Bex-Plex Enables Simultaneous Visualization of Protein Markers and Ribonucleic Acid Transcripts for Deep Spatial Profiling of Intact Tissue

Summary

Here, we present a protocol for combining iterative protein staining (IBEX) with RNAscope in situ hybridization technology for the deep spatial phenotyping of intact fixed frozen tissues for high-resolution spatial profiling applications.

Abstract

Here, we present a protocol to enable simultaneous spatial profiling of protein and ribonucleic acid (RNA) markers at single-cell resolution in intact tissues. One of the core issues surrounding solid tumor cancers is the inability to predict patient responsiveness to therapeutic intervention. Deciphering these complexities requires quantitative measurement of cellular behaviors, interactions, and phenotypes within intact tissues. Specifically, the proteome and transcriptome provide overlapping but nonredundant information that can be critical to designing rational immunotherapeutics. However, there is not currently a straightforward and cost-effective way to deeply profile both the transcriptional and functional state of all cells in the tumor microenvironment while retaining their critical spatial localization information. To this end, we have successfully combined the IBEX (iterative bleaching extends multiplexity) platform with HiPlex RNAscope to simultaneously visualize high-plex protein markers and high-plex ribonucleic acid (RNA) transcripts with single-cell resolution. This approach incorporates a modified RNA target retrieval step to preserve nuclear morphology and enable accurate image alignment across cycles. “Bex-Plex” enables us to perform an unmodified IBEX protocol on fixed frozen tissues followed by the HiPlex RNAscope protocol, in which we use a modified RNA target retrieval to ensure nuclear-level alignment with IBEX cycles. This protocol is optimized for fixed frozen tissues and enables integrated spatial analysis of transcriptional and functional cellular states. The power to profile at this single-cell level not only provides a deep well of information about the tissue itself but can also empower predictions about the nuanced cellular interactions that drive therapeutic responsiveness, providing a framework for studying spatial biology in tumor microenvironments and related systems.

Introduction

Since their introduction in 2011, cancer immunotherapies that decrease the suppression of cytotoxic T cells by blocking inhibitory immune checkpoint molecules such as PDL-1 and CTLA-4 have emerged as the gold standard for solid tumor treatment¹. However, despite large-scale use in patients, the clinical efficacies of these drugs vary widely because many solid tumor cancers either intrinsically present with or eventually generate resistance to these therapies². In many cases this is due to the extremely heterogeneous nature of solid tumors, even among the same cancer type³. A more in depth understanding of the ....

Protocol

The procedures outlined in this protocol were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego (protocol number S24049). Male and female mice 7-12 weeks of age on a C57BL/6 genetic background and housed under specific pathogen-free conditions were used for these experiments.

1. Tissue preparation

1. Euthanize experimental mice by approved primary and secondary measures (e.g. CO₂ euthanasia and cervical dislocation) and harvest tissues of interest into supplemented media.  
2. Prepare a fresh stock of tissue fixative by diluting a 4% stock ....

Results

Figure 1 : Overview of Bex-Plex workflow.
Fixed frozen tissues sectioned onto chrome alum-coated slides undergo up to 3 cycles of standard IBEX followed by up to three cycles of RNAScope HiPlex wherein the initial target retrieval step is p.......

Discussion

The Bex-Plex workflow integrates the iterative protein staining platform, IBEX, with RNAscope in situ hybridization to enable simultaneous detection of proteins and RNA transcripts at single-cell resolution. The primary goal of this protocol is to provide a reproducible method for integrated spatial analysis of protein and RNA expression within intact tissues. The ability to measure both protein and RNA expression in the same tissue has previously presented technical challenges: high costs, complex antigen retri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 ml microcentrifuge tubes	Biopioneer	MCNT-1.5F
200 proof EtOH	Fisher Scientific	BP28184
20X SSC buffer	Invitrogen	AM9765
24 well plates
Alexa Fluor 488 anti-mouse CD11b Antibody	Biolegend	101217
Alexa Fluor 523 anti-mouse CD3e Antibody	Invitrogen	58-0032-82
Alexa Fluor 647 anti-mouse Ly6G Antibody	Biolegend	127610
Alexa Fluor 700 anti-mouse CD68 Antibody	Biolegend	137025
BD Cytofix/Cytoperm fixation and permeablization solution	BD	554722
Chrome alum gelatin	Fisher	NC1692262
Confocal microscope (e.g Leica Stellaris)
Cryomolds	VWR	25608-922
Cryostat (e.g CryoStar NX50 Cryostat)	Thermofisher Scientific	957100
Cryostat blades (e.g Leica Low Profile Disposable Blades DB80LX)	Leica Biosystems	14035843496
DAPI	Thermofisher Scientific	62248
Dissection microscope (e.g Zeiss SteREO Discovery.V8)
Dissection tools
Dry Ice
Dunk tank for slide staining (e.g Simport Scientific EasyDip Slide Staining Jars)	Fisher Scientific	22-038-489
Fluoromount-G	Fisher	OB10001
Heated water bath
Histology brushes (e.g. Ted Pella Camel Hair Brushes)	Fisher Scientific	NC2023341
Hybridization oven (e.g. HybEZ II Hybridization System)	ACD	321710
Image analysis software of choice
ImmEdge Hydrophobic Barrier PAP Pen	Vector Laboratories	H-4000
Kimwipes	VWR	21905-026
Lithium borohydride	STREM Chemicals	93-0397
Micropipettes (P-10, P-20, P-200, P-1000)
Mini Microcentrifuge (e.g. Corning LSE Mini Microcentrifuge)	Corning	6770
Mouse BD Fc Block	BD	553142
No 1.5 Coverglass	VWR	48393-241
Pipette tips (P-10, P-20, P-200, P-1000)
RNAscope HiPlex Probe, mm-CD274-T7	ACD	420501-T7
RNAscope HiPlex Probe, mm-IL-10-T6	ACD	317261-T6
RNAscope HiPlex12 Reagents Kit v2 (includes detection kit, cleaving stock solution, protease reagents, target retreival reagents, and wash buffer)	ACD	324409
RNAscope Protease III	ACD	322337
RNase ZAP	Millipore Sigma	R2020-250
Simple ITK software: https://github.com/niaid/sitk-ibex
Slide staining tray/humidity chamber
Sucrose Bioxtra	Sigma	S7903-1KG
Superfrost Plus Microscope slides	VWR	48311-703
Tissue-Tek OCT	VWR	25608-930
Tween-20, Molecular Biology grade	Promega	H5152
UltraPure DNase/RNase free water	Fisher Scientific	10977023