Abstract 173: Advancements in 3D high-multiplex imaging using the stellaris confocal platform

Abstract In cancer immunology research, evaluating tissue changes and cell interactions is crucial for identifying specific cell types, assessing the activation status of immune cells, monitoring the expression of certain proteins and genetic markers, and characterizing the tumor microenvironment. Traditional imaging techniques often fall short in providing comprehensive spatial and functional insights due to their limitations in multiplexing and three-dimensional (3D) analysis. Here, we introduce an innovative high-multiplexing workflow on the STELLARIS confocal platform, utilizing a new functionality called SpectraPlex for advanced 3D imaging (Kunz L., et al. Nat. Methods, 2024). SpectraPlex significantly enhances the capabilities of spatial biology by streamlining the steps from panel creation to image acquisition and unmixing through advanced unmixing algorithms. In addition to the traditional single references and semi-blind unmixing for 15-plex experiments, we introduce a “group” approach that allows users to generate 15 references using only 3 samples, thereby saving preparation and imaging time and preserving a larger amount of samples for experimental steps. This workflow was validated on a cancer model where murine pancreatic ductal adenocarcinoma cells (KPC-4662) were injected into C57Bl/6 mice and allowed to grow for three weeks. Tumor tissues were harvested, fixed, and processed into 70-µm-thick sections for imaging. A 15-plex panel, including markers for T cells (CD4, CD8), B cells (B220), myeloid cells (CD11b, MHC II, CD11c), and other functional and structural markers (TCF1, FoxP3, PD1, Ki67, α-SMA, MHC I, E-cadherin, Tenascin C, CD31), was used to visualize the resulting tumor infiltration. High-resolution 3D imaging was performed on a STELLARIS confocal system, capturing comprehensive spatial information and enabling the identification of immune cell subtypes, functional states, and structural markers within the tumor microenvironment. All 15 markers were present and imaged in a single acquisition, allowing further probing in areas of interest. We also demonstrate how this general approach is easily adapted to investigate the immune landscape of murine lymph nodes (Roberti M. J., et al, Nat. Methods, 2024). The implementation of the 3D high-multiplexing workflow on the STELLARIS confocal platform represents a significant advancement in the ability to interrogate the tumor microenvironment in detail. The SpectraPlex toolbox not only streamlines the imaging process but also ensures high data quality and reliability, setting a new standard for future spatial biology research in cancer. Citation Format: L Alvarez, I Steinmetz, T Straka, F Hecht, M J. Roberti. Advancements in 3D high-multiplex imaging using the stellaris confocal platform [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 173.