Abstract
Adipose-derived stem cell extracellular vesicles (ADSC-EVs) represent a promising therapeutic modality, yet their oncological safety and biological influence on triple-negative breast cancer (TNBC) remain incompletely understood. This study investigated the effects of ADSC-EVs isolated from distal adipose tissue on the migration, proliferation, and gene expression of the TNBC cell line MDA-MB-231, while also examining inter-donor variability in EV function. EVs were isolated using size-exclusion chromatography in alignment with MISEV2023 recommendations and methods previously validated within the laboratory. Due to technical issues with qNano analysis, full concentration characterisation could not be performed, and EV dosing was instead standardised to established laboratory mean concentrations. Nonetheless, representative data confirmed successful EV recovery from long-term frozen conditioned media, demonstrating the feasibility of EV isolation from stored samples.
Functional assays revealed that ADSC-EV treatment modestly reduced early migratory capacity in wound-healing assays, although all groups achieved full closure by 48 hours. In contrast, CCK-8 assays showed a time-dependent increase in proliferation for both fibroblast and ADSC-EVs at 72 hours, suggesting a general EV-driven enhancement of metabolic activity rather than a donor-specific or cell-type–specific proliferative effect. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of selected EMT, proliferation, signalling and immune-evasion markers vimentin, marker of proliferation Ki-67, epidermal growth factor receptor, programmed death-ligand 1 (VIM, MKI67, EGFR, CD274) showed no significant transcriptional changes following EV treatment. This lack of detectable mRNA modulation, paired with observable phenotypic effects, supports a model in which ADSC-EVs primarily act through transient signalling, protein-level regulation or miRNA-mediated post-transcriptional mechanisms rather than altering steady-state transcript abundance.
Analysis of inter-individual variation revealed subtle donor-dependent differences in EGFR and MKI67 expression following EV treatment, consistent with known influences of donor BMI, adipose tissue depot, metabolic health and cellular ageing on EV cargo composition. Although the study was not powered to statistically resolve donor-specific effects, these findings highlight the biological heterogeneity intrinsic to human ADSC-EV preparations and underscore the importance of donor selection and EV standardisation in therapeutic development. Overall, ADSC-EVs derived from adipose tissue exerted modest and context-dependent effects on TNBC cells. These results reinforce the need for rigorous EV characterisation, expanded donor cohorts and deeper mechanistic studies targeting protein-level and miRNA-mediated pathways to clarify the therapeutic potential and safety profile of ADSC-EVs in oncology.