CITATION.cff

cff-version: 1.2.0
title: Pathology-oriented multiplexing enables integrative disease mapping
abstract: |
  The expression and location of proteins in tissues represent key determinants of health and disease. Although recent advances in multiplexed imaging have expanded the number of spatially accessible proteins1–3, the integration of biological layers (that is, cell structure, subcellular domains and signalling activity) remains challenging. This is due to limitations in the compositions of antibody panels and image resolution, which together restrict the scope of image analysis. Here we present pathology-oriented multiplexing (PathoPlex), a scalable, quality-controlled and interpretable framework. It combines highly multiplexed imaging at subcellular resolution with a software package to extract and interpret protein co-expression patterns (clusters) across biological layers. PathoPlex was optimized to map more than 140 commercial antibodies at 80 nm per pixel across 95 iterative imaging cycles and provides pragmatic solutions to enable the simultaneous processing of at least 40 archival biopsy specimens. In a proof-of-concept experiment, we identified epithelial JUN activity as a key switch in immune-mediated kidney disease, thereby demonstrating that clusters can capture relevant pathological features. PathoPlex was then used to analyse human diabetic kidney disease. The framework linked patient-level clusters to organ disfunction and identified disease traits with therapeutic potential (that is, calcium-mediated tubular stress). Finally, PathoPlex was used to reveal renal stress-related clusters in individuals with type 2 diabetes without histological kidney disease. Moreover, tissue-based readouts were generated to assess responses to inhibitors of the glucose cotransporter SGLT2. In summary, PathoPlex paves the way towards democratizing multiplexed imaging and establishing integrative image analysis tools in complex tissues to support the development of next-generation pathology atlases.
issn: 1476-4687
journal: Nature
url: https://doi.org/10.1038/s41586-025-09225-2
authors:
  - family-names: Kuehl
    given-names: Malte
  - family-names: Okabayashi
    given-names: Yusuke
  - family-names: Wong
    given-names: Milagros N.
  - family-names: Gernhold
    given-names: Lukas
  - family-names: Gut
    given-names: Gabriele
  - family-names: Kaiser
    given-names: Nico
  - family-names: Schwerk
    given-names: Maria
  - family-names: Gräfe
    given-names: Stefanie K.
  - family-names: Ma
    given-names: Frank Y.
  - family-names: Tanevski
    given-names: Jovan
  - family-names: Schäfer
    given-names: Philipp S. L.
  - family-names: Mezher
    given-names: Sam
  - family-names: Sarabia del Castillo
    given-names: Jacobo
  - family-names: Goldbeck-Strieder
    given-names: Thiago
  - family-names: Zolotareva
    given-names: Olga
  - family-names: Hartung
    given-names: Michael
  - family-names: Delgado Chaves
    given-names: Fernando M.
  - family-names: Klinkert
    given-names: Lukas
  - family-names: Gnirck
    given-names: Ann-Christin
  - family-names: Spehr
    given-names: Marc
  - family-names: Fleck
    given-names: David
  - family-names: Joodaki
    given-names: Mehdi
  - family-names: Parra
    given-names: Victor
  - family-names: Shaigan
    given-names: Mina
  - family-names: Diebold
    given-names: Martin
  - family-names: Prinz
    given-names: Marco
  - family-names: Kranz
    given-names: Jennifer
  - family-names: Kux
    given-names: Johan M.
  - family-names: Braun
    given-names: Fabian
  - family-names: Kretz
    given-names: Oliver
  - family-names: Wu
    given-names: Hui
  - family-names: Grahammer
    given-names: Florian
  - family-names: Heins
    given-names: Sven
  - family-names: Zimmermann
    given-names: Marina
  - family-names: Haas
    given-names: Fabian
  - family-names: Kylies
    given-names: Dominik
  - family-names: Wanner
    given-names: Nicola
  - family-names: Czogalla
    given-names: Jan
  - family-names: Dumoulin
    given-names: Bernhard
  - family-names: Zolotarev
    given-names: Nikolay
  - family-names: Lindenmeyer
    given-names: Maja
  - family-names: Karlson
    given-names: Pall
  - family-names: Nyengaard
    given-names: Jens R.
  - family-names: Sebode
    given-names: Marcial
  - family-names: Weidemann
    given-names: Sören
  - family-names: Wiech
    given-names: Thorsten
  - family-names: Groene
    given-names: Hermann-Josef
  - family-names: Tomas
    given-names: Nicola M.
  - family-names: Meyer-Schwesinger
    given-names: Catherine
  - family-names: Kuppe
    given-names: Christoph
  - family-names: Kramann
    given-names: Rafael
  - family-names: Karras
    given-names: Alexandre
  - family-names: Bruneval
    given-names: Patrick
  - family-names: Tharaux
    given-names: Pierre-Louis
  - family-names: Pastene
    given-names: Diego
  - family-names: Yard
    given-names: Benito
  - family-names: Schaub
    given-names: Jennifer A.
  - family-names: McCown
    given-names: Phillip J.
  - family-names: Pyle
    given-names: Laura
  - family-names: Choi
    given-names: Ye Ji
  - family-names: Yokoo
    given-names: Takashi
  - family-names: Baumbach
    given-names: Jan
  - family-names: Sáez
    given-names: Pablo J.
  - family-names: Costa
    given-names: Ivan
  - family-names: Turner
    given-names: Jan-Eric
  - family-names: Hodgin
    given-names: Jeffrey B.
  - family-names: Saez-Rodriguez
    given-names: Julio
  - family-names: Huber
    given-names: Tobias B.
  - family-names: Bjornstad
    given-names: Petter
  - family-names: Kretzler
    given-names: Matthias
  - family-names: Lenoir
    given-names: Olivia
  - family-names: Nikolic-Paterson
    given-names: David J.
  - family-names: Pelkmans
    given-names: Lucas
  - family-names: Bonn
    given-names: Stefan
  - family-names: Puelles
    given-names: Victor G.
date-published: 2025-07-18
identifiers:
  - type: doi
    value: 10.1038/s41586-025-09225-2
preferred-citation:
  title: Pathology-oriented multiplexing enables integrative disease mapping
  abstract: |
    The expression and location of proteins in tissues represent key determinants of health and disease. Although recent advances in multiplexed imaging have expanded the number of spatially accessible proteins1–3, the integration of biological layers (that is, cell structure, subcellular domains and signalling activity) remains challenging. This is due to limitations in the compositions of antibody panels and image resolution, which together restrict the scope of image analysis. Here we present pathology-oriented multiplexing (PathoPlex), a scalable, quality-controlled and interpretable framework. It combines highly multiplexed imaging at subcellular resolution with a software package to extract and interpret protein co-expression patterns (clusters) across biological layers. PathoPlex was optimized to map more than 140 commercial antibodies at 80 nm per pixel across 95 iterative imaging cycles and provides pragmatic solutions to enable the simultaneous processing of at least 40 archival biopsy specimens. In a proof-of-concept experiment, we identified epithelial JUN activity as a key switch in immune-mediated kidney disease, thereby demonstrating that clusters can capture relevant pathological features. PathoPlex was then used to analyse human diabetic kidney disease. The framework linked patient-level clusters to organ disfunction and identified disease traits with therapeutic potential (that is, calcium-mediated tubular stress). Finally, PathoPlex was used to reveal renal stress-related clusters in individuals with type 2 diabetes without histological kidney disease. Moreover, tissue-based readouts were generated to assess responses to inhibitors of the glucose cotransporter SGLT2. In summary, PathoPlex paves the way towards democratizing multiplexed imaging and establishing integrative image analysis tools in complex tissues to support the development of next-generation pathology atlases.
  type: article
  issn: 1476-4687
  journal: Nature
  url: https://doi.org/10.1038/s41586-025-09225-2
  authors:
    - family-names: Kuehl
      given-names: Malte
    - family-names: Okabayashi
      given-names: Yusuke
    - family-names: Wong
      given-names: Milagros N.
    - family-names: Gernhold
      given-names: Lukas
    - family-names: Gut
      given-names: Gabriele
    - family-names: Kaiser
      given-names: Nico
    - family-names: Schwerk
      given-names: Maria
    - family-names: Gräfe
      given-names: Stefanie K.
    - family-names: Ma
      given-names: Frank Y.
    - family-names: Tanevski
      given-names: Jovan
    - family-names: Schäfer
      given-names: Philipp S. L.
    - family-names: Mezher
      given-names: Sam
    - family-names: Sarabia del Castillo
      given-names: Jacobo
    - family-names: Goldbeck-Strieder
      given-names: Thiago
    - family-names: Zolotareva
      given-names: Olga
    - family-names: Hartung
      given-names: Michael
    - family-names: Delgado Chaves
      given-names: Fernando M.
    - family-names: Klinkert
      given-names: Lukas
    - family-names: Gnirck
      given-names: Ann-Christin
    - family-names: Spehr
      given-names: Marc
    - family-names: Fleck
      given-names: David
    - family-names: Joodaki
      given-names: Mehdi
    - family-names: Parra
      given-names: Victor
    - family-names: Shaigan
      given-names: Mina
    - family-names: Diebold
      given-names: Martin
    - family-names: Prinz
      given-names: Marco
    - family-names: Kranz
      given-names: Jennifer
    - family-names: Kux
      given-names: Johan M.
    - family-names: Braun
      given-names: Fabian
    - family-names: Kretz
      given-names: Oliver
    - family-names: Wu
      given-names: Hui
    - family-names: Grahammer
      given-names: Florian
    - family-names: Heins
      given-names: Sven
    - family-names: Zimmermann
      given-names: Marina
    - family-names: Haas
      given-names: Fabian
    - family-names: Kylies
      given-names: Dominik
    - family-names: Wanner
      given-names: Nicola
    - family-names: Czogalla
      given-names: Jan
    - family-names: Dumoulin
      given-names: Bernhard
    - family-names: Zolotarev
      given-names: Nikolay
    - family-names: Lindenmeyer
      given-names: Maja
    - family-names: Karlson
      given-names: Pall
    - family-names: Nyengaard
      given-names: Jens R.
    - family-names: Sebode
      given-names: Marcial
    - family-names: Weidemann
      given-names: Sören
    - family-names: Wiech
      given-names: Thorsten
    - family-names: Groene
      given-names: Hermann-Josef
    - family-names: Tomas
      given-names: Nicola M.
    - family-names: Meyer-Schwesinger
      given-names: Catherine
    - family-names: Kuppe
      given-names: Christoph
    - family-names: Kramann
      given-names: Rafael
    - family-names: Karras
      given-names: Alexandre
    - family-names: Bruneval
      given-names: Patrick
    - family-names: Tharaux
      given-names: Pierre-Louis
    - family-names: Pastene
      given-names: Diego
    - family-names: Yard
      given-names: Benito
    - family-names: Schaub
      given-names: Jennifer A.
    - family-names: McCown
      given-names: Phillip J.
    - family-names: Pyle
      given-names: Laura
    - family-names: Choi
      given-names: Ye Ji
    - family-names: Yokoo
      given-names: Takashi
    - family-names: Baumbach
      given-names: Jan
    - family-names: Sáez
      given-names: Pablo J.
    - family-names: Costa
      given-names: Ivan
    - family-names: Turner
      given-names: Jan-Eric
    - family-names: Hodgin
      given-names: Jeffrey B.
    - family-names: Saez-Rodriguez
      given-names: Julio
    - family-names: Huber
      given-names: Tobias B.
    - family-names: Bjornstad
      given-names: Petter
    - family-names: Kretzler
      given-names: Matthias
    - family-names: Lenoir
      given-names: Olivia
    - family-names: Nikolic-Paterson
      given-names: David J.
    - family-names: Pelkmans
      given-names: Lucas
    - family-names: Bonn
      given-names: Stefan
    - family-names: Puelles
      given-names: Victor G.
  date-published: 2025-07-18
  identifiers:
    - type: doi
      value: 10.1038/s41586-025-09225-2