Accelerating Immuno-Oncology Research With A Pre-Configured HORIZON™ Image Analysis Workflow For SPYRE™ Antibody Panels

Spatial biology is transforming how researchers study tissues by preserving the spatial context of molecular information. Hyperplex imaging technologies provide an unprecedented view of cellular organization and function by combining protein and RNA detection in the same tissue section. 

But while generating this data has become faster and more accessible, extracting meaningful insights remains a significant hurdle. 

Hyperplex images are rich in information and are very complex to interpret. Translating them into quantitative, single-cell data requires multiple analytical steps, each with its own challenges. As spatial biology continues to expand, reducing this analytical complexity is key to enable more labs to adopt spatial biology technologies. 

COMET™ Suite: A Fully-Integrated Workflow 

COMET™ is a high-throughput, multiplex imaging platform that enables spatially resolved, single-cell analysis of protein and RNA expression in tissue samples using sequential immunofluorescence. COMET™ enables researchers to generate high-quality multiomic, hyperplex datasets in a fully automated way. 

In parallel, SPYRE™ Antibody Panels simplify assay design by providing modular and ready-to-use antibodies tailored for immuno-oncology applications. 

However, the transition from image acquisition to biological interpretation still requires: 

• Reliable cell segmentation
• Quantitative feature extraction
• Cell phenotyping
• Spatial analysis 

These steps are available in HORIZON™ Image Analysis Software, designed specifically for hyperplex immunofluorescence images generated on COMET™, enabling a smooth transition from tissue staining and imaging to multiplex data interpretation. 

Together, COMET™, SPYRE™ and HORIZON™ create an end-to-end workflow that removes friction at every step from tissue staining to data analysis. 

Introducing A Standardized HORIZON™ Workflow for SPYRE™ Antibody Panel Analysis 

To support users in navigating the complexity of spatial data, HORIZON™ provides a ready-to-use analysis workflow template optimized for SPYRE™ Antibody Panels for immuno-oncology research. Instead of building pipelines from scratch, users can import the pre-configured workflow and start analyzing their data immediately.  

The standardized SPYRE™ analysis workflow integrates the following steps into a single automated pipeline: 

1. Background Subtraction 

Optional background subtraction to improve signal clarity while preserving the integrity of the data. Background channels are automatically selected using metadata available in COMET™ images. 

      2. Cell Segmentation 

HORIZON™ applies COMET™-optimized deep-learning segmentation models to segment nuclei and cells. With the SPYRE™ workflow, the most promising channels for segmentation are pre-selected from available SPYRE™ panel markers. 

      3. Feature Extraction 

Quantitative features are measured across cellular compartments of the tissue sample, enabling detailed characterization of marker expression for downstream cell phenotyping. 

     4. Feature Thresholding 

     5. Marker signal intensities are thresholded to separate cells which are positive for a marker from those which are negative. A default threshold value is suggested for markers included in the SPYRE™ Antibody Panel. The default value can be adjusted to better fit the specific staining. 

     6. Cell Phenotyping 

Cells are then classified based on their expression profiles, using a rule-based approach. The predefined classification framework provides foundational phenotyping based on the specific markers included in the SPYRE™ Antibody Panel. The classification decision tree is flexible and can be expanded to integrate additional markers and cell phenotypes of interest. 

        7. Spatial analysis and visualization 

Finally, spatial relationships between cells can be explored thanks to neighborhood analysis and interactive plots. 

All outputs, including segmentation annotations and numerical data, can be exported in industry-standard .geojson and .csv files for further downstream analysis if needed. 

Standardization And Flexibility in One Workflow 

A key aspect of the design of HORIZON™ is the balance between standardization and adaptability. 

Workflow templates provide a structured starting point, enabling consistent analysis across samples and experiments. At the same time, they can be easily adapted to: 

• Different panel designs 

• Variations in marker expression 

• Changes in channel naming or data structure 

This allows workflows to serve both as guidance for new users and as a foundation for more advanced analyses. 

Accelerating Time to Results With HORIZON™ 

By providing ready-to-use analysis templates, HORIZON™ helps reduce the time and effort required to move from multiplex image to biological interpretation. 

In practical terms, this means: 

• Fewer manual steps and reduced setup time 

• Increased reproducibility across experiments thanks to batch analysis 

• Greater accessibility for users without computational expertise 

As spatial biology continues to evolve, making data analysis more accessible will be critical for unlocking its full potential. 

By combining the streamlined panel design using SPYRE™ Antibody Panels, automated hyperplex staining and imaging on COMET™ and guided image analysis workflows in HORIZON™, researchers can adopt a more efficient and reproducible approach to spatial data analysis, making spatial biology a practical, scalable tool for everyday research.  

If you’re already using COMET™, you can start immediately: 

• Download the SPYRE™ workflow template and analyze your data in minutes (my.lunaphore.com) 

Or download the COMET™ brochure to find out more about the COMET™ Suite.