Spatial proteome profiling by immunohistochemistry-based laser capture microdissection and data-independent...

Area of Science:

• Proteomics
• Cancer Research
• Biomedical Imaging

Background:

• Spatially resolved proteome profiling is crucial for understanding tumor heterogeneity.
• Laser capture microdissection (LCM) enables spatial analysis but conventionally relies on subjective H&E staining.
• Limitations in cell-type identification hinder broad LCM application in cancer research.

Purpose of the Study:

• To develop an accurate and efficient workflow for cell type-resolved proteome analysis using immunohistochemistry (IHC).
• To optimize protein recovery from IHC-stained tissues for proteomic analysis.
• To establish a sensitive method for spatial proteomic profiling of specific cell populations within tissues.

Main Methods:

• Developed an IHC-based workflow (IHC-SISPROT) for targeted cell marking and LCM.
• Optimized a decrosslinking procedure for IHC tissues compatible with spintip-based sample preparation (SISPROT).
• Utilized data-independent acquisition proteomics for high-sensitivity and reproducible analysis.

Main Results:

• IHC-SISPROT demonstrated comparable performance to H&E staining-based proteomic analysis.
• Identified over 3500 proteins from small tissue sections (0.2 mm², 12 µm) of hepatocellular carcinoma (HCC).
• Quantified 6660 and 6052 protein groups from cancer cells and cancer-associated fibroblasts (CAFs) in HCC tissues, revealing cell-specific ligands, receptors, and intercellular communication pathways.

Conclusions:

• IHC-SISPROT is a sensitive and accurate method for spatial proteomic profiling.
• This approach overcomes limitations of traditional LCM by enabling precise cell-type identification.
• Reveals novel insights into cell-specific signaling and communication within complex tissue microenvironments.