COEXIST: Coordinated single-cell integration of serial multiplexed tissue images

Multiplexed tissue imaging (MTI) and other spatial profiling technologies commonly utilize serial tissue sectioning to comprehensively profile samples by imaging each section with unique biomarker panels or assays. The dependence on serial sections is attributed to technological limitations of MTI panel size or incompatible multi-assay protocols. Although image registration can align serially sectioned MTIs, integration at the single-cell level poses a challenge due to inherent biological heterogeneity. Existing computational methods overlook both cell population heterogeneity across modalities and spatial information, which are critical for effectively completing this task. To address this problem, we first use Monte-Carlo simulations to estimate the overlap between serial 5μm-thick sections. We then introduce COEXIST, a novel algorithm that synergistically combines shared molecular profiles with spatial information to seamlessly integrate serial sections at the single-cell level. We demonstrate COEXIST necessity and performance across several applications. These include combining MTI panels for improved spatial single-cell profiling, rectification of miscalled cell phenotypes using a single MTI panel, and the comparison of MTI platforms at single-cell resolution. COEXIST not only elevates MTI platform validation but also overcomes the constraints of MTI’s panel size and the limitation of full nuclei on a single slide, capturing more intact nuclei in consecutive sections and thus enabling deeper profiling of cell lineages and functional states. ### Competing Interest Statement The authors declare the following competing interests: G.B.M. is a SAB member or Consultant: for Amphista, Astex, AstraZeneca, BlueDot, Chrysallis Biotechnology, Ellipses Pharma, GSK, ImmunoMET, Infinity, Ionis, Leapfrog Bio, Lilly, Medacorp, Nanostring, Nuvectis, PDX Pharmaceuticals, Qureator, Roche, Signalchem Lifesciences, Tarveda, Turbine, Zentalis Pharmaceuticals. G.B.M. has Stock/Options/Financial relationships with: Bluedot, Catena Pharmaceuticals, ImmunoMet, Nuvectis, SignalChem, Tarveda, and Turbine. G.B.M. has Licensed Technology: HRD assay to Myriad Genetics, DSP patents with Nanostring. G.B.M. has Sponsored research with AstraZeneca. The other authors declare no competing interests.