Spatial transcriptomics with RNAscope HiPlex v2 enables visualization of immune cell interactions in FFPE tumor tissues at single-cell resolution
Imagine trying to understand a complex conversation in a crowded room by only listening to the average volume of all voices combined. This is the challenge scientists have faced when studying the tumor microenvironment - the complex ecosystem where cancer cells interact with immune cells, blood vessels, and other components.
Within this battlefield, different immune cell types engage in delicate dances of activation and suppression that determine whether a tumor grows or shrinks. Until recently, our view of these critical interactions has been largely limited, like watching a war movie with only wide-angle shots and no close-ups.
The development of formalin-fixed paraffin-embedded (FFPE) tissues decades ago gave researchers a way to preserve tumor samples for long-term storage, creating vast biobanks of cancer specimens from around the world. However, extracting meaningful information about gene activity from these preserved tissues while maintaining the spatial context of where these conversations happen has remained extraordinarily challenging. Traditional methods either provided spatial information for just one or two genes at a time or required destroying the tissue architecture that reveals how cells are organized and interacting.
Enter the RNAscope™ HiPlex v2 assay - a revolutionary technology that lets researchers visualize up to 12 different RNA targets simultaneously in the same tissue section while preserving precious samples. This breakthrough represents a powerful lens through which we can finally read the hidden conversations between cancer and immune cells in exquisite detail, opening new possibilities for understanding cancer progression and developing more effective immunotherapies 1 .
RNAscope HiPlex v2 enables visualization of up to 12 RNA targets simultaneously in FFPE tissues, preserving spatial context while providing single-molecule resolution.
To appreciate why RNAscope HiPlex v2 represents such a significant advance, it helps to understand the limitations of previous technologies. Traditional methods for studying gene expression in tissues typically fell into two categories:
The problem with these approaches is that cellular function in the tumor microenvironment is determined not by single genes working in isolation, but by complex networks of genes working together in concert. Understanding whether an immune cell will attack or ignore a cancer cell requires knowing the combination of genes it's expressing - is it producing cytotoxic molecules that can kill tumor cells, or inhibitory receptors that shut down its attack functions?
In situ hybridization (ISH) techniques have long allowed researchers to visualize where specific RNA molecules are located within intact tissue sections. The original RNAscope technology, built on a proprietary "double Z" probe design, provided exceptional sensitivity and specificity, enabling researchers to see individual RNA molecules as distinct dots under a microscope 4 . This was a major advance over previous ISH methods that often struggled with background noise and false signals, but it was still fundamentally limited in how many different genes could be visualized simultaneously.
The need for multiplexing - detecting multiple targets in the same sample - became increasingly urgent as single-cell RNA sequencing studies revealed astonishing diversity in immune cell populations within tumors. Scientists discovered that what appeared to be a single cell type (such as "T cells") actually consisted of numerous subtypes with different functions, locations, and gene expression patterns, all mixed together in the tumor microenvironment 6 8 . Understanding this complexity required a method that could detect multiple gene signatures while preserving spatial relationships.
The RNAscope HiPlex v2 assay represents a sophisticated solution to the multiplexing challenge, employing an elegant iterative detection approach that combines specialized chemistry with computational image analysis. The process can be visualized as a multi-layered painting where each layer reveals additional details without disturbing the previous ones.
Probes bind to target RNA molecules
Signal amplification without background noise
Fluorescent imaging of 4 targets
Fluorophores removed for next round
Process repeats for multiple rounds until all targets are detected
At the heart of the system are cleavable fluorophores - fluorescent dyes that can be attached and later removed without damaging the underlying RNA targets or tissue structure. The process begins with the application of probes targeting the first set of four genes, each labeled with a different fluorescent channel corresponding to Alexa Fluor 488, DyLight 550, DyLight 650, or Alexa Fluor 750. After imaging, these fluorophores are cleaved away, making room for the next round of detection targeting four new genes 1 7 .
This cycle of hybridization, imaging, and cleavage repeats until all 12 targets have been detected across multiple rounds. The final step involves using specialized registration software to align all the images from different rounds, creating a composite visualization where all 12 RNA targets can be seen in their precise spatial relationships to each other 1 . What makes this particularly powerful is that each dot represents a single RNA molecule, providing quantitative data at single-molecule resolution, not just general expression patterns.
| Sample Type | Maximum Targets | Key Applications |
|---|---|---|
| FFPE | 12 targets | Translational research, biomarker discovery, immune profiling |
| Fresh Frozen | Up to 48 targets with HiPlexUp | Neuroscience, developmental biology, comprehensive cell typing |
| Fixed Frozen | Up to 48 targets with HiPlexUp | Complex phenotyping, rare cell detection, signaling pathway analysis |
Another crucial advantage is the technology's compatibility with FFPE tissues 1 , which represent the vast majority of clinical specimens stored in hospital archives worldwide. This means researchers can apply this powerful method to thousands of existing samples with associated clinical data, potentially uncovering relationships between spatial gene expression patterns and patient outcomes to identify new prognostic biomarkers.
To illustrate the power of this technology, let's examine how a research team might use the RNAscope HiPlex v2 assay to profile the immune landscape in human lung cancer samples. This hypothetical experiment follows established protocols and targets based on real-world applications described in the scientific literature 1 7 .
| Target Gene | Cell Type/Function | Expression Pattern |
|---|---|---|
| CD3E | Pan-T cell marker | High in T cell regions |
| CD8A | Cytotoxic T cells | Variable, often near tumor boundary |
| CD4 | Helper T cells | Diffuse distribution |
| FOXP3 | Regulatory T cells | Focal, often near tertiary lymphoid structures |
| PDCD1 (PD-1) | T cell exhaustion | High in tumor-infiltrating T cells |
| CD274 (PD-L1) | Immune checkpoint | Tumor cells and myeloid cells |
The power of this approach lies not just in detecting these individual markers, but in revealing their spatial relationships. For instance, the technology might reveal that some tumors contain CD8+ T cells expressing high levels of PD-1 that are located immediately adjacent to PD-L1+ macrophages, suggesting a potential mechanism of immune suppression. Meanwhile, other regions of the same tumor might show CD163+ macrophages clustered near cancer cells that lack T cell infiltration, suggesting a different immunosuppressive niche.
Analysis of the results would extend beyond simple presence/absence of cell types to sophisticated spatial metrics such as neighborhood analysis, distance measurements, and interaction patterns. These spatial relationships are not just academic curiosities - they can have profound clinical implications.
Provide amplification enzymes, buffers, and detection reagents for the assay
Target-specific probes that bind to RNA of interest; required for each gene
Verify assay performance; include species-specific positive controls
Aligns and merges images from multiple detection rounds into a single composite
Provides controlled temperature conditions for hybridization steps
Fluorescent microscope capable of imaging multiple channels with high sensitivity
When designing a HiPlex panel, researchers must carefully consider fluorophore assignment based on expression levels and practical considerations. The green channel (Alexa Fluor 488) is recommended for high expressors since it's most visible to the human eye, while the far-red channels (DyLight 650 and Alexa Fluor 750) work well for low expressors since they have minimal tissue autofluorescence in these ranges 1 7 .
The development of RNAscope HiPlex v2 represents more than just technical innovation - it marks a fundamental shift in how we approach the study of cancer and the immune system.
By allowing researchers to visualize complex gene expression patterns within the architectural context of intact tissues, this technology bridges the critical gap between single-cell molecular profiling and traditional histopathology.
"We love the RNAscope HiPlex assay! The capabilities allow us to visualize several different cell types and/or states in the same slice of tissue which provides critical information on spatial distribution and relationships" 1 .
This sentiment captures the excitement in the field about finally being able to see the full picture of cellular interactions rather than just isolated fragments.
The implications extend far beyond basic research. As spatial profiling technologies continue to evolve, they hold tremendous promise for:
The journey to fully understand the complex conversations happening within tumors is far from over, but technologies like RNAscope HiPlex v2 provide前所未有的清晰度 to eavesdrop on these discussions. As we continue to map these intricate cellular relationships, we move closer to the goal of truly personalized cancer therapy that leverages each patient's unique immune microenvironment to fight their disease.
| Feature | RNAscope HiPlex v2 |
|---|---|
| Multiplexing Capacity | 12 targets (up to 48 in frozen) |
| Resolution | Single RNA molecule |
| Tissue Requirement | Single section for all targets |
| Spatial Context | Preserved and quantitatively analyzable |
| Sensitivity | High, minimal background |
| Compatibility | Optimized for FFPE |