Identification of Proteomic Markers in Head and Neck Cancer Using MALDI–MS Imaging, LC–MS/MS, and Immunohistochemistry

Abstract

Purpose The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI–MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level. Experimental design MALDI–MSI is used on snap frozen and formalin‐fixed and paraffin‐embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) to analyze m/z values localized in tumor and nontumor regions. Peptide identification is performed using LC–MS/MS and immunohistochemistry (IHC). Results In both FFPE and frozen tissue specimens, eight characteristic masses of the tumor's epithelial region are found. Using LC–MS/MS, the peaks are identified as vimentin, keratin type II, nucleolin, heat shock protein 90, prelamin‐A/C, junction plakoglobin, and PGAM1. Lastly, vimentin, nucleolin, and PGAM1 are verified with IHC. Conclusions and Clinical Relevance The combination of MALDI–MSI, LC‐MS/MS, and subsequent IHC furnishes a tool suitable for characterizing the molecular heterogeneity of tissue. It is also suited for use in identifying new representative biomarkers to enable a more individualized therapy.

Microdissection—An Essential Prerequisite for Spatial Cancer Omics

“…Hoffmann et al. [ 74 ] used MALDI MSI to investigate tumor tissue heterogeneity. The focus of their investigations was on biomarkers useful for detecting malignant cells in squamous cell carcinoma.…”

“…[ 17,80 ] For all identification done by MS/MS the results should be confirmed by immunohistochemically staining with an appropriate antibody. [ 81 ]…”

Spatial proteomics revealed a CX3CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder

“…The following steps of on-section tryptic digestion and application of CHCA matrix were performed as described previously. 52 Measurements were performed using the UltrafleXtreme mass spectrometer from Bruker. The parameters were as follows: reflective positive ion mode, mass range: 700-4000 m/z, spatial resolution: 50 µm 2 , laser beam size: medium, shots per position: 300-500, sampling rate: 2.5 GS/s.…”

Relocation of macrophages maintains the barrier function of the urothelium and protects against persistent infection

“…Tissue sections were washed with ethanol (70% and 95%) for 2 minutes. The following steps of on-section tryptic digestion and application of CHCA matrix were performed as described previously (Hoffmann et al, 2019). Data acquisition and analysis: Measurements were performed using the UltrafleXtreme mass spectrometer from Bruker.…”