# Protocol for spatial proteomic profiling of tonsil cancer microenvironments using multiplexed imaging-powered deep visual proteomics

## TL;DR

This paper introduces a detailed protocol for analyzing the tumor microenvironment in tonsil cancer using advanced imaging and proteomics techniques.

## Contribution

The novel contribution is a workflow combining multiplexed imaging and deep visual proteomics for spatially resolved proteomic profiling of FFPE tissues.

## Key findings

- The protocol enables automated 22-plex immunofluorescence staining and imaging of FFPE tissue sections.
- It facilitates single-cell isolation and proteomic analysis using ultra-sensitive mass spectrometry.
- The method is optimized for studying tumor-immune interactions and identifying biomarkers.

## Abstract

Here, we present a protocol for spatial proteomic profiling of the tumor microenvironment in tonsil cancer using multiplexed imaging-powered deep visual proteomics (mipDVP). We describe steps for automated 22-plex immunofluorescence staining and imaging on formalin-fixed paraffin-embedded (FFPE) tissue sections, automated single-cell laser microdissection, and single-cell-type mass spectrometry. This workflow enables the spatially resolved isolation of distinct cell populations for proteomic analysis. We optimized this protocol for studying tumor-immune interactions, where it facilitates the systematic identification of biomarkers and functional cellular networks.

For complete details on the use and execution of this protocol, please refer to Zheng et al.1

•Protocol for spatial proteomics of FFPE tissues•Steps for tumor microenvironment spatial analysis using 22-plex fluorescence imaging•Instructions for single-cell isolation using automated laser microdissection•Guidance on single-cell-type proteomic profiling using ultra-sensitive mass spectrometry

Protocol for spatial proteomics of FFPE tissues

Steps for tumor microenvironment spatial analysis using 22-plex fluorescence imaging

Instructions for single-cell isolation using automated laser microdissection

Guidance on single-cell-type proteomic profiling using ultra-sensitive mass spectrometry

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for spatial proteomic profiling of the tumor microenvironment in tonsil cancer using multiplexed imaging-powered deep visual proteomics (mipDVP). We describe steps for automated 22-plex immunofluorescence staining and imaging on formalin-fixed paraffin-embedded (FFPE) tissue sections, automated single-cell laser microdissection, and single-cell-type mass spectrometry. This workflow enables the spatially resolved isolation of distinct cell populations for proteomic analysis. We optimized this protocol for studying tumor-immune interactions, where it facilitates the systematic identification of biomarkers and functional cellular networks.