Spatial Proteomics for Clinical Research: A Platform Perspective

By Tad George, PhD · Senior Vice President, Biology R&D · RareCyte

Many spatial proteomics platforms are built to maximize information from a limited number of samples for discovery applications but fall short of the demands of clinical research programs. Explore the four requirements for supporting clinical research.

For precision therapeutics programs, drug-induced change in the tumor microenvironment (TME) often determines whether a therapy succeeds. What has happened to target expression and the state of the tumor? Which immune cells are recruited, and what states are they in? How are they organized within and around the tumor?

These perturbations reveal drug mechanism of action, provide insight into drug efficacy, inform whether to progress to later stage clinical trials, and can impact patient enrollment and stratification decisions. Spatial proteomics, with its ability to measure multiple proteins simultaneously in tissue with spatial context, is well-suited to resolve the activities in the TME.

Spatial proteomics for clinical research

Clinical researchers make critical data-driven decisions that ideally result in the right drugs progressing through optimally-designed trials. Confident decision-making thus requires essential information content provided by custom proteomics panels with the statistical power afforded by large volume studies and trials, all built on a foundation of high-quality data.

While many spatial platforms are designed to maximize information on a few samples for discovery research applications, most cannot meet the constellation of requirements demanded by clinical research programs.

In practice, clinical research for precision therapeutics places four key demands on a spatial proteomics platform: 

• Biomarker depth to resolve the tumor microenvironment
• Workflow capacity to process statistically relevant sample numbers on the program's timeline
• Rapid and flexible development of high-performance assays
• Accurate and reproducible quantitative results for confident decisions about which drugs advance and how trials are designed

Prior to the existence of the Orion™ spatial proteomics platform, researchers were faced with unacceptable tradeoffs that limited application of spatial proteomics in clinical research. The sections below discuss each of these four fundamental requirements and the demands they place on spatial platforms.

Requirement 1: Multiplex depth to resolve the tumor microenvironment

Resolving the TME requires measuring a range of biomarkers to not only identify tumor cells, immune cell subsets, and stromal populations, but also to measure markers of activation, exhaustion, proliferation, and immune checkpoint expression by those cells. In practice, this means utilizing a panel that addresses at least 10 protein markers applied to the same tissue section to classify the different sub-populations. This exceeds the capacity of traditional IHC and multiplex immunofluorescence (mIF) approaches that have been historically used in translational and clinical research settings.

As RareCyte Principal Scientist Edward Lo, PhD, puts it, "mechanism of action studies require panels in the 10–18 marker range, with enough markers to define cell types and capture functional states simultaneously, on the same tissue section, from the same cells." 

Requirement 2: Workflow capacity to meet clinical timelines

Clinical programs typically involve large sample cohorts, often prospectively collected, placing fundamentally different operational demands on a platform than small volume discovery studies require. 

While cyclic immunofluorescence (IF) approaches can provide very high plex data, the iterative and serial staining and imaging workflow does not provide the throughput or capacity to run large cohort studies on a timeline required by clinical research programs.

Orion’s paradigm-shifting 20-channel single-round approach has unlocked plex at capacity for two main reasons: 1) high plex staining and imaging is performed in a single round, dramatically increasing throughput; and 2) staining samples in batches and in parallel to imaging creates a high-capacity workflow rather than a serial bottleneck. 

Requirement 3: Custom panels you can build and validate reliably

As programs progress from discovery through clinical research, emphasis is placed on translating discovery-level insights into validated proteomic panels that measure the essential biomarkers needed to resolve the microenvironment.

The conventional empirical validation process can be time and labor-intensive, especially for platforms with challenging labeling procedures or that require staining order optimization for each new custom panel. In contrast, Orion’s reagent ecosystem facilitates rapid development of custom panels built on a foundation of validated antibody conjugates. The extensive menu of conjugates are all validated to work in multiplex with the same antigen retrieval condition and in interchangeable combinations. Furthermore, the small molecule organic dyes utilized by the Orion platform are attached to antibodies with simple amine conjugation chemistry that can be performed by any lab.

This matters most during biomarker development, where teams narrow broad exploratory findings into a focused panel of the most clinically relevant markers, followed by analytical validation of the panel for use in a trial. Because panels are quick to assemble and adjust, teams can evaluate more marker combinations and converge on the final panel faster, then run it as a fixed, reproducible assay for trial deployment.

Requirement 4: Data quality for confident clinical decision-making

Clinical researchers utilize large cohort studies and clinical trials to validate the mechanism, safety, dosage, and effectiveness of novel therapeutic interventions. As a result, premium data quality is of paramount importance as it provides a foundation for trustworthy results.

The Orion platform is designed to maximize data quality for large volume studies. The use of antibodies directly conjugated to ultra-bright, photostable fluorochrome tags combined with high-powered laser excitation ensures sensitivity to low abundance biomarkers. Single-round high-plex staining with direct conjugates preserves tissue and antigen integrity, and eliminates concerns for residual signal between rounds and/or non-specific staining associated with secondaries and amplification schemes, all of which can be an issues for platforms that employ iterative and/or complex staining protocols. Furthermore, reproducibility is facilitated by the simplicity of direct conjugate staining, the batch staining workflow, and the long shelf life of Orion reagents, enabling studies to be performed with the same lot of reagents.

Tissue preservation is especially consequential in clinical research, where samples are often limited, heterogeneous, and difficult to replace. Repeated processing cycles can cause tissue to move, fold, or detach, and can alter antigenicity from round to round. A workflow that touches the tissue once protects both the morphology and the sample's usability for downstream analysis.  

As Jennifer Bordeaux, PhD, of Navigate BioPharma Services — a CRO that has deployed Orion in clinical trial testing — describes it, single-round high-plex imaging is "an optimal method to analyze small clinical biopsy samples without compromising tissue integrity, which is a huge win for generating data in these precious clinical samples."

Meeting these requirements at once

Prior to the Orion spatial proteomics platform, clinical researchers designing pilot studies, pre-clinical models, and early-phase trials faced an uncomfortable compromise between plex, throughput, and data quality. Orion removes this hurdle through its single-round, high plex approach, introducing a paradigm shift in the application of spatial proteomics to clinical research. Today, researchers are actively using the platform to analyze drug mechanisms of action and measure biomarkers of therapeutic response in pre-clinical and Phase I/II trials. By scaling both panel and study sizes without compromising data quality, Orion delivers the statistical power and actionable information content necessary for robust decision-making throughout clinical research.

Dr. Jennifer Bordeaux's remarks above are drawn from a recent webinar with RareCyte and Navigate Biopharma on bringing scalable, reproducible spatial proteomics into clinical trials.
Watch the webinar on-demand →

If you're evaluating how to incorporate spatial proteomics into your program, RareCyte's spatial biology team can walk you through the Orion platform.
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