Breast cancer-related heterogeneity reflected on circulating and vesicular miRNAs: high-throughput results and proof-of-concept for selecting tumor-derived extracellular vesicles

Author(s)

Alexis German Murillo Carrasco

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2023-12-04

Examining board members

Roger Chammas; Edna Teruko Kimura; Camila Motta Venchiarutti Moniz

• Advisor: Roger Chammas

Abstract

Breast cancer has recently become the most frequently diagnosed cancer type worldwide, with a particular emphasis on women diagnosed before age 40, known as early-onset (young diagnosed) breast cancer. While classification based on immunohistochemical profiles serves as a broad screening, prognosis, and treatment prediction system, BC patients often require additional information for precise treatment. Clinical routines also demand minimally invasive methods to obtain patients\' data and monitor their treatment response. In light of these challenges, this study proposed the collection of tumor characteristics from blood samples (liquid biopsies), with emphasis on the accumulation of microRNAs (miRNAs) regulatory molecules that are found in the cell-free circulating material, such as extracellular vesicles (EVs). In this thesis, we explored the miRNome of both circulating (cf-miRNA) and vesicular (EV-miRNA) content of BC patients classified into main immunohistochemical groups (Luminal A, Luminal B, Luminal HER2, HER2+, and TNBC). Two separate cohorts of BC samples were run in EV-miRNA experiments to assess technical protocols to ensure consistent results. Upon analyzing miRNA expression data, it became evident that cf-miRNA is susceptible to contamination from other circulating sources, such as hemolysis. However, both cf-miRNA and EV-miRNA proved to be informative sources for discerning miRNA expression differences between BC subtypes. For instance, hsa-miR-197-3p distinguished TNBC patients, and hsa-miR-5001-5p differentiated Luminal HER2 patients, as observed in both cf-miRNA and EV-miRNA datasets. Among vesicular cargo, hsa-miR-411-5p characterizes Luminal HER2 patients, whereas hsa-miR-1266-5p, hsa-miR-584-5p, hsa-miR-2053, hsa-miR-525-5p, and hsa-miR-642a-5p were indicative of young TNBC patients. In addition to these specific markers, various deregulated miRNAs were identified, some associated with tumor-related pathways and others linked to anti-tumor processes. This discovery led to the hypothesis that different EV subpopulations might exist in the bloodstream. We then performed experiments using a glycan-binding protein derived from jackfruit (Artocarpus integrifolia) to identify specific targets (tumor-related Tn antigen and its derivatives) in formalin-fixed paraffin-embedded tissues and in plasma. BC cell lines, and EVs isolated from BC tumors were tested using Jacalin and recovered through incubation with D-galactose (the monosaccharide which binds in the carbohydrate recognition domain of Jacalin). This approach successfully separated 10% of total EVs, demonstrating the feasibility of selecting plasma EV fractions using surface glycoproteins and affinity strategies (AU)

• FAPESP's process: 19/05583-0 - Enrichment of STn/Tn+ tumor-derived extracellular microvesicles for evaluating miRNA-based biomarkers in Breast Cancer patients
• Grantee: Alexis Germán Murillo Carrasco
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)