitine. Stearoylcarnitine was identified using on-tissue ion mobility MS/MS, see reference 41. S-6