An Integrated Spatial Multi-Omics Workflow for Sequential RNA and Protein Profiling in FFPE Tumor Tissue

Area of Science:

• Spatial biology
• Multi-omics
• Cellular niche analysis

Background:

• Understanding complex cellular niches, such as tertiary lymphoid structures (TLS), is crucial.
• Spatially resolved, multi-omic approaches are needed to link transcriptional and protein expression within individual cells.

Purpose of the Study:

• To present an integrated spatial multi-omics workflow for sequential mapping of gene and protein markers.
• To apply this workflow to colorectal liver metastases (CRLM) and matched normal liver tissues.
• To enable robust profiling of functional cellular states and spatial mapping of chemokine expression.

Main Methods:

• Sequential mapping of hundreds of genes using Xenium In Situ platform.
• Mapping of over 40 protein markers using Imaging Mass Cytometry™ (IMC™) technology.
• Application on formalin-fixed, paraffin-embedded (FFPE) tissue sections with computational co-registration for single-cell data integration.

Main Results:

• The workflow maintains tissue morphology and assay sensitivity.
• Observed correlation between β-catenin proteomic levels and malignant cell states.
• Characterized immune cell phenotypes in lymphoid aggregates and identified RNA-protein discrepancies for checkpoint molecules (PD-L1, TIM-3, IDO).

Conclusions:

• The technical framework enables robust profiling of functional cellular states and spatial mapping of chemokine expression.
• The approach offers more sensitive detection of clinically relevant targets compared to single-modality methods.
• This integrated spatial multi-omics workflow advances the understanding of complex cellular niches like TLS.