MCF-7 breast cancer cell line derived extracellular vesicles and their effects on the production of the inflammatory cytokine interleukin-8 by immortalized Monocytic THP-1 cells
Cardiff Metropolitan University
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Background: Breast cancer is the most common cancer in the United Kingdom with around 54,800 incidents diagnosed in 2014 alone. While Breast Cancer is well understood, treatment proves challenging due to the lack of knowledge surrounding the tumour microenvironment (TME) and the metastasis process. Although, we do know that cytokines and inflammatory mediators are heavily involved, one of which, IL8, has been linked to increased angiogenesis and invasiveness of breast cancer tumours. It has been suggested that the process of IL8 secretion within the TME could be controlled and influenced by extracellular vesicles (EV’s) secreted from tumour cells and therefore this study aimed to investigate the link between THP-1 monocytes and MCF-7 derived EV’s at various time points and in different stages of immune activity, achieved via use of lipopolysaccharide (LPS). Methods: Extracellular vesicles were isolated from MCF-7 (immortalised breast cancer cell line) via ultracentrifugation at 200,000 x g in an ultracentrifuge, the supernatant removed, re-suspended and stored at -80oC. Protein concentration of the isolates was estimated using Bradford protein assay and Nanodrop™. Characterization of the EV’s was performed via Nanosight™ nanoparticle tracking (NTA). The MCF-7 samples were diluted and analysed via three sandwich ELISA’s, the first looked at EV’s at their physiological levels while the second and third ELISA utilised slight protocol adjustments such as dilutions and reduction in number of time points. Results: The BPA and Nanodrop gave data that was not significantly different, suggesting they are both competent for protein determination of extracellular vesicles. The ELISA data showed that IL8 concentration (pg/ml) from the combined immortal THP-1 cells and MCF-7 EV’s when stimulated with LPS was lower than when THP-1 cells independently were stimulated with LPS (48 hour time point with EV’s at a concentration of 40μg/ml) and that this was a statistically significant result (P < 0.05 under t-tests). Conclusions: These data indicates that MCF-7 extracellular vesicles can regulate the IL8 concentration of the environment by an unknown mechanism, consequently influencing the IL8 secretion from immortal THP-1 cells and therefore a much more complex function in tumour development than originally considered. Suggesting that EV’s can potentially influence the IL8 concentration and therefore the ability to potentially transform cellular functions. If this mechanism(s) can be identified it could potentially lead to more advanced diagnosis, staging and treatment of breast cancers.
BSc in Biomedical Science
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