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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Retrieve or prepare formalin-fixed, paraffin-embedded tissue from human adult-type diffusely infiltrating gliomas.
   NOTE: In this experiment, paraffin-embedded blocks of biopsies from three patients with gliobl....

Results

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.......

Discussion

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.......

Materials

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