How to unlock the complexity of tumor microenvironments

The interplay between tumor microenvironments and immune cell infiltrates holds the key to unlocking next-generation therapies for cancer. However, traditional immunohistochemistry protocols often fall short, limited by the number of markers they can analyze and the risk of tissue and signal loss. Enter the Orion platform, a tool delivering spatial biology and cellular phenotyping with high speed and resolution, revealed in a study by Navigate Biopharma Services presented at AACR 2024. This multiplex imaging instrument from RareCyte employs single-round immunofluorescence staining and imaging of standard formalin-fixed paraffin-embedded (FFPE) or fresh-frozen tissue, enabling subcellular quantification of biomarker targets across an entire slide within a few hours.

The study sought to harness the power of spatial insights through a single-step staining workflow using a cocktail of 15 antibody-conjugated fluorophores against FFPE tumor tissue microarrays. These arrays were imaged and the protein biomarkers quantitated using the Orion instrument.

In our exploration of this study, we hear from two of the researchers, Jennifer Bordeaux and Richard Van Krieken, from the Digital Pathology team at Navigate BioPharma Services.

Getting to know the Orion

The Orion platform from RareCyte streamlines high-plex staining and scanning to support fast turn-around and large-scale clinical studies. Its single-round design ensures sample integrity and enables subsequent H&E imaging for region selection. With the ability to analyze up to 20 markers per round, it accommodates diverse sample types. Staining across the entire slide preserves spatial context, facilitating comprehensive assessment by pathologists. Overall, Orion's capabilities help uncover spatial biology insights in large cohort studies.

Understanding the study design

The study set out to address critical gaps in current immunohistochemistry protocols, particularly the limitations imposed by the number of markers that can be simultaneously analyzed. Van Krieken discussed the challenges they faced with traditional assays, highlighting the need to expand the scope to fully capture the intricacies of the immune landscape without compromising tissue integrity. "Typically, our assays look at up to seven markers, but there's a big interest in pushing that higher, into the 10 to 20 range, where you can fully characterize the immune landscape," he said.

Bordeaux added, “I would emphasize the ability to look at these 15 markers on a single slide with a single scan was why we moved forward with the collaboration and this concept study with RareCyte and the Orion specifically."

The custom panel developed by RareCyte included markers crucial for evaluating the immune landscape across various cancers, such as CD3e, CD4, CD8a, CD20, CD68, CD163, FOXP3, Ki67, Pan-CK, PD1, PDL1, CD45, Granzyme B, and LAG3. This panel allowed for the evaluation of immune markers on pan-tumor and melanoma tissue microarrays, covering a diverse range of cancers including Hodgkin’s lymphoma, ovarian cancer, esophageal carcinoma, hepatic cell carcinoma, and head and neck cancer.

Serial sections were stained with both the 15-plex Orion platform and traditional 6-plex clinical grade assays, enabling a comprehensive comparison. Correlation scores were determined between the Orion and traditional assays, showcasing the efficacy of the Orion platform in capturing a broad spectrum of biomarkers in a single workflow.

The performance of the Orion platform was assessed through rigorous quantitative and qualitative measures. Linear regression analysis demonstrated remarkable similarity between the 6-plex clinical grade assays and the 15-plex Orion workflow, showcasing comparable results in terms of cell proportions and fluorescence intensity. The correlation coefficients (R2) ranged from approximately 0.7 to 0.98, with slopes close to unity, indicating a high degree of concordance between the Orion platform and traditional assays. “We were able to get numbers that were within generally the range of what we’re looking for,” said Van Krieken.

Insights into tumor microenvironments

One of the outcomes of the study was the ability of the Orion platform to capture rare cell populations with clarity. Images captured by the Orion platform revealed intricate interactions between immune cells within the tumor microenvironment, shedding light on potential mechanisms of resistance and immune surveillance. From cytotoxic T-cells to exhausted Tregs and macrophages, the Orion platform provided a comprehensive view of the complex dynamics within tumors. “The resolution of the cells themselves were absolutely amazing,” said Van Krieken.

Implications for clinical practice

The implications of these findings are profound as they help offer a solution for comprehensive biomarker analysis. Unlike traditional TSA-based fIHC multiplexing, which is limited to the detection of six biomarkers on a single slide, the Orion platform enables the rapid quantitation of up to 20 biomarkers on a single FFPE slide. This helps enhance the efficiency of biomarker analysis and provides an opportunity to assess higher combinations of potentially clinically relevant biomarkers and spatial interactions in cancer patients.

The study presented at AACR 2024 signals rapid advancement in spatial biology. By helping provide insights into immune cell infiltrates and tumor dynamics, the Orion platform gives way for deeper understanding the unique spatial biology of individual patients. As we explore further into the spatial complexities of cancer, the Orion platform leads the charge towards enhanced treatment strategies and improved patient prognoses.

To learn about Navigate BioPharma Services’ spatial biology capabilities click here

To learn about Orion panels and biomarker reagents click here

To learn more about the Orion platform or to request pricing, click here