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

Summary

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 µm.

Abstract

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 µm and 300 µm diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Introduction

Advances in multiplexing techniques continue to provide better characterization tools for targets present in tumors. The tumor microenvironment (TME) is a complex system of tumor cells, infiltrating immune cells, and stroma, where spatial information is critical to better understand and interpret mechanisms of interaction between biomarkers of interest¹. With emerging techniques such as the GeoMx Digital Spatial Profiler (DSP) and 10x Visium, multiple targets can be detected and quantified simultaneously within their spatial context. The use of immunofluorescence protocols that facilitate tissue visualization can further improve the spatial pro....

Protocol

All human tissues were acquired from commercial biobanks or accredited tissue banks under warranty that appropriate Institutional Review Board approval and informed consent were obtained.

NOTE: The protocol is performed using the Discovery Ultra and the GeoMx Digital Spatial Profiler. See the Table of Materials for details about reagents, equipment, and software used in this protocol.

1. Automated visualization protocol for spatial proteomics assays

1. Program autostainer to apply fluorescent visualization antibodies
   1. In the autostainer software, click on the ....

Results

Automated visualization protocol to guide ROI selection
In this paper, we present the use of an automated, custom TSA-based IF protocol to visualize the tissue and select specific ROIs. Visualization panel development using melanoma and human normal skin as control tissues consisted of epitope stability testing, fine-tuning of marker intensities, and bleedthrough assessment through leave one out controls. To test if epitope stability of the antibodies is affected by repetitive elution steps, FAP and .......

Discussion

To date, directly conjugated fluorescent antibodies in a manual protocol are most commonly used as visualization panels for spatial proteomics or spatial transcriptomics assays^9,10. However, the use of directly conjugated fluorescent antibodies can be challenging for less abundant markers, limiting the selection of suitable antibodies. This protocol shows that labeling of visualization markers can be automated on an automated staining platform using TSA technolog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Tris buffered saline (TBS)	Cell Signaling Technologies	12498S	Diluted to 1x TBS in DEPC treated water
Antibody X (not disclosed)			antibody blinded due to confidentiality
DEPC-treated water	ThermoFisher	AM9922	Another can be used
DISCOVERY Cell Conditioning ( CC1)	Ventana	950-500
DISCOVERY Cy5 Kit	Ventana	760-238	Referred as Cy5
DISCOVERY FAM Kit	Ventana	760-243	Referred as FAM
DISCOVERY Goat Ig Block	Ventana	760-6008	Referred as Gt Ig Block
DISCOVERY OmniMap anti-Ms HRP	Ventana	760-4310	Referred as OMap anti-Ms HRP
DISCOVERY OmniMap anti-Rb HRP	Ventana	760-4311	Referred as OMap anti-Rb HRP
DISCOVERY Rhodamine 6G Kit	Ventana	760-244	Referred as Rhodamine 6G
DISCOVERY ULTRA Automated Slide Preparation System	Ventana	05 987 750 001 / N750-DISU-FS	Referred as autostainer on the manuscript
FAP [EPR20021] Antibody	Abcam	Ab207178
GeoMx Digital Spatial Profiler	NanoString	GMX-DSP-1Y	Referred as spatial profiling platform on the manuscript
Humidity chamber	Simport	M920-2	Another can be used
Pan-Cytokeratin [AE1/AE3] Antibody	Abcam	Ab27988
ProLong Gold Antifade Mountant	ThermoFisher	P36934
Python	Python		Statistical analysis
Reaction Buffer (10x)	Ventana	950-300
Statistical analysis software	GraphPad	Prism 7	Statistical analysis
SYTO 64	ThermoFisher	S11346
ULTRA Cell Conditioning (ULTRA CC2)	Ventana	950-223
Ventana Antibody Diluent with Casein	Ventana	760-219	Referred as specified diluent on the manuscript
Ventana Primary antibody dispenser	Ventana		Catalog number depends on dispenser number