Abstract
Autologous fat grafting (AFG) is a favourable surgical option for oncologic breast reconstruction as it provides a natural cosmetic outcome with a low surgical risk. However, graft rates are variable, which limits AFG’s clinical utility. Enriching grafts with extracellular vesicles (EVs) from adipose-derived stem cells (ADSCs) may promote AFG retention by modulating the tissue microenvironment to reduce fibrosis, promote vasculogenesis, and promote a favourable inflammatory state. To adequately examine the functional role of ADSC-EVs in this context, we carried out several in vitro experiments to determine the effect of ADSC-EVs on different cell types prevalent in the breast microenvironment, using both single cell type and multicellular models.
Lipoaspirates were collected from patients undergoing AFG and ADSCs were isolated enzymatically and cultured. To fulfil international scientific guidelines, ADSCs were characterised using a flow cytometry panel and demonstration of multipotent capabilities. ADSC-EVs were then isolated from the culture media and characterised using tunable resistive pulse sensing, Western blot, and transmission electron microscopy. We demonstrated successful isolation and characterisation of ADSCs and their associated EVs as a step towards investigating their potential functional role on different cell types in the breast cavity.
These methodologies were then used to determine the functional impact of ADSC-EVs on stromal cells in single cell type in vitro models. Fibroblasts and endothelial cells were cultured with isolated ADSC-EVs to assess proliferation, migration, and tube formation. We determined that ADSC-EVs modulate fibroblast proliferation and migration under unstimulated, pro-inflammatory, and pro-fibrotic conditions. We also report that ADSC-EVs increase tube formation and decrease migration of endothelial cells. This study demonstrated that ADSC-EVs modulate both fibroblast and HUVEC activity in vitro, demonstrating potentially beneficial functional changes for AFG retention.
As many cellular functions and processes are directly reliant on the local inflammatory environment, we next investigated the immunomodulatory role of ADSC-EVs on primary human macrophages in vitro. Monocyte derived macrophages were isolated, polarised towards an M0-like, M1-like, or M2-like phenotype, and cultured with ADSC-EVs. Using flow cytometry and high dimensional analysis, we identified 10 distinct metaclusters, each of which represents a unique macrophage phenotype. We determined that ADSC-EVs shift M1-like and M0-like macrophages towards metaclusters that express lower levels of functional markers, while not directly impacting marker expression on M2-like macrophages. This is suggestive of ADSC-EVs facilitating a re-balancing of macrophages towards an anti-inflammatory phenotype.
Lastly, following the results of our single cell type in vitro models, we aimed to determine how co-culture in 3D multicellular models affects endothelial cell tube formation in the presence of ADSC-EVs. Endothelial cells were cultured in 3D multicellular models with ADSC-EVs and either fibroblasts or macrophages. Endothelial cell tube formation was visualised using a confocal microscope. We report that ADSC-EVs are taken up by endothelial cells, macrophages, and fibroblasts, and that they cause an increase in tube formation when endothelial cells are cultured within models with either macrophages or fibroblasts. ADSC-EVs were also shown to increase co-localisation of macrophages and endothelial cells. The results of this study further suggest that ADSC-EVs facilitate pro-reparative effects.
Together, the findings from this thesis indicate that ADSC-EVs facilitate beneficial effects on relevant cells in both single cell type and multicellular models in vitro. At the cellular level, ADSC-EVs demonstrate great promise as a future therapeutic agent for improving AFG retention for breast cancer survivors.