miR-183-5p-enriched Endothelial Extracellular Vesicles: Key players in pre-metastatic niche formation in breast cancer ?

Abstract

Breast cancer is one of the most prevalent malignancies among women worldwide and remains a leading cause of cancer-related mortality. Although early-stage disease is associated with favorable outcomes, prognosis declines drastically once the tumor progresses to a metastatic state. Elucidating the mechanisms that drive pre-metastatic niche (PMN) formation is therefore essential for the development of novel strategies for preventing and treating metastasis. Recently, the laboratory of molecular angiogenesis discovered that extracellular vesicles (EVs) released by endothelial cells in a mammary tumour environment participate in the recruitment of macrophages into the tumour, leading to an immunomodulatory phenotype permissive for tumour growth. Using RNA-Seq approaches, we identified several microRNAs (miRNAs) found in endothelial EVs sharing common targets involved in the regulation of the immune system, such as miR-183-5p. The objectives of this thesis are to investigate the potential role of miR-183-5p in PMN formation using both in vitro and in vivo breast cancer models, with particular emphasis on extracellular matrix (ECM) remodeling and immune modulation in the lung. These processes are recognized as critical determinants of PMN establishment and subsequent metastatic progression. To address these objectives, ECM remodeling was investigated using mouse embryonic fibroblasts (MEF) through a series of functional assays, including proliferation, adhesion, and invasion analysis, complemented by immunofluorescence detection of activation markers in MEFs treated with miR-183-5p-enriched endothelial EVs. In parallel, immune modulation was examined using bone-marrow-derived macrophages (BMDMs) via adhesion assays and immunostainings to assess M1/M2 polarization, with additional validation supported by flow cytometry (FACS) and quantitative PCR (qPCR) analyses. Our findings indicate that treatment with miR-183-5p-enriched EVs induces the activation of MEFs, as evidenced by enhanced adhesion and upregulation of α-smooth muscle actin (αSMA), a key protein involved in ECM remodeling. Furthermore, miR-183-5p promotes immune modulation by skewing BMDMs toward an M2-polarized phenotype. Notably, both in vitro immunostainings in BMDMs and in vivo qPCR analyses on the lungs and tumors of 4T1 tumor-bearing mice support the involvement of the aryl hydrocarbon receptor (AhR) pathway in mediating these effects. Taken together, these results support the hypothesis that miR-183-5p contributes to ECM remodeling and immune suppression, thereby promoting PMN formation. This work highlights miR-183-5p as a potential therapeutic target for metastasis prevention and provides a foundation for future mechanistic and translational investigation.