Changes in Circulating Tumor Cells and Endothelial Cells: The Role of Shear Stress and Extracellular Vesicles in Cellular Interactions

• Grant number: 25/00135-0
• Support Opportunities: Regular Research Grants
• Start date: June 01, 2025
• End date: May 31, 2028
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Vanessa Morais Freitas
• Grantee: Vanessa Morais Freitas
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Breast cancer is the most common type of tumor among women, with the triple-negative subtype (TNBC) being the most aggressive, associated with high mortality rates and lower survival in metastatic cases. Metastasis occurs through the spread of the tumor via circulation, involving processes such as intravasation and extravasation of tumor cells through the endothelium. Studies suggest that extracellular vesicles (EVs) released by tumor cells play a crucial role in preparing the metastatic niche. These EVs modulate the tumor microenvironment, increasing the permeability of endothelial cells (ECs) and allowing tumor cells to pass through the junctions between these cells. Additionally, blood flow exerts shear stress (FSS, fluid shear stress), which influences the adhesion and extravasation of tumor cells.Preliminary data from our group showed that small EVs (sEVs) derived from TNBC cells accumulate on the cell-cell junctions of human umbilical vein endothelial cells (HUVEC) under FSS. Moreover, mass spectrometry analyses comparing endothelial cells in static conditions and under FSS identified a group of proteins located in the plasma membrane and cell-cell junctions that are more abundant in cells exposed to FSS. The aim of this study is to identify the cell-cell junction proteins that interact with tumor-derived sEVs and investigate how this interaction affects the adhesion of circulating tumor cells (CTCs) to the EC monolayer under FSS.To achieve this, HUVECs will be treated with tumor-derived sEVs, and cell surface proteins, including those bound to the sEVs, will be biotinylated to avoid contamination with vesicles from the endosome-lysosome system in the cytoplasm. Subsequently, cell lysates will be purified on streptavidin-agarose columns, and proteins associated with CD63 will be co-immunoprecipitated, followed by analysis through Western blot and/or mass spectrometry. Additionally, microfluidic assays will be performed to evaluate the adhesion, extravasation, and viability of circulating tumor cells in ECs treated or not with sEVs.The expected results should complement the group's previous studies, elucidating an innovative mechanism by which sEVs modulate vascular permeability and promote metastasis in TNBC. These findings may open new avenues for the development of therapies targeting this interaction and blocking the preparation of the metastatic site. (AU)