Abstract A number of risk factors have been identified in relation to breast cancer (BC). Among these, environmental factors, particularly those related to nutritional status, have been shown to play a significant role in the biology of BC. Thus, there is a growing interest in assessing the potential benefits of diet and life style in the prevention and treatment of BC. Among dietary interventions, the Mediterranean Diet (MD) has been suggested to be the most efficacious in reducing the risk of BC and enhancing patient outcomes. Numerous studies have attempted to address the relationship between diet and circulating Extracellular Vesicles (EVs), small membrane-bound particles that represent a novel axis of intercellular communication in BC. It has been reported that dietary compounds may influence the biogenesis, release, and composition of the endogenous EVs. However, there is still a knowledge gap regarding the accurate phenotyping of diet-induced EVs and their potential role in mediating the effects of nutritional status on BC. Here, we investigated the impact of MD adherence on the composition and function of serum circulating EVs isolated from BC patients. BC patients have been classified by their level of MD adherence (Low: ≤5; Medium: 6-9; High: ≥10), as assessed by the MEDAS questionnaire. We isolated and fully characterized EVs from the serum of BC patients, in accordance with the MISEV 2023 guidelines, by Transmission Electron Microscopy, Nanotracking particle analysis (NTA) and immunoblot. The biological effects of BC patient-derived EVs on cell, proliferation, stemness, motility and metabolism in two different ERα-positive BC cell lines, MCF-7 and T-47D, were evaluated. The expression profiles of EV-packaged miRNAs were investigated by small RNA-sequencing analysis. NTA analysis revealed a correlation between elevated MD adherence and alterations in the number and dimensions of circulating EVs in BC patients. In addition, we demonstrated that the EVs isolated from BC patients with High MD adherence were less effective in sustaining cell viability and growth in both MCF-7 and T-47D as evidenced by anchorage-dependent (clonogenic assay) and anchorage-independent (soft-agar) assays, respectively. The boyden-chamber transmigration assay revealed that EVs isolated from BC patients with High MD adherence were less effective in promoting the migration of BC cells in the two employed experimental models. Finally, given that migratory potential is among the most significant characteristics of BC stem cells, the mammosphere forming efficiency assay was conducted. We found that EVs isolated from BC patients with High MD adherence exhibited a reduced capacity to form mammospheres in ER+ BC cells compared to EVs isolated from BC patients with Low MD adherence. Accordingly, BC cells treated with high MD adherence EVs showed a reduced cellular bioenergetics as measured by oxygen consumption rate, in terms of basal and maximal respiration, compared to cells treated with low MD adherence EVs. The analysis of EV-miRNA profile revealed 172 miRNAs, 18 of which were differentially expressed (∣fold change∣≥1.5) in Low vs High MD adherence EVs. These deregulated miRNAs are mainly involved in controlling proliferation, migration and metabolism in BC. These data demonstrated that High MD adherence modulates the EV composition (e.g. the miRNA profile) and exerts beneficial effects on BC cell development, growth and progression. Although these results are still preliminary, they underscore the importance of healthy dietary patterns in modulating BC biology and open new avenues for identifying potential therapeutic targets, to be exploited in patients with poor dietary habits. Citation Format: L. Gelsomino, D. Cristofaro, G. Filice, F. Giordano, I. Barone, D. Bonofiglio, G. Arpino, G. Salvatore, S. Andò, S. Catalano, C. Giordano. Mediterranean Diet and Breast Cancer Biology: Novel Insights into the Role of Extracellular Vesicles abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-10.
Gelsomino et al. (2026) studied this question.