Abstract 2084: Development of a novel tri-omic spatial biology assay that provides in-depth immune profiling of the tumor immune microenvironment

Spatial biology has advanced our understanding of cancer biology and shed light on the immense transcriptomic and proteomic heterogeneity that exists within the tumor-immune microenvironment (TiME). To date, most analytical and diagnostic approaches only provide one type of biological readout per tissue section, and the generation of true multi-omic data has remained challenging. We developed a workflow that allows the integration of multiple omic modalities on a single section. The workflow is centered around a new signal removal strategy, EpicIF™ technology, which effectively and rapidly removes fluorophores while leaving tissue and probe chemistries unaffected. Using this workflow on the CellScape™ platform, we iteratively performed spatial proteomic, transcriptomic, and interactomic assays all on the same tissue sections. Formalin-fixed, paraffin-embedded tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1, which were then treated with oligonucleotide-modified secondary probes designed for an in situ Proximity Ligation Assay (isPLA) to detect PD1 and PD-L1 interactions. Next, the detection of RNA targets was accomplished by adding 12 HCR™ HiFi RNA-ISH probes, which were visualized by addition of target-specific fluorescently labeled HCR™ Gold amplifiers (RNA-FISH). Lastly, spatial proteomic labeling was conducted with VistaPlex™ multiplex immunofluorescence (mIF) panels targeting 30+ biomarkers. We deployed this tri-omic assay on samples of primary CNS lymphoma (PCNSL), a rare and aggressive form of B-cell lymphoma that remains an area of unmet clinical need, with over half of diagnosed patients succumbing to the disease. Unlike diffuse large b-cell lymphoma (DLBCL), its morphologically identical systemic counterpart, PCNSL shows sensitivity to PD1 checkpoint inhibition. We investigated if our assay could describe unique interactions between the PD1-PDL1 axis and the lymphoma TiME in PCNSL (n=24), in comparison to DLBCL (n=152) cases. High-resolution imaging with the CellScape platform allowed for precise detection and quantification of isPLA, RNA-FISH and mIF signals on the same slide. The combined assay showed differential biomarker expression and interaction profiles associated with immune-regulatory processes in the interrogated tumor samples, revealing remarkable inter- and intra-tumoral heterogeneity. This spatial multi-omics approach allows for a more comprehensive insight into the complex interplay of immune and non-immune cell populations in the TiME. Citation Format: Arne Christians, Jannik Boog, Daniel Jimenez Sanchez, Sara Bodbin, Agata Wicher, Subham Basu, Aneesh Acharya, Randy Chen, Hong Liang, Shruti Shridar, Tan Char Loo, Ng Siok Bian, Michelle Poon, Anand Jeyasekharan, Oliver Braubach. Development of a novel tri-omic spatial biology assay that provides in-depth immune profiling of the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2084.