Residual (fresh) breast cancer tissue was collected by the Tissue Biobank of the University of Liege, directly frozen in liquid nitrogen, and then stored at − 80 °C. The standardized protocol was approved by the Ethics Committee of the University Hospital Center of Liege. Informed consent was obtained from the participant included in this study. A cryosection of 12-μm thickness was thaw-mounted on a polyethylene naphthalate (PEN) membrane slide (Leica Microsystems, Wetzlar, Germany) and stored at − 80 °C until analysis.
Mass spectrometry imaging
All solvents, if not stated otherwise, were purchased from Biosolve (Dieuze, France). First, the membrane slide was desiccated for 30 min at room temperature. Several fiducial markers were applied next to the tissue using water-based Tipp-Ex (BIC, Paris, France) for later co-registration purposes. Five milligrams per milliliter α-cyano-4-hydroxycinnamic acid (Sigma Aldrich, St. Louis, MO, USA) in 70% acetonitrile and 0.2% trifluoroacetic acid was sprayed in eight layers using an HTX-TM sprayer (Chapel Hill, NC, USA) onto the tissue section with a constant flow rate of 0.1 ml/min and at a speed of 1300 mm/min. The breast cancer section was measured with a MALDI HDMS SYNAPT G2-Si (Waters, Manchester, UK) which is compatible with non-conductive PEN membrane slides. The experiment was performed in positive mode and at 70-μm spatial resolution within a mass range of m/z 350–1600 in which mostly lipids are detected. Red phosphorus was used for external calibration.
Staining and optical images
Directly after the MSI experiment, a digital high-resolution image of the slide with matrix was obtained with a microscopic slide scanner (Mirax Desk, Zeiss, Jena, Germany), subsequently referred to as the optical image.
Then, the matrix was removed with 70% ethanol and stained for hematoxylin and eosin (H&E) used as standard protocol (Milli-Q water 3 min, hematoxylin 90 s, tap water 3 min, eosin 30 s, tap water 3 min, 100% ethanol 1 min, xylene 30 s). The H&E-stained tissue section was not covered with a cover slip but immediately scanned with the same microscopic slide scanner and stored at − 80 °C until LMD. This resulted in a digital optical image, subsequently referred to as the H&E image.
Both digital high-resolution images were downscaled 1:4 for better handling using the scan viewer software (Pannoramic Viewer, 3DHISTECH Ltd., Budapest, Hungary) which resulted in a pixel size of 2.076 μm for x and 2.084 μm for y (12,235 × 12,189 dpi).
Co-registration, data analysis, and image processing
These images were imported together with the MSI data into MATLAB R2017b (MathWorks, Natick, MA, USA) for co-registration of the images (MSI, optical and H&E; Fig. 1), data analysis, and image processing with the Image Processing toolbox (v.10.1). All image co-registrations were performed using affine geometric transformation (command fitgeotrans). Every spectrum of the MSI data was normalized to its total ion current. Tumor-associated spectra were clustered using non-negative matrix factorization (NNMF) where each pixel is assigned to the component with the highest score. After image processing of the segmentation results (see Electronic Supplementary Material (ESM), Figs. S3 and S4), the coordinates of the regions belonging to the segments were recalculated with respect to the coordinates of the fiducial markers in the optical image and written into an XML file for compatibility with subsequent LMD system.
Laser microdissection was performed using a Leica LMD 7000 (Leica Microsystems, Wetzlar, Germany). This system supports the import of external coordinate information of areas to be cut out in form of an XML file. Areas were dissected from the H&E-stained tissue sections with the following settings: wavelength 349 nm, power 20, aperture 45, speed 15, specimen balance 0, head current 100%, and pulse frequency 501 Hz. The microdissected regions were collected in 0.5-ml centrifuge tubes and stored at − 80 °C until further analysis.
For every MSI segment, a total of 0.3-mm2 dissected material was prepared and analyzed by an optimized LC-MS/MS protocol for bottom-up proteomics of very small samples (~ 2000 cells) using an ultraperformance liquid chromatography (UPLC) 2D nanoACQUITY (Waters, Corp., Milford, USA). This was done as described previously , but without paraffin removal and antigen retrieval due to the use of fresh-frozen tissue sections.
Protein identification and label-free quantification (LFQ) were performed using MaxQuant v.188.8.131.52 with the following settings: UniProt reviewed human database, trypsin digestion with maximum two missed cleavage sites, methionine oxidation as variable modification and carbamidomethyl cysteine as fixed modification, a minimal peptide length of seven amino acids, at least two peptides per protein (of which at least one is unique), and a maximum false discovery rate of 1%. The label-free intensities were normalized using the MaxLFQ algorithm .
Data analysis was performed with Perseus v.184.108.40.206 . Proteins identified as “reverse”, “only identified by site”, or “potential contaminants” hits were removed. The LFQ intensities were log2-transformed and z-scored before performing a hierarchical clustering of the proteins with the following settings: Euclidean distance, complete linkage, based on a preprocessing with k-means with 300 clusters, 10 iterations, and 1 restart. Under- and overexpressed proteins were selected based on z-scores being exclusively ≤ − 1 or ≥ + 1, respectively. The gene IDs corresponding to the under- and overexpressed proteins were then imported into the PANTHER v.13.1 gene ontology classification system .