Quantification of exosomal survivin in the tumour microenvironment with enzyme-linked immunosorbent assay (ELISA)

Thesis

Exosomes facilitate intercellular communication within the tumour microenvironment (TME), affecting cancer progression and immune interactions. Survivin, an inhibitor of apoptosis (IAP), is highly expressed in cancer and linked to unfavourable clinicopathologic parameters. Survivin has been shown to be released via exosomes by cancer cells, making serum survivin levels insufficient to reflect its true amount. While exosomal survivin can be measured with Western blot and flow cytometry, these methods are unsuitable for high-throughput clinical use. Moreover, practical consensus on exosome isolation remains lacking. This study aimed to develop a method to quantify exosomal survivin in biological samples using ELISA, combining polyethylene glycol (PEG) precipitation to concentrate exosomes and a non-ionic detergent-based lysis of exosomal membranes. We optimised ELISA to detect survivin in cancer biological samples, including cancer cell culture conditioned media and patient sera. Colorectal carcinoma (HCT116) and breast adenocarcinoma (MDA-MB-231) cell lines were used to generate conditioned media, and serum samples were collected from 15 cancer patients (ovarian and prostate) and 5 healthy controls. Two methods of exosome concentration were performed and compared i.e., centrifugal filtering and PEG precipitation. 1% NP-40 was identified as the optimal detergent for exosome lysis. ELISA revealed significantly higher survivin levels in cancer biological samples compared to controls. In serum, survivin detection was significantly improved after PEG precipitation and NP-40 treatment. Results were validated by Western blot. This study demonstrates a practical ELISA-based method for quantifying exosomal survivin by combining PEG precipitation with NP-40 lysis. This method provides a potential clinical applications for exosomal biomarkers, particularly but not limited to survivin, in cancer diagnostics and monitoring.

Authors: Wijaya, W

• Alternative title: Quantification of exosomal survivin in cancer biological samples with ELISA
• DOI: 10.5287/ora-jvazrdpvm
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Subjects: Polymer precipitation; Biochemical markers; Cancer; Inhibitors of apoptosis; Extracellular vesicle; Enzyme-linked immunosorbent assay; Survivin; Exosome; BIRC5