dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples

Abstract

We employed laser microdissection to selectively harvest tumor cells and stroma from the microenvironment of formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. The captured HNSCC tissue fractions were analyzed by quantitative mass spectrometry-based proteomics using a data independent analysis approach. In paired samples, we achieved excellent proteome coverage having quantified 6,668 proteins with a median quantitative coefficient of variation under 10%. We observed significant differences in relevant functional pathways between the spatially resolved tumor and stroma regions. Our results identified extracellular matrix (ECM) as a major component enriched in the stroma, including many cancer associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from very low starting input in a scalable format that is useful to decipher the alterations in tumor and the stromal microenvironment. Correlating such results with clinical features or disease progression will likely enable identification of novel targets for disease classification and interventions.

Figure 1.

Representative (A) H&E stained FFPE tissue section, and (B) image after LMD capture of the tumor regions. Base peak chromatograms showing the overlay of triplicate runs of an enriched (C) tumor and (D) paired stroma sample. (E) Graphical representation of number of proteins identified with 2 or more peptide matches in the enriched tumor and stroma samples. The median is indicated by a line. (F) Schematic representation of CV value spread of quantitation for each sample, median (bold line) and upper and lower quartile (thin line) are indicated.

Figure 2.

Functional association of upregulated proteins. (A) Unsupervised hierarchical cluster analysis of stroma and tumor region proteins based on abundance, and (B) Volcano plot of protein abundance. In volcano plot the box dot plots of significantly changed proteins between the regions are indicated, and selected candidates are in red. Selected enriched functional pathways (String analysis) in stroma (B) and tumor (C) are shown with enrichment score and false discovery rate (FDR).

Figure 3.

High confidence STRING network (A) functional and physical association of upregulated CAF markers in stroma, physical association of upregulated stromal (B) heparin binding and (C) GAG binding proteins.