Saurav, MD Faruk Abdullah (2018) Identification of exosomal proteins in primary human bronchial tracheal epithelial cell HBTE and the H358, THP1 and MCF7 cell lines. PhD thesis, University of Greenwich.

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Abstract

Background: Early detection of cancer is of paramount importance for successful treatment. Unfortunately, this is currently complex and time consuming. New sources of biomarkers are needed to improve. Exosomes are nano sized extracellular vesicles released by almost all cells have gained much interest as a cancer biomarker source due to their ability to transfer genetic materials, stability and ease of availability.

Aims: The aim of this study is to investigate the potentials of exosomes as a source of biomarkers of cancer in general and lung cancer in particular. To achieve this, exosomes from three difference cancer cell lines (lung cancer H358, leukaemia THP1 and breast cancer MCF7) and a primary lung cell HBTE were isolated and their protein contents were analysed to establish whether cancer specific proteins are present.

Methods and Materials: Exosomes were isolated and characterized by scanning (SEM) and transmission electron microscopy (TEM), dynamic light scattering (DLS) and Western Blot analysis. The exosome number and protein profile were analysed at different cellular growth stages using Exo-Elisa based on exosomal marker CD63 and mass spectrometry (MS) respectively. LC-MS based proteomic approach has been used to compare the proteomic profile of exosomes from three cancer cells. Finally, comparative proteomic study and gene expression analysis were carried out between exosomes from lung cancer cell (H358) and its counterpart normal cell (HBTE)

Results and Discussions: Successful isolation of exosomes from H358, THP1 and MCF7 was confirmed based on their size distribution (ranging from 96.54±28.53nm to 128.06±17.74nm) and by the presence of exosomal markers (CD63, CD81, CD9 and Hsp70). The number of exosomes released/cell was shown to increase with time ranging from 14500 on day 1 to 18000 at day 15. Interestingly, MS analysis revealed that, alpha-2-macroglobulin (A2M) and pregnancy zone protein was present only in stationary phase indicative of the oxidative stress.
Comparative proteomic study by LC-MS identified a total of 613 proteins commonly found across three cell lines. A large proportion of membranous proteins were identified including integrins, catenins, cadherins, and cathepsins. Most of which are involved in molecular signalling, cellular growth and transport, supporting the role of exosomes in cellular communication. Several adhesion molecules such as integrins, laminlins, catenins as well as cadherins and cathepsins were also identified as differentially expressed between different types of cancer derived exosomes.
Proteomic profiling of HBTE cell derived exosomes revealed 1011 proteins which only partially overlapped with those identified in H358 exosomes. A total of 205 proteins were specific for the cancerous lung cell line derived exosomes. Of particular interest was the identification of CTNNB1, an adhesion molecule known to be present in several other lung cancers, making it an ideal candidate for biomarker for non-small cell lung cancer, bronchioalveloar carcinoma. Gene expression analysis revealed that this protein is expressed at cellular level in both cancerous and normal lung cells, albeit found in a significantly higher level in H358 (p≤0.05).

Conclusion: This is the first report to show successful isolation and characterization of exosomes from H358 and HBTE. Comparative proteomic analysis of the newly isolated exosomes not only revealed that exosomes are a good source of lung cancer biomarkers but identified a potential candidate. CTNNB1 was selectively found in lung cancer cell derived exosomes but not in the counterpart normal cell (HBTE) and was selected for further investigation.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Biomarkers of cancer; exosomes; cancer detection; lung cancer;
Subjects:	R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	08 Apr 2019 12:11
URI:	http://gala.gre.ac.uk/id/eprint/23486

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