Abstract 176: Simultaneous spatial characterisation of copy number change and immune cell profiling using DNA FISH and multiplexed immunofluorescence

Abstract Background: Recent advances in spatial characterization of protein expression has greatly improved our understanding of tissue architecture, cell-cell interactions and their function. In this work, we aim to further improve cell characterization by combining the detection of DNA and proteins in the same sample in an automated and cyclic manner. Single molecule DNA FISH can identify cells with genetic aberrations linked to different tumor types or aggressiveness, however as a stand-alone method this technique lacks spatial context. We set out to use the microfluidic Phenocycler-Fusion platform from Akoya Biosciences to detect single molecule DNA FISH signals, as well as multiplexed protein detection, in order to gain multiple layers of spatial information from one sample and the possibility to explore clonal heterogeneity within tumor sections. Methods: The Phenocycler-Fusion involves the use of antibodies conjugated to oligonucleotide barcodes. In each cycle, the microfluidics instrument adds up to three reporters labelled with distinct fluorophores (AF488/AF750, Atto550, AF647), these are complementary to individual barcodes. The fluorophores are then imaged and the reporters are eluted ready for the next round of reporter addition, imaging and elution. Our integrated workflow for detecting DNA and protein involves hybridization of primary DNA FISH probes downstream of antibody staining before the sample is loaded onto the Phenocycler-Fusion instrument for cyclic addition of secondary fluorescent DNA FISH probes and reporters. The data is processed so that images from each cycle are collated into a single image meaning that DNA and protein signals can be spatially analyzed simultaneously. Results: The protocol was developed using fixed cells to identify the optimal conditions for combined DNA and protein detection and we successfully executed the same protocol in fresh frozen tissue sections. We have obtained proof of concept data illustrating the possibility of imaging chromosomal regions simultaneously with immune cell and structural markers such as CD45, pan-cytokeratin and Ki67. We have also shown that DNA targets can be detected across imaging cycles along with protein detection and thereby demonstrate the multiplexing capacity of our integrated protocol on the Phenocycler-Fusion platform in a fully automated manner. The combined method can be optimized and applied to various cancer samples to investigate the tumor microenvironment along with chromosomal aberrations or copy number variations of specific genes. Overall, these promising results show the possibility of using a commercially available system for detecting multiplexed DNA and protein simultaneously across a large imaging area. Citation Format: Eleanor M. O'Roberts, Pranauti Panshikar, Xiaoze Li-Wang, Quentin Verron, Shahrzad Shirazi Fard, Magda Bienko, Charlotte Stadler. Simultaneous spatial characterisation of copy number change and immune cell profiling using DNA FISH and multiplexed immunofluorescence [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 176.