Deep multiplexed 50-marker imaging of circulating tumor cells expands actionable biomarker profiling for precision oncology

Mann, T. J.; Zheng, Y.; Khan, T.; Huang, T.; Nindra, U.; Ma, Y.; James, A.; Neumann, D. P.; Kohane, F. V.; Gunawan, I.; Vafaee, F.; Chua, W.; de Souza, P.; Roberts, T. L.; Becker, T. M.; Lock, J. G.

doi:10.1101/2025.10.23.684075

bioRxiv · 10.1101/2025.10.23.684075

Deep multiplexed 50-marker imaging of circulating tumor cells expands actionable biomarker profiling for precision oncology

Mann, T. J.·Zheng, Y.·Khan, T.·Huang, T.·Nindra, U.·Ma, Y.·James, A.·Neumann, D. P.·Kohane, F. V.·Gunawan, I.·Vafaee, F.·Chua, W.·de Souza, P.·Roberts, T. L.·Becker, T. M.·Lock, J. G.

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Abstract

Liquid biopsy-derived circulating tumor cells (CTCs) offer a minimally invasive avenue for precision oncology by enabling longitudinal monitoring of actionable molecular and cell-phenotypic biomarkers. However, conventional immunofluorescence imaging-based CTC profiling captures just 4-5 markers per cell, restricting crucial insights into oncogenic and resistance drivers, as well as tumor cell heterogeneity. Here we present an integrated pipeline employing deep multiplexed imaging to profile up to 50 molecular markers per CTC, capturing expression, phosphorylation, and subcellular localization of diverse biomarkers. Validated in a multi-stage prostate cancer resistance model and applied to prostate cancer patient-derived CTCs, this approach enhances CTC classification and reveals inter- and intra-patient heterogeneity correlating with therapy response. Machine learning identified therapeutically actionable signatures, suggesting patient-specific treatment strategies. This deep multiplexed imaging pipeline advances the utility of CTCs in guiding personalized cancer therapy by providing comprehensive molecular and phenotypic insights through minimally invasive liquid biopsies. Highlights- Multiplexed immunofluorescence imaging of circulating tumor cells captures up to 50 molecular markers per cell with subcellular localisation. - Single-cell and subcellular analyses identify quantitative signatures correlating with patient outcome, as well as therapeutically actionable biomarkers. - This method transforms the utility of CTCs for future use in patient stratification and for guiding personalised therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/684075v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@a36ff4org.highwire.dtl.DTLVardef@b06c1org.highwire.dtl.DTLVardef@1e8a572org.highwire.dtl.DTLVardef@c60753_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

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Mann, T. J., Zheng, Y., Khan, T., Huang, T., Nindra, U., Ma, Y., James, A., Neumann, D. P., Kohane, F. V., Gunawan, I., Vafaee, F., Chua, W., de Souza, P., Roberts, T. L., Becker, T. M., Lock, J. G.. 2025-10-24. Deep multiplexed 50-marker imaging of circulating tumor cells expands actionable biomarker profiling for precision oncology. https://doi.org/10.1101/2025.10.23.684075

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