Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Summary

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Abstract

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system's image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

Introduction

The importance of the numerous cell types surrounding clonal tumor populations is a crucial element in the categorization of carcinogenesis. Interest in elucidating this tumor microenvironment (TME) composition and interactions has risen continuously following the establishment of immune-based therapy as part of the cancer treatment arsenal. Therefore, treatment strategies have shifted from a tumor-centric approach to a TME-centric one¹.

Efforts to elucidate the roles of immune cells in tumor surveillance and cancer development have increased strikingly in recent years^2,3. In medical research, a plethora of methods, including cytometry-based methods and singleplex and multiplex imaging technologies, arose as part of this attempt to decipher the unique interactions of multiple elements of TMEs.

Pioneering methods such as flow cytometry (invented in the 1960s), fluorescence-activated cell sorting, and mass cytometry are focused mainly on identifying and quantifying TME components⁴. Even though cytometry-based quantitative techniques allow for immune landscape phenotyping, determining the cellular spatial distribution is impossible. Conversely, methods such as standard singleplex immunohistochemistry preserve the tissue architecture and enable researchers to analyze cellular distribution, though a reduced number of targets in a single tissue section is a limitation of these methods^5,6. Over the past several years, multiplexed imaging technologies for single-cell resolution such as multiplex immunofluorescence, barcoding fluorescence imaging, and imaging mass spectrometry have emerged as comprehensive strategies for acquiring information on simultaneous marker staining using the same tissue section⁷.

Here we present a technology that couples metal tagged antibodies and secondary ion mass spectrometry and enables single-cell resolution quantification, marker co-expression (phenotyping), and spatial analysis using formalin-fixed, paraffin-embedded (FFPE), and fresh frozen (FF) tissue samples^8,9. FFPE samples are the most widely used materials for tissue archiving samples and represent a more readily available resource for multiplexed imaging technologies than fresh frozen samples¹⁰. Additionally, this technology offers the possibility of reacquiring images months after. Herein, we discuss our staining and image processing protocols using FFPE tissue samples.

Protocol

Tissue samples were obtained for research purposes in accordance with the Institutional Review Board of The University of Texas MD Anderson Cancer Center, and samples were further de-identified.

1. Antibody selection

1. Define the research questions to choose the best available antibody clones. Use the Human Protein Atlas, published research, and antibody manufacturer websites as research resources.
   NOTE: The selection of adequate human tissue controls and the same human tissue controls to be stained in the different steps is critical to make sure that the markers are stained correctly, in the right place, and in the correct cellular compartment. A small tissue microarray (TMA) including normal tissue and neoplastic tissues is always recommended for staining validation.
2. Use only carrier-free antibodies to design the panel.
   NOTE: The use of carrier-free antibody formulations is required if a commercial carrier-free antibody is not available; purification kits can be used to adjust the antibody to the correct formulation. Protein additives such as bovine serum albumin are known to diminish conjugation capacity.
3. Test and titrate each antibody using standard chromogenic immunohistochemistry to determine the subcellular pattern of a marker's expression; membrane, cytoplasmic, or nuclear staining; and expression in specific cells in control tissues.
   NOTE: However, each antibody must be tested in pH 6 citrate and pH 9 ethylenediaminetetraacetic acid (EDTA), and whether the panel will be stained with pH 6 citrate or pH 9 EDTA must be determined. The use of pH 9 EDTA is recommended to obtain good signals, especially when working with this methodology.
4. Choose the optimal staining concentration according to the pattern of expression in positive controls.
5. Assign each antibody to one of the available metal isotopes according to the immunohistochemical marker abundance, subcellular localization, and cellular co-expression with the other targets.
6. Stain the antibody with the corresponding assigned metal and compare with the expression in the same control tissue used previously.
   NOTE: Antibody metal conjugation starts with antibody preparation, reduction, and subsequent conjugation (Supplementary File 1). Each metal-loaded polymer tube is sufficient for labeling 100 µg of an antibody.

2. Antibody panel design

1. Create small batches of antibody staining. Test up to five markers in a cocktail.
   NOTE: Testing antibodies already conjugated and analyzed using immunohistochemistry in batches facilitates the early identification of channel interferences and provides the opportunity to reconjugate the antibodies with another metal. It also provides an opportunity to evaluate adequate marker co-expression.
2. Stain each small batch with four different antibody concentrations (0.05 µg/mL, 0.2 µg/mL, 1 µg/mL, and 4.0 µg/mL).
   NOTE: Comparing different titers, identify the antibody concentration that results in the correct location and highest expression with minimum background staining (best signal-to-noise ratio).

3. FFPE tissue sectioning

1. Select gold-coated slides (specific for this purpose) and keep them close to the microtome. Prepare the microtome by inserting a new blade into the holder. Set the section thickness to 4-5 μm.
2. Put FFPE blocks on ice before sectioning. Prepare a tissue floatation bath by heating distilled water or deionized water (diH₂O) to 40-45 °C.
   NOTE: Using diH₂O or distilled water reduces the possibility of contamination.
3. Start sectioning. Place the tissue section in the water using tweezers and let it flatten. Use the slides to remove tissue sections from the water.
4. Place the slides in a slide rack and let them dry at room temperature overnight.

4. FFPE antibody staining

NOTE: The staining process takes place on two separate days.

CAUTION: The solutions employed in this protocol are potentially corrosive and represent hazards to the skin and eyes. Wearing gloves, a lab coat, and protective eye and face equipment is advised and part of our institution's biosafety policy.

1. Reagent preparation (day 1)
   1. Dilute 50 mL of 20x Tris-buffered saline with Tween (TBS-T) into 950 mL of diH₂O to make 1 L of TBS-T.
   2. Dilute 8 mL of 10x Tris-ethylenediaminetetraacetic acid (EDTA) into 72 mL of diH₂O to make a 1x heat-induced epitope retrieval (HIER) solution.
   3. Dilute donkey serum in 1x TBS-T to a final concentration of 5% to make blocking buffer. Store the solution at 4 °C until ready to use.
2. HIER preparation: Pretreatment (PT) module
   CAUTION: Wear chemical protective gloves when handling any reagents or parts immersed in any reagents used in the PT module.
   1. Dilute 140 mL of 10x phosphate-buffered saline (PBS) into 1,260 mL of diH₂O to make 1.4 L of 1x PBS. Alternatively, dilute seven PBS tablets into 1.4 L of diH₂O.
   2. Fill a tank with 1.4 L of PBS and place the prepared slide chamber container with 100 mL of 1x HIER solution into the tank.
   3. Preheat the tank in a PT module to 75 °C.
3. Excess paraffin removal
   1. Bake slides at 70 °C in an oven for at least 20 min.
      NOTE: Some tissues or sections may need longer baking times. The recommended baking time for brain tissue or a TMA is 1 h. This can be extended to 16 h (overnight) or according to lab experience.
   2. Place baked slides in a slide holder and perform the following washing steps on a shaker under a fume hood. Wash the slides in the following solution: Xylene 2x for 30 s (metal-free), 100% alcohol 2x for 30 s (metal-free), 95% alcohol 2x for 30 s (metal-free), 80% alcohol for 30 s (metal-free), 70% alcohol for 30 s (metal-free), and diH₂O 2x for 30 s (metal-free).
   3. Keep the slides in a fresh diH₂O container until ready for antigen retrieval.
      NOTE: The slides should not be dried until the end of the procedure.
4. Antigen retrieval
   1. Place the slides in a preheated slide chamber containing 1x HIER buffer inside the PT module. Place the cover on the tank. Close and latch the lid.
   2. Press the Menu button on the PT module to open the main menu and press the Setup cycle (time and temperature) button to create a custom program.
   3. Run the PT module at 97 °C for 40 min. Afterward, the PT module will automatically cool to 65 °C.
   4. Remove the slides from the PT module once the 65 °C temperature is reached. Keep the slides at room temperature for 30 min.
      NOTE: The PT module will not open until the temperature cools to 65 °C. Do not touch the golden surface of the slides during this process, and always use gloves.
5. Antibody blocking
   1. Set a shaker to 70 rpm.
   2. Wash the slides by dipping them in 1x TBS-T for 5 min 2x.
   3. Using a hydrophobic barrier pen, draw a border around the tissue section at least 1 mm away from the slide edges and let it dry for 15-30 s.
      NOTE: Take care to prevent the pen fluid from touching the tissue section to avoid any tissue interference with the staining. Do not press the pen down too hard while drawing the barrier to avoid potential leaks. For optimal staining and image acquisition results, the tissue sections are placed in the middle of the gold-coated slides in a frame no larger than 15 mm x 30 mm to allow enough space to draw the barrier without touching the tissue or the guide dots placed on the outer edge of the slide.
   4. To remove any pen residue, dip the slides in TBS-T.
   5. In a moisture chamber at room temperature, incubate the tissue section with 100-200 μL of blocking buffer for 20 min. Leave the tissue incubating in the blocking buffer until an antibody master mix is ready.
6. Antibody panel preparation
   NOTE: The final volume of the antibody panel cocktail varies according to the estimated tissue section surface area. To cover an area of 20 mm x 20 mm, prepare 100-200 μL of the cocktail.
   Do the following to prepare an antibody panel cocktail of metal isotope-conjugated antibodies:
   1. Confirm the final volume of the cocktail that equals the antibody cocktail volume added to the blocking buffer volume.
   2. Confirm the specifications for all antibodies, dilutions, and/or antibody concentrations in a panel spreadsheet for the project.
   3. Spin down all the antibody-containing tubes at 10,000 x g for 5 min. Add individual antibodies to the blocking buffer and mix very well. Do not disturb the bottom of the antibody tube when pipetting it.
   4. Filter the antibody panel by prewetting a 0.1 μm centrifugal filter device with 100 μL of blocking buffer. Spin the filter at 10,000 x g for 2 min and remove the blocking buffer flow-through with a pipette.
   5. Transfer the antibody panel into a spin column and spin the filter at 10,000 x g for 2 min. Discard the spin column and use the flow-through as the filtered antibody panel.
      NOTE: Perform the staining immediately after making the cocktail to prevent metal exchange. If storage of the antibody cocktail for an extended time is needed, it can be subjected to lyophilization.
7. Antibody staining
   1. Carefully remove the blocking buffer from the slides by tipping each slide on its side and gently tapping the edges against a task wiper.
   2. Place the slides in a moisture chamber and add 100-200 μL of antibody master mix to the tissue (avoid contact with tissue and the creation of air bubbles).
   3. Add positive and negative control slides to the staining batch.
   4. Incubate the slides at 4 °C overnight (14-16 h).
8. Reagent preparation (day 2)
   1. Dilute 100 mL of 10x low-barium PBS, pH 7.4, in 900 mL of diH₂O to make 1x PBS.
   2. Prepare 350 mL of a working stock solution of 2% glutaraldehyde in low-barium PBS (340 mL of low-barium PBS + 10 mL of 70% glutaraldehyde).
      NOTE: Perform the dilution under a hood. Glutaraldehyde is a very viscous liquid. Use a 1 mL pipette and add 1 mL of low-barium PBS to the glutaraldehyde tube every time until it becomes liquid enough to pour out of the tube.
   3. Dilute 10x Tris, pH 8.5, in 900 mL of diH₂0 to make 1x Tris.
9. Fixation and dehydration
   1. Remove the antibody master mix from each slide by tipping the slide on its side and gently tapping the edge against a task wiper.
   2. Wash the slides with light to moderate agitation in the following buffers: 1x TBS-T, 3x times for 5 min each (metal-free); filtered 2% glutaraldehyde for 5 min; filtered 1x tris, pH 8.5, 3x for 30 s; filtered diH₂O 2x for 30 s; 70% alcohol for 30 s (metal-free); 80% alcohol for 30 s (metal-free); 95% alcohol for 30 s (metal-free); and 100% alcohol for 30 s (metal-free).
   3. Gently tap the edge of each slide against a task wiper to remove excess alcohol and allow residual alcohol to evaporate at room temperature (~5-10 min).
   4. Dry slides in a desiccator for at least 1 h prior to image acquisition or keep the slides in a vacuum chamber for long-term storage.

5. Image acquisition

1. Slide setup
   NOTE: Before scanning using the instrument, the slides must be defined and set up using the web-based image management application following the steps described below.
   1. On the slide page in the web-based image management application, click Accession New Slide and enter the desired details (name, slide identification, location, and description).
   2. Create scanning sections by clicking Add Section. Fill in the information for each section (name of the project, slide name, block, and position). To save the new slides/sections created, click Submit.
   3. Under the resources tab, open the panels page and select the panel to be used. For new panels, click Create New Panel.
   4. After selecting the panel, click on Sections. Add the sections created in step 2. by clicking Edit Section Assignment. Save by clicking Submit.
2. Instrument setup
   NOTE: This instrument (see Table of Materials) is operated using a specific control software program (see Table of Materials).
   1. Log in to the control software. Click Wake Up if the instrument is in sleep mode.
      NOTE: At least 1 h before the operation, warming up the instrument is recommended, which helps with beam focus stabilization.
   2. To load a slide, click on Exchange Sample and then click Continue to open the door. Put the slide in the loading slot with the sample facing up and the label on the right. Click Continue to close the door.
      NOTE: If the slide is not loaded correctly, a warning sound and notification to adjust the slide will appear.
   3. Select the project and then select the slide name for the loaded sample.
   4. Visualize the panoramic image on the slide optical image pane.
3. Field of view (FOV) selection and acquisition
   1. Click on the Mode menu and select SED (Secondary Electron Detector). Click on the imaging mode menu and pick QC −300 μm.
   2. Click on Jog Stage to control the FOV location and then click on the arrows to navigate and select the position of the FOV.
   3. Click Add FOV, set 400 μm x 400 μm as the field size, and select fine as the imaging mode and 1 ms of dwell time. Click Confirm.
      NOTE: The set dwell time will depend on the desired resolution. The dwell time increases as the resolution increases. The acquisition time may vary depending on multiple factors, including detector break-in period and length of operation. Our mean acquisition time for one FOV is about 17 min. Use the instrument nonstop for up to 8 h, which allows for scanning of about 28 FOVs. The quality of the acquired images decreases after many consecutive hours of use.
   4. After creating an FOV list, proceed to image focus and adjust the stigmation by moving the beam to a tissue region that will not be imaged. Adjust the parameters until a clear central image is obtained.
      NOTE: To optimize image acquisition, keeping the tissue section centralized on the slide is ideal. While focusing, a clear image of only the central area can be obtained. If the tissue section is too large, the edges will be out of focus, and the image will be blurred.
   5. Click on Start Run. Acquired images will be automatically uploaded and stored in the web-based image management application.

6. Image preparation

1. Image isobaric correction
   NOTE: The purpose of isobaric correction is to remove spillover signal between channels resulting from the presence of isotopes of equal or very similar mass whose differences in mass cannot be detected using the mass analyzer.
   1. In the web-based image management application, click on the green Download icon to save the FOVs (TIFF images).
   2. Download the most updated version of the image processing software (see Table of Materials) available on the about tab in the web-based image management application and save it in a file. Open the .zip archive file and follow the installation steps. Open the software once the installation is complete.
   3. Select the folder containing the images to be corrected by clicking on the File icon on the input pane of the image processing software. An FOV list will be loaded.
   4. Click on the Filter icon on the input pane to apply default corrections. For each channel, visualize images obtained before and after correction on the right side of the screen.
   5. Click on the Floppy disk icon on the output pane to save the corrected image.
2. Image filtering: Voronoi tessellation
   NOTE: Voronoi tessellation diagrams illustrate a partition of space containing seed points into Voronoi cells. The aim is to estimate the cell density and define cell boundaries. Using the Voronoi tessellation calculation, a mask is generated and applied to the original image for filtering.
   1. Click on the Filtering tab and select Voronoi Tesselation on the filtering parameters menu.
   2. On the input pane, select the MassCorrected.tiff image. Click on the Filter icon on the input pane.
   3. Click on the Floppy disk icon on the output pane to save the filtered image. The two resulting files will have the suffixes -Filtered.tiff and -Filtered.json.
      NOTE: The image named MassCorrected-Filtered.tiff then can be uploaded to a third-party digital analysis software program or open-source software program.

Results

Tonsil and lung adenocarcinoma TMA tissue sections (5 mm thick) were obtained and placed on the middle of gold-coated slides following the specifications regarding tissue size and secure margins of the slides. Free glass margins of 5 mm and 10 mm between the edge of the tissue and the lateral and inferior borders of the glass slides, respectively, are necessary for optimal staining. The tissue sections were baked overnight in an oven prior to staining to assure proper adherence of the section to the slide. The antibody p...

Discussion

The comprehensive elucidation of the complex, intricate interactions among the multiple components of a TME remains a pivotal objective of cancer research. Manufacturers have introduced numerous multiplexed assays as part of this effort, especially over the past 5 years. Multiplexed spatial analysis is a versatile and powerful tool that facilitates the simultaneous categorization of several targets while preserving structural morphology in tumor samples. Spatial analysis techniques can be performed using fluorescence ima...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100% Reagent Alcohol	Sigma-Aldrich	R8382
95% Reagent Alcohol	Sigma-Aldrich	R3404
80% Reagent Alcohol	Sigma-Aldrich	R3279
70% Reagent Alcohol	Sigma-Aldrich	R315
20X TBS-T	Ionpath	567005
10X Low-Barium PBS pH 7.4	Ionpath	567004
10X Tris pH 8.5	Ionpath	567003
4°C Refrigerator	ThermoScientific	REVCO
Aerosol Barrier Pipette Tips P10	Olympus	24-401
Aerosol Barrier Pipette Tips P20	Olympus	24-404
Aerosol Barrier Pipette Tips P200	Olympus	24-412
Aerosol Barrier Pipette Tips P1000	Olympus	24-430
Centrifugal Filter Ultrafree-MC	Fisher Scientific	UFC30VV00
Deionized H2O	Ionpath	567002
Donkey serum	Sigma-Aldrich	D9663
EasyDip Slide Staining Jar, Green	Electron Microscopy Sciences	71385-G
EasyDip Slide Staining Jar, Yellow	Electron Microscopy Sciences	71385-Y
EasyDip Slide Staining Kit (Jar+Rack), White	Electron Microscopy Sciences	71388-01
EasyDip Stainless Steel Holder	Electron Microscopy Sciences	71388-50
Glutaraldehyde 70% EM Grade	Electron Microscopy Sciences	16360
Heat Induced Epitope Retrieval (HIER) buffer: 10X Tris with EDTA, pH 9	Dako	S2367
Heat resistant slide chamber	Electron Microscopy Sciences	62705-01
Hydrophobic barrier pen	Fisher	50-550-221
MIBI/O software	Ionpath	NA
MIBIcontrol software	Ionpath	NA
MIBIslide	Ionpath	567001
MIBIscope	Ionpath	NA
Microcentrifuge	Eppendorf	5415D
Microtome	Leica	RM2135
Moisture Chamber (Humid Chamber)	Simport	M922-1
Phosphate Buffered Saline (PBS) Tablets	Fisher Scientific	BP2944100
PT Module	Thermo Scientific	A80400012
Rapid-Flow Sterile Disposable Filter Units	Fisher Scientific	097403A
Shaker	BioRocker	S2025
Spin column (Ultrafree-MC Spin Filter, 0.5mL 0.1μm )	MillQ	UFC30VV00
Slide oven	Fisher Scientific	6901
Vaccum Cabinet Desiccator	VWR	30621-076
Task-whipe	Kimberly Clark	34155
Xylene	Sigma-Aldrich	534056-4L