Hepatocellular carcinoma (HCC) exosomes are found to be responsible for cancer progression, metastasis, and angiogenesis via cellular communications. However, studying cancer derived exosomes in vitro is limited, due to the way cell lines are grown in medium supplemented with foetal bovine serum (FBS) that contains naturally occurring exosomes. Bovine-derived exosomes can cause artefacts and interfere with interpretation of results. The aim of this study was to investigate the release of exosomes from human liver cancer cell line HepG2 (HepG2-Exo) under different conditioned media with modified FBS to deliver the best approach for exosome production. Thus, two media were developed for growing HepG2 cell line which are M1 and M2 using dulbecco's modified eagle medium (DMEM), where M1 supplemented with 10 % (v/v) FBS, and M2 with 10 % (v/v) exosome depleted FBS (Dep-FBS). However, after cells reached confluency, those two media were removed and replaced with serum free media (only DMEM), to create M3 and M4, respectively. This resulted in collecting four categories of media: M1, M2, M3, and M4. Consequently, four groups of exosomes were obtained (Exo(M1), Exo(M2), Exo(M3), and Exo(M4).In regard to cell culture, findings confirmed that M2 was the best approach for cultivating HepG2 and collecting exosomes, as cell viability was enhanced and contamination with FBS-exosomes was minimal, compared to M1. However, analysis of the different HepG2-Exo groups showed significant difference in protein concentration, percentage of fluorescence, exosome marker detection, tetraspanins expression, particle count, metabolic and lipidomic profiling, RNA sequencing, and gene expression. This difference indicates that the effect of media composition is inevitable on cell-derived exosome which may cause misinterpretation of the effect of the exosomes of interest. Consequently, biological assessment and metabolomic profiling of HepG2-Exo effect on different cancer and normal cell lines was carried out: A375 (melanoma), A549 (lung cancer), and PNT2A (normal prostate epithelium). The biological assays revealed that HepG2-Exo induced the proliferation, migration, adhesion, and invasion of A549 at 50 µg/ml. While metabolome analysis showed that HepG2-Exo at 100 µg/ml, induced significant changes in the cell metabolome of A375.The outcomes of this project provided an effective approach in developing successful cell culture for exosome collection without concern over contamination from FBS-derived exosomes and brought attention to the critical effect of media in exosome studies. Moreover, this project has highlighted the effect of HepG2-Exo on other cell lines and the potential of HepG2-Exo to be applied to the development of future lung cancer therapeutics.
|Date of Award||18 Dec 2020|
- University Of Strathclyde
|Supervisor||Valerie Ferro (Supervisor) & Rothwelle Tate (Supervisor)|