Highly multiplexed 3D profiling of cell states and immune niches in human tumors

Diseases such as cancer involve alterations in cell proportions, states and interactions, as well as complex changes in tissue morphology and architecture. Histopathological diagnosis of disease and most multiplexed spatial profiling relies on inspecting thin (4-5 µm) specimens. Here we describe a high-plex cyclic immunofluorescence method for three-dimensional tissue imaging and use it to show that few, if any, cells are intact in conventional thin tissue sections, reducing the accuracy of cell phenotyping and interaction analysis. However, three-dimensional cyclic immunofluorescence of sections eightfold to tenfold thicker enables accurate morphological assessment of diverse protein markers in intact tumor, immune and stromal cells. Moreover, the high resolution of this confocal approach generates images of cells in a preserved tissue environment at a level of detail previously limited to cell culture. Precise imaging of cell membranes also makes it possible to detect and map cell-cell contacts and juxtracrine signaling complexes in immune cell niches.

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Publication Details

Journal
Nature Methods
Published
2025-09-29
DOI
https://doi.org/10.1038/s41592-025-02824-x
Citations
11
Primary Topic
Cell Image Analysis Techniques
Type
article
Field-Weighted Citation Impact
17.97
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article

Highly multiplexed 3D profiling of cell states and immune niches in human tumors

Paula Montero Llopis, Sandro Santagata, Gëorge F. Murphy, Y. Lu et al.
11 citations
Nature Methods
Cell Image Analysis Techniques
17.97
article

Highly multiplexed 3D profiling of cell states and immune niches in human tumors

Paula Montero Llopis, Sandro Santagata, Gëorge F. Murphy, Y. Lu, Zoltan Maliga, Felix Zhou, Gaudenz M. Danuser, Clarence Yapp, Peter Karl Sorger, Alex Y. H. Wong, Ajit J. Nirmal, Juliann B. Tefft, Christine G. Lian, Zhiguo Shang
article en
11 citations

Abstract

Diseases such as cancer involve alterations in cell proportions, states and interactions, as well as complex changes in tissue morphology and architecture. Histopathological diagnosis of disease and most multiplexed spatial profiling relies on inspecting thin (4-5 µm) specimens. Here we describe a high-plex cyclic immunofluorescence method for three-dimensional tissue imaging and use it to show that few, if any, cells are intact in conventional thin tissue sections, reducing the accuracy of cell phenotyping and interaction analysis. However, three-dimensional cyclic immunofluorescence of sections eightfold to tenfold thicker enables accurate morphological assessment of diverse protein markers in intact tumor, immune and stromal cells. Moreover, the high resolution of this confocal approach generates images of cells in a preserved tissue environment at a level of detail previously limited to cell culture. Precise imaging of cell membranes also makes it possible to detect and map cell-cell contacts and juxtracrine signaling complexes in immune cell niches.

Nature MethodsVol. 22(10)
Brigham and Women's Hospital (US), Harvard University (US), Southwestern Medical Center (US), Center for Systems Biology (US), Southwestern Medical Center (US), The University of Texas Southwestern Medical Center (US)
Openalex Percentile: Top 1%
Cell Image Analysis Techniques
17.97
DOIOpen Access PDFOpenAlex
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