Mcd viewer

FFPE lung tissue–section slides (5 μm thick) were stained with metal-conjugated Abs (anti–human CD68, CD4, CD8, and CD15; Fluidigm) after antigen retrieval. Intercalator-Ir (Fluidigm) was used to stain DNA. Slides were ablated on the Fluidigm Hyperion Imaging System using CyTOF7 software (Fluidigm) and visualized using an MCD Viewer (Fluidigm). Images were processed for publication using FIJI (56 (link)) to despeckle and sharpen the images.

Imaging mass cytometry with the Hyperion mass cytometry system was performed as described before (36 (link)). In short, antibodies were conjugated to purified lanthanide metals (Fluidigm, CA, United States; Table 1) using the MaxPar X8 antibody labeling kit and protocol (Fluidigm). 4 μm FFPE sections were deparaffinized, rehydrated and antigen retrieval (high pH—pH9 Thermo Fisher Scientific) was performed by boiling the sections in the microwave. Sections were incubated for 30 min with Superblock solution, after which, excess Superblock was tapped off. Sections were incubated with antibodies according to Table 1. Following, sections were incubated with Intercalator-Ir (125 μM, Fluidigm) for 5 min. The slides were then dried under an airflow and stored at room temperature until ablation. Prior to acquisition, the Hyperion mass cytometry system (Fluidigm) was autotuned using a three-element tuning slide (Fluidigm) according to the tuning protocol provided by the Hyperion imaging system user guide (Fluidigm). Regions of interest were selected based on hematoxylin and eosin stains and pan-cytokeratin IHC, after which areas of 1,000 × 1,000 μm were ablated and acquired at 200 Hz. Data was exported as MCD files and visualized using the Fluidigm MCD™ viewer.

Literature was reviewed to identify markers relevant to the tumor and premetastatic niche. Metal conjugated antibodies were obtained with custom conjugations with Fluidim Maxpar Antibody Labeling Kit (Supplementary Table S2). Antibodies were validated on neck area and LNs on 5 µM formalin-fixed paraffin-embedded tissue sections. Heat-induced antigen retrieval (Tris-EDTA buffer at pH 9), blocking with 3% bovine serum albumin, and titrated antibody staining concentrations were employed. Slides were imaged using the Fluidigm Hyperion Tissue Imager system with a 1-µm² laser ablation spot size at a frequency of 200 Hz and ablation energy of 3 dB. Area of analyzed tissue varied from 400 um² up to 1000 um² with multiple areas imaged per specimen. Image analysis was performed with MCD viewer (Fluidigm) and HistoCAT [48 (link)].

For IMC histology, tissue sections were de-paraffinized and rehydrated, and antigen retrieval was performed in pH 8, 1 mM EDTA buffer at 96 °C for 20 min. Sections were cooled at room temperature and rinsed in tap water and TBS (20 mM Tris with 500 mM NaCl, pH 7.5). Tissue was blocked for 3 h at room temperature with 0.3% BSA, 25% FBS and 0.1 mg/mL FC receptor binding inhibitor in TBS-T (TBS + 0.05% Tween-20). All antibodies (Additional file 1: Table S2) were diluted in 0.3% BSA in TBS-T and applied to the tissue for overnight incubation at 4 °C. Sections were then rinsed in TBS-T and TBS, and counterstained with 125 nM Maxpar® Intercalator-Ir (Fluidigm) in PBS for 1 h at room temperature. Sections were rinsed in TBS-T, TBS, and two washes of distilled water before air-drying at room temperature. Antibody-labeled tissue areas (1000 × 1000 μm) were raster-ablated using a Hyperion™ Laser Scanning Module (Fluidigm) with a 1 μm diameter spot size at 200 Hz. This process was coupled to a Helios™ Mass Cytometer (Fluidigm) for lanthanide metal detection [43 (link)]. Images for each antibody channel were acquired on CyTOF Software (Fluidigm, version 6.7). MCD Viewer (Fluidigm, version 1.0) was used to export raw 16-bit tiff images for computational analyses on histoCAT (version 1.75) [36 (link)]. For visualization purposes, images were processed in MCD Viewer and ImageJ [38 (link)].

Data from Hyperion mass cytometry system were converted to TIFF format using the Fluidigm MCD™ Viewer for data visualisation. The preprocessing of IMC images for individual cell segment followed the protocol of ‘ImcSegmentationPipeline’ (https://github.com/BodenmillerGroup/ImcSegmentationPipeline). We processed image segmentation and cell measurement with CellProfiler (version 4.2.1) and Ilastik (version 1.3.3) software tools. The downstream analysis of preprocessed IMC images, including but not limited to: object construction, dimension reduction and visualisation, cell clustering and phenotyping, and spatial analysis, was based on the ‘imcRtools’ R packages.²⁵