Study of protein composition in the invasion area of squamous cell carcinoma by proteomics based on mass spectrometry

Author(s)

Carolina Moretto Carnielli

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-01-08

Examining board members

Adriana Franco Paes Leme; Alexandre Keiji Tashima; Diana Noronha Nunes; Daniel Guariz Pinheiro; Mariane Tami Amano

• Advisor: Adriana Franco Paes Leme

Abstract

Head and neck cancers are a group of cancers that involve the oral cavity, pharynx and larynx. The majority of head and neck cancers are squamous-cell carcinoma (SCC), which has high morbidity. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Recent evidence suggests that the invasive tumor front area of carcinomas, ie, the tumor-host interface, have different molecular profile and distinct morphological features compared to other areas of the tumor and may be considered as a region of interest to identify prognostic markers. Thus, the aim of this study is to identify the proteins present in the stroma of the invasive tumor front compared to the stroma of the inner tumor. The combination of laser microdissection (LMD) and mass spectrometry (MS) has been considered as an approach with high robustness to protein identification of specific regions of tumor tissues. Twenty samples of CEC of tongue tissues fixed in paraffin and in formalin were used to isolate the stroma from the invasive tumor front and from the inner tumor by LMD, followed by protein extraction, protein digestion and liquid-chromatography coupled to mass spectrometry in tandem by discovery-based proteomics (LC-MS/MS). Statistical analysis and correlation analysis with clinicopathological data revealed five spatially distinct proteins between the stroma regions. The abundance of COL6A1 (Collagen alpha-1(VI) chain), FSCN1 (Fascin-1), ITGAV (Integrin alpha-V), MB (Myoglobin) and THBS2 (Thrombospondin-2) were associated to clinical stage, lymph node metastasis and lymph node recurrence (p-valor<0.05, R<-0.7 or 0.70.5). The location of COL6A1 and MB were confirmed by immunohistochemistry in a new set of 96 patient samples of oral SCC (OSCC) and association to lymph node capsular infiltration, margin status and local recurrence. Once lymph node metastasis is the main poor prognostic factor in OSCC, the selected proteins were evaluated in saliva samples of patients with metastasis (n=10) and without metastasis (n=23). Targeted proteomics analysis based on SRM method (Selected Reaction Monitoring) demonstrated that COL6A1 and ITGAV were found with lower abundance in patients with lymph metastasis and able to distinguish between the two groups of samples by ROC analysis with high sensitivity (COL6A1: 0.739; ITGAV: 0.696) and high specificity (COL6A1: 0.9; ITGAV: 0.8). Taken together, by the association of LMD and MS (DDA and SRM) approaches, this project demonstrate for the first time proteins spatially organized in the stroma of different regions of the tumor, the invasive tumor front and the inner tumor, that are also present in saliva samples of OSCC patients and able to distinguish between those with metastasis and without metastasis, thus, with potential prognostic value. These results indicate new candidate prognosis markers for OSCC that may guide therapeutic strategies in clinical routine (AU)

• FAPESP's process: 13/16483-0 - Study of protein composition in the invasion area of squamous cell carcinoma by proteomics based on mass spectrometry
• Grantee: Carolina Moretto Carnielli
• Support Opportunities: Scholarships in Brazil - Doctorate