Abstract 5421: Characterizing functional cell migration states with highly multiplexed immunofluorescence imaging

Abstract

Abstract Cancer cell migration is a necessary step in the metastatic cascade, but there are heterogeneous cellular mechanisms to achieve this behavior. This study leveraged live-cell tracking and end-point spatial proteomics to better characterize what underlying molecular states drive migration heterogeneity. U2OS osteosarcoma stably expressing the FUCCI cell cycle reporter system were tracked for 20 hours on a Leica DMI8 widefield microscope. Immediately after, cells were fixed and prepared for highly-multiplexed immunofluorescence imaging on a Canopy CellScape instrument. Initial results from these experiments demonstrate that transferring the sample between these two systems is feasible and results in minimal loss of cells despite the multiple staining cycles. Unlike standard marker panels intended to differentiate between cell types, the initial panel used in this study includes markers that differentiate between migration modes. Even within one cell line, unsupervised clustering identified cell clusters for vimentin high/E-cadherin low and E-cadherin high/vimentin low, indicated an epithelial to mesenchymal transition (EMT) axis in the dataset. Furthermore, the spatial relationships between the cell neighborhoods show largely heterogeneous distributions of cell states, with clusters of more homogeneous cell states arising in more densely-packed regions of the coverslip. Further analysis of how single cell trajectories correlate to these cell states, as well as how cell cycle dynamics impact both migration and molecular cell states are in progress. Results from these analyses will shed new light on the “go vs. grow” hypothesis by directly testing how cell speed, persistence, and overall invasion correspond to cell cycle stage and progression. Furthermore, the high-resolution detail from the multiplexed imaging will allow for subcellular profiling of dynamic proteins, such as beta catenin, and directly link it to a larger protein and migration state. Overall, this experimental approach represents an important bridging between functional measurements and end-point omics technologies that will be crucial to gain actional insights amid the boom in multiomics. Citation Format: William Dee Leineweber, Alice Finkelstein, Emma Lundberg. Characterizing functional cell migration states with highly multiplexed immunofluorescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5421.