Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Digital Spatial Profiling, or DSP, offers an efficient method for spatially stratified protein quantification. Its implementation enables us to characterize protein expression across distinct regions of a tumor and the microenvironment. A key feature of DSP is its multiplexing capability, enabling high throughput data processing.

The ability to define customized regions of interest additionally provides a spatial dimension to data analysis. DSP can be applied to any sample in which spatially quantifying protein, or RNA expression may help elucidate a pathologic, or physiologic process. This is especially relevant in oncology, where regional target variability may correlate with malignant growth, or invasion.

Demonstrating the procedure will be Scott Palisoul, a histotechnologist, and Rachael Barney, a genomic technologist. To prepare slides, begin by taking several two-millimeter cores from each biopsy, and putting them in a single tissue microarray block. Cut four-micron sections from the block, and mount them to glass slides.

Place each slide inside the slide holder gasket, and incubate it at 60 degrees Celsius for 30 minutes. In the semi-automated IHC system, fill the container in position one with 150 microliters of wash buffer per slide, plus five milliliters of dead volume, and leave the lid open. Fill the same amount of blocking solution in the container in position two.

Load the reagent container tray onto the machine. Select the Process IHC, followed by blocking solution name in the dropdown box of the Marker field. Once finished for all slides, click on Close.

Then click on Print Labels, and check All slide labels not yet printed, and click on Print. Affix labels to the tops of the slides. Load the slides onto the slide tray, ensuring the sample and label face upward.

Press the LED button to lower the tray, and allow the instrument to begin the scanning and recognition of slides. Click on the Start button to begin the experiment. When the run is complete, press the LED button when it blinks green.

Remove the tray from the instrument, and carefully lift the cover tiles from each slide. Place the slides in the 1X phosphate buffered saline. Remove excess buffer, and outline each tissue section with a hydrophobic pen to create a hydrophobic barrier.

Make an antibody PC oligo solution by adding eight microliters of each antibody per slide and using Buffer W to reach a final volume of 200 microliters. Then apply the antibody PC oligo solution to the slides and incubate the slides overnight at four degrees Celsius. After incubation, place the slides in a humidified tray, and wash thrice for 10 minutes in 1X TBST.

Post-fix in 4%paraformaldehyde for 30 minutes at room temperature, followed by washing twice for five minutes in 1X TBST. Add SYTO 13 nuclear stain for 15 minutes at room temperature, followed by washing twice with 1X TBST. Hover the mouse over Data Collection in the Control Center and select New, or Continue Run.

Place the slide in the slide holder with the label toward the user. Lower the slide tray clamp, ensuring that the tissue is visible in the elongated window. Add six milliliters of Buffer S.Follow the prompts in the Control Center.

Zoom between different axes with the x and y sliders to delineate a region for scanning. Select Scan, and allow the scan to proceed until the entire defined target area has been imaged. Generate a 20 times image.

Define the ROIs by selecting three equally sized circular ROIs of a diameter of 250 micrometers for each tissue core. Approve the ROIs by clicking on the Exit Scan Workspace button. Then wait for the UV light to cleave the oligos from the antibodies.

After completing the cleaning instrument process, select New Data collection to continue with the current slides, or plate. Open the finalized plate window by double clicking on the Plate icon area. Enter the hybridization code pack lot number, and click on Update.

Then click on Finalize. Detach the slide holder and collection plate by following the prompts. Store the slides in TBST, or cover them with an aqueous medium and cover slip for long-term storage.

Seal the plates using a permeable seal and dry the aspirates at 65 degrees Celsius in a thermal cycler with the top open. Add seven microliters of diethyl pyrocarbonate-treated water and mix. Incubate at room temperature for 10 minutes, and then spin down quickly.

Prepare the probe buffer mix by choosing appropriate probe R and probe U equations applying the equation as described in the text protocol. Add 84 microliters of probe buffer mix to each hybridization code pack. In a fresh 96-well hybridization plate, add eight microliters of each hybridization code master mix into all the 12 wells of the indicated row.

Transfer seven microliters from the DSP collection plate to the corresponding well in the hybridization plate. Heat seal, spin the plate, and incubate it overnight at 67 degrees Celsius. After incubation, cool the plate on ice, and perform a quick spin.

Pool the hybridization products from each well into a strip tube, gently pipetting each well five times. Spin down, and load the strip tubes into the analysis system. On the DSP instrument, save the CDF file onto a USB drive, and transfer the data to the digital analyzer.

On screen, press Start Processing. Select High Sensitivity followed by Next. Press Select All for the number of wells with samples, then Finish, followed by Next on the email notification.

Then click on Start. Once the prep station is done, seal the cartridge and transfer it to the digital analyzer. Press Start Counting.

Select Stage Position. Press Load Existing CDF file, and select previously uploaded file. Press Done.

Select Stage Position again, press Done, then Start to run the program. Save the ZIP file of the reporter count conversion files from the analysis system to a USB drive. Insert the drive into the DSP machine.

In the DSP Control Center, hover over Data Collection, and then click on Upload Accounts. Select the relevant ZIP file. Click on Records in the DSP Control Center.

To view scans in the queue, select Add Selected Scans to Queue, then My Analysis Queue. Select New Study from Queue by hovering over the Data Analysis option in the Control Center. DSP experiment was performed on samples of glioblastoma, and the results were visualized as a heat map.

Rows represent protein targets, and each column corresponds to a region of interest. Expression level is depicted by a color range varying from blue, showing low expression, to red, which denotes high expression. Variability of color within a row reflects regional protein heterogeneity, and suggests a possible spatial association with differential protein expression.

In this experiment, S100 and CD-56 were universally high, because they are neural markers. Markers with the most variability include B7-H3, KI-67, CD-44, and fibronectin, which are known to be associated with tumor proliferation, migration, and metastasis. From a vast array of candidate targets, DSP can identify those exhibiting significant regional variation.

This lays the foundation for investigating factors associated with variability and their relevance to disease.