Identifying immune cell phenotypes and their function by using high-throughput spatial multiomics

Tumor microenvironment (TME) is a complex milieu of multiple cell types mainly including tumor cells, immune cells, endothelial cells and stromal cells. Immune cell profile within the TME can determine success of immunotherapies, indicate therapeutic efficacy as well as determine any potential therapeutic toxicity. Spatially interrogating these immune cells is crucial in studying immune-immune and immune- tumor interactions. To address this, we have developed a high throughput 
RNAscope™ assay on the BOND RX to spatially visualize RNA and protein markers on the same slide. 

We utilized the new RNAscope Multiomic LS assay that can detect up to 6 RNA and protein targets on the same slide. The TSA-based amplification strategy offers signal boost for both RNA and protein targets. To optimize protein detection, the workflow is completely protease-free. Here, we demonstrate the use of our pre-conjugated antibody panel which includes CD8, CD4, FoxP3 and PanCK to visualize tumor infiltrating lymphocytes (TILs). We also utilized unconjugated primary 
antibodies for CD68 and CD163 to detect tumor macrophages in multiple tumor tissues.
The RNAscope Multiomic LS assay was successfully able to interrogate different subpopulation of immune cells with high quality RNA and protein signal while maintaining tissue morphology.

• With the TIL panel antibodies, T cell subpopulations were successfully visualized along with activating cytokine signatures.
• Using conjugated secondary antibodies macrophage markers such as CD68 and CD163 were used to detect tumor-associated macrophages with secreted cytokine and chemokine RNAs.
• HALO analysis was able to provide further insights into the T cell phenotypes , their prevalence in tumor regions and their activation states in cervical tumor tissues.
• Overall, the RNAscope Multiomic LS assay on the BOND Rx provides the flexibility to combine RNA and protein markers of choice to interrogate the tumor microenvironment
   

This poster was presented at the AACR 2025 meeting.