Abstract
Background: Breast Cancer is the most common cancer amongst wāhine in Aotearoa, with approximately 3,300 diagnoses every year. Breast reconstruction after breast cancer treatment (e.g. mastectomy) is an important part of the overall treatment process. Autologous Fat Grafting (AFG) is an increasingly popular breast reconstruction option with minimal risks. However, a major caveat of AFG is only 30% of the original fat graft retained in the recipient site. Fat graft retention is regulated by the tissue microenvironment involving complex cross-talk between Adipose derived stem cells (ADSCs) in the donor site and fibroblasts cells in the breast cavity. The release of extracellular vesicles (EVs) from ADSCs is one signalling pathway between these two sites and investigating this dynamic further could help to improve fat graft retention.
Aim: The aim of this study was to investigate the role of ADSC-EVs on human dermal fibroblast wound healing activity in vitro.
Methods: Adipose tissue samples were collected from three patients undergoing an AFG procedure at Wellington Regional Hospital and digested using collagenase. EVs were isolated from ADSCs using size exclusion chromatography (SEC) on a qEV10/35nm column with an automated fraction collector. ADSC-EVs were added to fibroblast cells (CCD-1128Sk) for 48 hrs. Following incubation fibroblast proliferation was measured using Cell counting kit-8, wound migration using a scratch-wound assay and mRNA expression levels using RT-qPCR at 0, 24 and 48 hr time points in ADSC-EVs co-cultures. Dummy EVs which were collected from ADSC media with no cells were used as a negative control against ASDC-EVs. Statistical Analysis of results were compared using a paired t-test and deemed significant if (p <0.05).
Results: ADSC-EVs show trends towards promoting a pro-inflammatory environment with IL-6 trending towards an increased expression in ADSC-EV co-cultures (6.556 +/- 6.512; p= 0.2775) and a decrease in ACTA2 expression (0.5328 +/- 0.3200, p= 0.1272). Based on standard deviation assessment, ADSC-EV co-cultures exhibited numerically larger levels of variation in gene expression compared to DEVs. Cell proliferation was not significantly different between ADSC-EV (0.8319 +/- 0.340) and DEV (0.8067 +/- 0.2757) co-cultures (p= 0.6290). EVs had no significant effect on cell migration compared to DEVs at 24 and 48 hr time points (p= 0.8976, 0.4931 respectively).
Conclusion: ADSC-EVs show a trend towards promoting a pro-inflammatory environment in human dermal fibroblast cells, potentially influencing fibroblast function. ADSC-EVs also showed large levels of variation in gene expression compared to DEVs. This suggests that primary ADSC-EVs from patients have diverse effects and further investigation is warranted.