Three-dimensional imaging mass cytometry for highly multiplexed molecular and cellular mapping of tissues and the tumor microenvironment

Technical Report https://doi.org/10.1038/s43018-021-00301-w Three-dimensional imaging mass cytometry for highly multiplexed molecular and cellular mapping of tissues and the tumor microenvironment Laura Kuett1,2,22, Raúl Catena1,3,22, Alaz Özcan 1,20, Alex Plüss1,21, Cancer Grand Challenges IMAXT Consortium*, Peter Schraml4, Holger Moch 4, Natalie de Souza1,5 and Bernd Bodenmiller 1,2 ✉ A holistic understanding of tissue and organ structure and function requires the detection of molecular constituents in their original three-dimensional (3D) context. Imaging mass cytometry (IMC) enables simultaneous detection of up to 40 antigens and transcripts using metal-tagged antibodies but has so far been restricted to two-dimensional imaging. Here we report the development of 3D IMC for multiplexed 3D tissue analysis at single-cell resolution and demonstrate the utility of the technology by analysis of human breast cancer samples. The resulting 3D models reveal cellular and microenvironmental heterogeneity and cell-level tissue organization not detectable in two dimensions. 3D IMC will prove powerful in the study of phenomena occurring in 3D space such as tumor cell invasion and is expected to provide invaluable insights into cellular microenvironments and tissue architecture. T issues and organs are complex ecosystems consisting of numerous cell types arranged in a manner that is inextricably related to function. Understanding tissue function and pathology thus requires knowledge of constituent cells and their states, extracellular matrix proteins and vasculature in the context of their native 3D arrangement. Historically, tissues have been studied using microscopy and recently developed methods have enabled various types of 3D tissue analysis (Supplementary Table 1). Confocal 3D microscopy enables analysis of tissue sections at subcellular resolution but is limited to a depth of about 100 µm1. Multi-photon confocal and light-sheet microscopes allow for 3D reconstructions of up to 1-mm tissue depth at single-cell resolution2,3. As these 3D microscopy methods rely on fluorescent reporters that show high spectral overlap, the number of epitopes that can be measured simultaneously is limited. To enable multiplexed tissue analysis, cyclic immunostaining and chromogenic approaches have been used4–7 and such methods have also been implemented in 3D8,9. In addition to fluorescence-based approaches, mass spectrometry-based imaging of epitopes and transcripts is becoming broadly used. In mass spectrometry-based technologies, mass tags, such as a molecule of a defined mass or metal isotopes, are used as reporters on affinity reagents10,11,12. IMC allows simultaneous detection of up to 40 antigens13 and nucleic acid sequences14 in formalin-fixed paraffin-embedded (FFPE) tissues15, in frozen tissue sections16 and in cultured cells17. Currently, however, none of these methods combines multiplex detection of many targets with 3D tissue imaging, which is necessary for visualization of single cells together with larger structures such as blood vessels. Here we describe an extension of IMC to the analysis of tissues in 3D. With the 3D IMC method, the volume and depth of a tissue that can be analyzed is limited mainly by the measurement time. The full pipeline, from sample processing to cell-level computational analysis of a complete 3D model, can be performed in 1 week15,18. We demonstrate how 3D IMC enables the study of tumor architecture by combining the analysis of tissue volumes with single-cell information. We show that spatial heterogeneity of marker expression and preferential cell–cell interactions become apparent with 3D models and that spatially contained events could be captured within a single 3D model. Overall, we demonstrate that the detailed models generated with 3D IMC facilitate comprehensive, single-cell-resolution analysis of cellular microenvironments and tissue architecture. Results Generation of 3D models from IMC data. Our 3D IMC approach relies on serial sectioning of a tissue cylinder punched from a paraffin-embedded tissue. We optimized our sample processing methods and settled on using an ultramicrotome with a diamond knife designed for FFPE sectioning to minimize deformations that are known to be caused during tissue cutting, handling of thin slices, and further experimental procedures19. We chose to cut 2-µm-thick sections to provide a compromise between capturing single cells across multiple slices and the difficulty of handling ultra-thin slices, thereby making the approach accessible to a broader user base. Tissue sectioning is followed by tissue hydration and heat-induced epitope retrieval (Extended Data Fig. 1). After acquiring two-dimensional (2D) IMC data, we assemble 3D tissue models and derive single-cell marker profiles using a computational Present address: Department of Quantitative Biomedicine, University of Zurich, Zurich, Switzerland. 2Institute of Molecular Health Sciences, ETH Zurich, Zürich, Switzerland. 3Leica Geosystems part of Hexagon, Heerbrugg, St. Gallen, Switzerland. 4Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland. 5Institute of Molecular Systems Biology, ETH Zurich, Zürich, Switzerland. 20Present address: Department of Immunology, University Hospital Zurich, University of Zurich, Zurich, Switzerland. 21Present address: Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland. 22These authors contributed equally Laura Kuett, Raúl Catena. *A list of members and their affiliations appears in the Supplementary Information. ✉e-mail: 1 Nature Cancer | www.nature.com/natcancer Technical Report Wet laboratory a Paraffin ultramicrotomy Tissue Diamond knife NATurE CAncEr Section collection and annotation Antigen retrieval IMC Metal antibody staining 2-µm sections Laser ~1 mm2 h–1 Single cell segmentation 3D voxel model In silico processing Image registration Visualization and single cell analysis Stack processing Raw data measurement y x z Voxel size 1 µm × 1 µm × 2 µm b VWF CD31 4 30 µm 488 µm 652 µm panCK vWF CD31 SMA CD68 CD8α CD20 Fig. 1 | Experimental and computational workflow for 3D IMC. a, Small rods or blocks of FFPE tissue are cut into 2-µm sections with an ultramicrotome and a modified diamond knife. Sequential sections were collected on regular microscopy slides. Typically, 20 to 40 sections were placed on each glass slide. After rehydration, tissues were subjected to antigen retrieval, followed by staining with metal-labeled antibodies. All sections were analyzed by IMC. Data were processed computationally to order sections according to the annotation. Images are aligned and cells are segmented with a 3D watershed algorithm. Finally, a full 3D model can be analyzed both at the voxel and cell level. b, Examples of raw data voxel rendering for the indicated markers in a representative example from one out of the two breast (...truncated)