Name: digital spatial profiling for characterization of the microenvironment in adult-type diffusely infiltrating glioma
Uploaded: 2022-09-13T12:50:00
Description: Watch this Scientific Journal Video about Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma at JoVE.com

The authors acknowledge the support of the Laboratory for Clinical Genomics and Advanced Technology in the Department of Pathology and Laboratory Medicine of the Dartmouth Hitchcock Health System. The authors also acknowledge the Pathology Shared Resource at the Dartmouth Cancer Center with NCI Cancer Center Support Grant 5P30 CA023108-37.

The protocol outlined below follows the guidelines of the Dartmouth-Hitchcock Human Research Ethics Committee. Informed consent was obtained from the patients whose tissue samples were included in this study. See the Table of Materials section for details related to all materials, reagents, equipment, and software used in this protocol.
1. Slide preparation17
<ol>
	<li>Retrieve or prepare formalin-fixed, paraffin-embedded tissue from ...

<ol>
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Given the diversity of proteins that could potentially influence the aggressiveness of gliomas and the notion that several of these proteins remain undiscovered, a high-throughput protein quantification method is an ideal technologic approach. Additionally, given that spatial data in oncologic samples often correlates with differential expression18, incorporating spatial profiling into the protein quantification approach allows for more effective analysis.
The high-thro...

Diffusely infiltrating gliomas are the most common type of malignant brain tumor in adults and are invariably lethal. The propensity for glioma cells to migrate extensively in the brain is a major therapeutic challenge. The mechanism by which they spread involves directed migration and unchecked invasion. Invasive glioma cells have been shown to exhibit tropism and migration along white matter tracts1, with recent research implicating demyelination of these tracts as an active, protumorigenic feature2. Invasion is mediated by an epithelial-to-mesenchymal transition, in which glioma cells acquire mesenchymal properties by reducing the expression of genes encoding extracellular matrix proteins and cell adhesion molecules, amplifying migration and facilitating propagation through the tumor microenvironment3,4,5.
At the molecular level, disruption of several proteins that confer cellular stability and interface with immunogenic components has been demonstrated6. Infiltrative gliomas are known to undergo suppression of proteins with anti-apoptotic (e.g., PTEN) properties7. They also overexpress proteins that promote evasion of the host immune response (e.g., PD1/PDL1)8. The dysregulation of these complex pathways enhances tumorigenicity and increases malignant potential.
Within samples of invasive glioma, the aim was to evaluate the differential expression of proteins key to cell growth, survival, and proliferation, and to microenvironment structural integrity between invasive and non-invasive components. Additionally, we sought to study the differential regulation of proteins with an active immunogenic role, offering insight to the mechanism by which compromised host immune defenses may enhance the proliferative and invasive potential of gliomas. This is especially relevant given the recent breadth of research demonstrating how immune markers and drivers of dysregulation in malignancy can serve as targets of immunotherapy. Identifying viable therapeutic targets among the many proteins involved in immunosurveillance and reactivity requires a highly sensitive and comprehensive approach.
Given the wide array of candidate proteins that can be studied, we sought a method akin to immunohistochemistry but with enhanced data processing efficiency. Within the field of cancer biology, DSP has emerged as a powerful technology with important advantages over alternative tools for proteomic analysis and quantification. The hallmark of DSP is its high-throughput multiplexing capability, allowing for simultaneous study of several different proteins within a sample, marking an important distinction from standard but lower-plex technologies such as immunohistochemistry (IHC)9,10. The multiplex feature of DSP does not compromise its fidelity as a quantitative and analytical tool, as demonstrated by studies comparing DSP to IHC. When used for proteomic quantification of non-small cell lung cancer specimens, for example, DSP has been shown to have similar results to IHC11. Additionally, DSP offers customizable regional specification, in which users can manually define regions within which to perform proteomic analysis. This presents an advantage over whole-section multiplex methods10,12. In a single round of processing, DSP thus offers multiple layers of analysis by surveying several protein targets across multiple regions of interest.
DSP has applications in several different pathological settings. DSP is especially advantageous in oncologic analysis, as spatial variation can correlate with cellular transformation and differential protein expression. For example, DSP has been used to compare the proteomic profile of breast cancer to the adjacent tumor microenvironment. This carries important implications for understanding the natural history of this tumor and its progression, as well as potential response to treatment13. Additional contexts illustrating the versatility of DSP include spatial quantification of protein diversity in prostate cancer14, association of immune cell marker expression with disease progression in head and neck squamous cell carcinoma15, and demonstration of an epithelial-mesenchymal gradient of protein expression distinguishing metastatic from primary clear cell ovarian cancer16. By implementing DSP, we characterize the spatial topography of proteins that could impact tumorigenesis and invasion of gliomas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BOND Research Detection System</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>DS9455</td>
			<td>Open detection system containing open containers in a reagent tray</td>
		</tr>
		<tr>
			<td>BOND Wash</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>AR950</td>
			<td>10X concentrated buffer solution for washing fixed tissue</td>
		</tr>
		<tr>
			<td>Buffer W</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Cy3 conjugation kit</td>
			<td>Abcam, Cambridge, UK</td>
			<td>AB188287</td>
			<td>Cy3 fluorescent antibody conjugation kit</td>
		</tr>
		<tr>
			<td>GeoMx Digital Spatial Profiler (DSP)</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>System for imaging and characterizing protein and RNA targets</td>
		</tr>
		<tr>
			<td>GeoMx DSP Instrument BufferKit</td>
			<td>NanoString, Seattle, WA</td>
			<td>100471</td>
			<td>Buffer kit for GeoMX DSP (including buffers for sample processing and preparation)</td>
		</tr>
		<tr>
			<td>GeoMx Hyb Code Pack_Protein</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300401</td>
			<td>Controls for running GeoMX DSP experiemtns</td>
		</tr>
		<tr>
			<td>GeoMx Immune Cell Panel (Imm Cell Pro_Hs)</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300101</td>
			<td>Protein module with targets for human immune cells and immuno-oncologic targets</td>
		</tr>
		<tr>
			<td>GeoMx Pan-Tumor Panel (Pan-Tumor_Hs)</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300105</td>
			<td>Protein module with targets for multiple human tumor types and for markers of epithelial-mesenchymal transition</td>
		</tr>
		<tr>
			<td>GeoMx Protein Slide Prep FFPE</td>
			<td>NanoString, Seattle, WA</td>
			<td>121300308</td>
			<td>Sample preparation reagents for GeoMX DSP protein analysis</td>
		</tr>
		<tr>
			<td>IDH1-R132H antibody</td>
			<td>Dianova, Hamburg, Germany</td>
			<td>DIA-H09</td>
			<td>Monoclonal antibody against human IDH1 R132H</td>
		</tr>
		<tr>
			<td>LEICA Bond RX</td>
			<td>Leica Biosystems, Wetzlar, Germany</td>
			<td>contact company</td>
			<td>Fully automated IHC stainer</td>
		</tr>
		<tr>
			<td>Master Kit--12 reactions</td>
			<td>NanoString, Seattle, WA</td>
			<td>100052</td>
			<td>Materials and reagents for use with the nCounter Analysis system</td>
		</tr>
		<tr>
			<td>nCounter Analysis System</td>
			<td>NanoString, Seattle, WA</td>
			<td>contact company</td>
			<td>Automated system for multiplex target expression quantification (to be used with GeoMx DSP)</td>
		</tr>
		<tr>
			<td>TMA Master II</td>
			<td>3DHistech Ltd., Budapest, Hungary</td>
			<td></td>
			<td>To create the tissue microarray block</td>
		</tr>
	</tbody>
</table>

digital spatial profiling for characterization of the microenvironment in adult-type diffusely infiltrating glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Figure 4 shows the representative results from a DSP experiment performed on samples of glioblastoma. A heat map is presented, illustrating one of the methods by which to capture data visually using the DSP software. Rows represent protein targets, and each column corresponds to a region of interest. A color range of blue to red denotes low to high expression, respectively. Variability of color within a row reflects regional protein heterogeneity and suggests a possible spatial association w...

Watch this Scientific Journal Video about Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma at JoVE.com

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

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

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.

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...