|dc.description.abstract||Breast Cancer is the most commonly diagnosed cancer among women in New Zealand, with an estimated 3000 women diagnosed in New Zealand every year. The most common form of treatment is surgery, and following treatment, the approximate 5-year survival rate is 90%. A key part of the treatment process for breast cancer survivors is breast reconstruction surgery, as it improves the physical, physiological, and social aspects of every-day life. Autologous fat grafting is a relatively new breast reconstruction option that is becoming increasingly attractive, as this has the ability to create a natural cosmetic outcome with minimal surgical risk. The major limitation associated with fat grafting is that the percentage of grafted tissue volume that is retained post-surgery is hugely variable, prompting further breast reconstruction in some cases. Adipose derived stem cells (ADSCs) are a key cell being investigated in the donor tissue due to their high proliferative abilities, coupled with their potential to differentiate into adipocytes, indicating that they are important in the regeneration of adipose tissue. In the breast cavity, macrophages are thought to play an important role in graft retention as they are important in inflammation and wound healing. It is thought that improving the relationship between ADSCs in the donor tissue and stromal cells in the recipient site, such as macrophages, could be important for improving retention rates. One way in which these cells can interact is through the release of extracellular vesicles (EVs) from ADSCs into the surrounding environment.
The overall aim of this study was to establish methodologies for in vitro ADSC and macrophage culture, for characterization of ADSC-derived EV (ADSC-EV) output during culture, and to determine the impact of ADSC-EVs on macrophage polarization. Adipose tissue samples were collected from patients undergoing fat grafting and ADSCs were isolated and cultured. ADSC cell specificity was confirmed using flow cytometry as well as successful differentiation into adipocytes and osteoblasts, indicating the successful establishment of methods to culture ADSCs from autologous fat graft samples.
ADSC-EVs were isolated from the media of cultured ADSCs using size exclusion chromatography and characterised using tunable resistive pulse sensing. We demonstrate a mean size 150 – 190 nm across patient samples, which is within the expected size range of EVs. Mean concentrations also ranged from 2.3x108 and 7.4x108 particles/mL as expected. Western blot analysis on proteins known to be present in EVs further confirmed presence of EVs in the collected samples. Together, these techniques indicated the successful isolation of EVs from cultured ADSCs.
Monocytes were isolated from healthy volunteers, cultured into M0 macrophages and further polarized towards M1 and M2 macrophage phenotypes. Successful polarization into M0, M1-like or M2-like macrophages was confirmed by assessing the morphology of cultures. Flow cytometry using known M1 and M2 markers, and RT-qPCR on pro- (TNFα, IL1-β) and anti-inflammatory (IL-10, TGFβ) markers were also used to assess successful polarization. However, low cell counts made these methods difficult for assessing polarization and further optimization is required.
In a further experiment, ADSC-EVs were added to M0, M1-like and M2-like macrophage cultures at the time of polarization. To analyse the impact of ADSC-EVs on polarization state, RT-qPCR was conducted to assess macrophage expression of pro- (TNFα, IL1-β) and anti-inflammatory (IL-10, TGFβ) markers. The addition of ADSC-EVs to macrophage cultures altered the expression of pro- and anti-inflammatory markers in each of the polarization states, and could thus potentially influence polarization status of macrophages. The results of this study warrant further investigation to determine the role of ADSC-EVs on macrophage polarization. However, the preliminary data here provides an exciting platform for future research into the potential role of ADSC-EVs in fat graft retention via their interaction with cells in the breast cavity.||