Abstract A031: Harnessing the potential of circulating tumour cells for precision diagnostics via multiplexed imaging of the levels and subcellular localizations of over 50 cell identity, state and signaling markers per cell
Authors: Lock John G , Mann Tim J , Zheng Ye , Khan Tanzila , Neumann Daniel , James Alexander , Becker Therese , Roberts Tara 
Published: November 2024

Abstract
Due to their longitudinal accessibility through liquid biopsy, circulating tumour cells (CTCs) hold huge potential as sources of molecular information capable of underpinning precision diagnostics in cancer. Yet key methodological limitations have thus far constrained the practical utility of CTCs as diagnostic analytes, including: a) reliance on positive CTC selection strategies that can bias against distinctive CTC subpopulations (e.g. low EpCAM, low size, low density), and; b) standard fluorescence imaging methods that capture only 4-6 molecular markers per cell. Since 3-4 markers are required to identify CTCs with even moderate confidence, such low marker plexity acutely restricts derivation of additional molecular insights about the cellular states and molecular signals that may be driving disease. Yet these insights are essential to guide selection of optimal therapies, to adapt therapeutic strategies as cycles of resistance arise, and even to stratify patients to the trials needed to expand the therapeutic arsenal. To overcome these challenges and harness the potential of CTCs for precision diagnostics, we have developed a comprehensive pipeline for CTC isolation, storage and batch processing via multiplexed immunofluorescence imaging of 50+ molecular markers per cell. These markers illuminate CTC identity, state (epithelial vs mesenchymal vs stemness), fate (proliferation, death, senescence) and signalling (spanning ∼10 alternate ‘driver’ signalling pathways), providing truly unprecedented molecular insights per CTC, across heterogeneous CTC populations, and regarding potential therapeutic targets. Downstream single-cell quantitative image analyses enable robust but adaptable computational parsing of CTC identity, removing false-positive and false-negative instances otherwise resulting from standard 4-plex CTC definitions (e.g. DAPI, CD45, EpCAM, pan-Cytokeratin) and complementing our upstream use of negative selection methods that capture the full, unbiased diversity of CTCs. The remaining 40+ markers facilitate integrated analyses of relationships between CTC state, fate and signalling pathways, capturing not only protein and phospho-protein expression levels per cell, but also variations in subcellular localisation that constitute additional layers of functional regulation not captured by most omics methods. Excitingly, when combined with machine learning, our approach can discern therapy- resistant cell subpopulations with over 98% accuracy in models of prostate cancer therapy resistance. We are now assessing CTC diversity across patients ranging from treatment naïve to advanced castrate-resistance prostate cancers. Overall, our unique high-plexity analysis now enables interrogation of detailed states, signals and dependencies in each patient CTC, providing the foundations for next-generation precision diagnostics to continuously match individual patients to optimal therapies at each stage of their disease journey. We believe this marks a significant step towards harnessing the true potential of CTCs for precision diagnostics.
Citation Format: John G Lock, Tim J Mann, Ye Zheng, Tanzila Khan, Daniel Neumann, Alexander James, Therese Becker, Tara Roberts. Harnessing the potential of circulating tumour cells for precision diagnostics via multiplexed imaging of the levels and subcellular localizations of over 50 cell identity, state and signaling markers per cell [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A031.