Potential markers in fibroblasts and extracellular vesicles of oral leukoplakia can antecipate malignant transformation to oral squamous cell carcinoma

Author(s)

Jamile de Oliveira Sá

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2021-11-26

Examining board members

Adriana Franco Paes Leme; André Zelanis Palitot Pereira; Vanessa Morais Freitas; Patricia Pintor dos Reis; Márcio Ajudarte Lopes

• Advisor: Adriana Franco Paes Leme

Abstract

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck region with 377,713 new cases estimated in 2020 (Globocan, 2020). Despite advances in research and local and systemic therapies, no significant improvement in survival rates has been achieved, with an overall 5-year survival rate of around 40-50%. Therefore, early diagnosis and treatment of lesions at risk of developing into cancer, such a oral leukoplakia (OL), are essential to increase the patient survival. In this context, there is an urgent need to identify markers of malignant transformation to aid in diagnosis and prognosis, as well as, in guiding treatment decisions. The communication between cells in the pre-and tumoral microenvironment (TM) is decisive for the progression of the tumor. The main cells involved in this process are the fibroblasts, known as carcinoma-associated fibroblasts (CAFs). Especially, the extracellular vesicles (EVs) released by CAFs can modulate a fibrotic and immunosuppressive TM, favoring the cell growth, invasion, and metastasis. However, little is known about the signaling processes promoted by fibroblast-derived EVs during the oral malignant transformation. Thus, to identify proteins associated with malignancy, this study characterized populations of fibroblasts isolated from control tissue (NAFs), oral leukoplakia (LAFs), and CAFs and compared the proteomic profile of the cells and their EVs using mass spectrometry-based proteomics. Initially, the characterization of the cell phenotypes was performed by RT-qPCR, revealing decreased expression of SLP1-, associated with antigen presentation, in LAFs and CAFs when compared to NAFs. CAFs showed a myofibroblastic signature, with differential expression of ACTA2+, POSTN+, TAGLN+, PDPN+, THY1-, CXCL12- and SLP1- in comparison with NAFs and/or LAFs. The EVs had similar mean size and number between groups, 116.0 ±18.1 nm, and 1.09E+12 ± 4.14E+11 particles, respectively. A total of 1,529 proteins in cells and 756 in EVs were identified, being 19 and 46 statistically significant proteins, respectively. The protein abundance profile of cells and EVs determined the patients’clusters, which indicated clinical characteristics associated with malignancy and tumor progression. Furthermore, it also led to the recognition of three distinct patterns between NAFs, LAFs, and CAFs, mainly characterized by pathways that modulate the immune system and regulate the cytoskeleton and cell migration. Among the significant proteins that showed clinical correlation, three proteins in cells and one protein in VEs showed differential abundance in LAFs and CAFs relative to NAFs. Particularly, the increased abundance of AK1 in LAF-cells and CAF-cells correlated with the size of OLs lesions, and the decreased abundance of TNFRSF11B in LAF-ev and CAF-ev was associated with the absence of abnormal size variation of cell nuclei in OLs and absence of angiolymphatic invasion in OSCC. Therefore, AK1 and TNFRSF11B are potential candidates for anticipating the transition to malignancy, being able to assist in the diagnosis and prognosis of OLs, as well as to guide individual treatment decisions (AU)

• FAPESP's process: 18/15728-3 - Association of extracellular vesicles derived from fibroblasts with the progression of Oral Squamous Cell Carcinoma
• Grantee: Jamile de Oliveira Sá
• Support Opportunities: Scholarships in Brazil - Doctorate