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Related Experiment Video

Updated: May 3, 2026

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection
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Protocol for spatial proteomic profiling of tonsil cancer microenvironments using multiplexed imaging-powered deep

Xiang Zheng1, Andreas Mund2, Matthias Mann3

  • 1Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, 2200 Copenhagen, Denmark; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.

STAR Protocols
|June 19, 2025
PubMed
Summary

We developed a spatial proteomic profiling protocol for tonsil cancer tumor microenvironments. This method uses multiplexed imaging-powered deep visual proteomics (mipDVP) to analyze cell types and interactions.

Keywords:
Biotechnology and bioengineeringCancerMicroscopy

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Area of Science:

  • Oncology
  • Proteomics
  • Immunology

Background:

  • The tumor microenvironment (TME) is crucial for cancer progression and treatment response.
  • Understanding spatial cell-cell interactions within the TME is key to identifying therapeutic targets.
  • Current proteomic methods often lack spatial resolution, limiting insights into TME heterogeneity.

Purpose of the Study:

  • To present a novel protocol for spatial proteomic profiling of the tumor microenvironment in tonsil cancer.
  • To enable detailed analysis of tumor-immune interactions within FFPE tissue sections.
  • To facilitate the identification of novel biomarkers and functional cellular networks in cancer.

Main Methods:

  • Development of an automated 22-plex immunofluorescence staining and imaging workflow.
  • Implementation of automated single-cell laser microdissection for precise cell isolation.
  • Integration of single-cell-type mass spectrometry for proteomic analysis.
  • Optimization of the workflow for formalin-fixed paraffin-embedded (FFPE) tissues.

Main Results:

  • Successful spatial proteomic profiling of the tonsil cancer TME.
  • Spatially resolved isolation of distinct cell populations for proteomic analysis.
  • Facilitation of systematic identification of biomarkers and functional cellular networks.
  • Demonstration of the protocol's utility in studying tumor-immune interactions.

Conclusions:

  • The presented multiplexed imaging-powered deep visual proteomics (mipDVP) protocol offers a powerful approach for spatial proteomic analysis of FFPE tissues.
  • This workflow enables a deeper understanding of cellular heterogeneity and interactions within the tumor microenvironment.
  • The protocol is optimized for identifying biomarkers and therapeutic targets in tonsil cancer and other malignancies.