dia-PASEF proteomic analysis of HNSCC tumor and stroma enriched sections from FFPE samples prepared with laser capture microdissection

Introduction

Head and neck squamous cell carcinoma (HNSCC), an epithelial cancer is the most common type of head and neck cancer. HNSCC cells first invade the basement membrane of native epithelium, and in >50% cases proceed to lymph node metastasis, which is associated with poor survival. Overall, the response to available treatments has been moderate. The genomic and transcriptomic landscape of HNSCC (The Cancer Genome Atlas) has been defined, but pinpointing the genetic aberrations linked to tumor phenotypes remains elusive. Deep proteome analysis of tumor and matched normal adjacent tissues (NATs) has been performed (Clinical Proteomic Tumor Analysis Consortium). Proteomic comparison of the cancer cells and its neighboring microenvironment may help identify novel targets for early detection, and intervention of HNSCC.

Methods

In this study, laser capture microdissection (LCM) was used to collect tumor and stroma enriched sections from formalin-fixed paraffin-embedded (FFPE) tissues. The samples were processed and digested with trypsin. Data independent LC-MS/MS analysis was performed using the timsTOF HT mass spectrometer connected to nanoElute 2 LC system. Each sample was analyzed in triplicate using a 32-minute gradient (500 ng peptide per injection, 40 min total run time), resulting in a throughput of 24 samples per day. The data analysis was performed using the directDIA+ workflow (Spectronaut 18 software) and the Uniprot-Human-reviewed database (20,383 protein entries).

Preliminary Data

We have completed analysis of 20 samples, and in total 8,300 protein groups were identified corresponding to ~96,000 precursors. Excellent technical reproducibility was observed between sample runs with median CV of <8% at protein quantitation level. In paired tumor and stroma samples 6,676 proteins were quantified across all samples. All stroma samples clustered together and clearly separated from tumor group by hierarchical analysis. Volcano plot analysis extracted the proteins that are significantly more abundant in stroma vs. tumor enriched samples. GO functional and pathway enrichment analysis of these proteins identified several functional groups relevant to stromal and tumor regions, e.g., relative high abundance of growth factor binding, collagen binding, heparin binding proteins, and ECM structural constituents in the stromal region. Overall, in this study we were able to achieve excellent proteome coverage in LCM FFPE samples. This is especially important for stromal samples where available amount of protein is very low. The methodology allows for comparative deep proteome analysis of tumor and its adjacent microenvironment in a scalable format.