Periodic Reporting for period 1 - ExoSensor (A Nano-photoelectric Exosome Biosensor for Point-of-care Diagnosis of Early-stage Cancer)
Berichtszeitraum: 2017-10-11 bis 2019-10-10
Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Exosomes are small extracellular vesicles (nano-sized), has double layer membranes and aqueous core. Cancer cell-derived exosomes are potential non-invasive biomarkers for early cancer diagnosis. Their important roles in cancer studies and limited accesses to large quantity of biological samples necessitate the need for development of sensitive, reliable and rapid cancer detection method, i.e. liquid biopsy. The main objectives of the project are to develop an exosome biosensor (ExoSensor) for rapid and specific detection of exosomal cancer biomarkers towards POCT diagnosis of pancreatic cancer, offering a universal technology for clinical cancer diagnosis through quantitatively monitoring the protein marker of cancer-derived exosomes, in a simple way.
Pancreatic cancer (PC) was used as a model disease in this study. PC cells were culture in normal tissue culture flasks and integra bioreactors for comparison. Exosomes were isolated using in-house developed ultracentrifugation onto sucrose cushion method. The size distribution and particle number/concentration of the isolated exosomes were measured by nanoparticle tracking analysis (NTA). The exosomal surface proteins such as CD63, CD9, CD81, were characterised using bead-based flow cytometry. The morphology of exosomes were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The total protein contents in exosomes were measured by micro-BCA assay. Particle:protein (P:P) ratio was used to assess the purity of the exosome samples from contaminating proteins co-precipitating from conditioned media during isolation. CD63 was used as a model surface exosomal marker for the proof-of-concept study of biosensing assays (Figure 1). Exosomes were conjugated to latex beads first. The CD63 surface marker was recognised by specific reagent, which was labelled with enzyme. Colorimetric detection was achieved via substrate (colourless) to product (coloured) deposition catalysed by enzyme in a special reaction solution. This biosensor was further used to detect HER2 derived exosomes from the serum of breast cancer patients, which proved the feasibility of this technology to be used for direct serum detection. Then a biosensor for a new biomarker A on PC derived exosomes was developed and further used for the diagnosis of PC. Clinical samples from PC patients were detected in a blind test for the validation of the ability of new marker positive (+) exosomes to differentiate high-grade and low-grade PC patients. Results show the isolated exosomes were of high quality for downstream applications. The developed CD63 biosensor was highly sensitive and specific. The biosensor can be used for testing whole serum samples in its current format, as evidenced by biosensor for exosomal HER2 detection. The biosensor for new marker A can be used for rapid screening of PC samples.
A simple, rapid and cost-effective colorimetric biosensor was developed for sensitive and selective detection of cancer-derived exosomes in small volume liquid biopsies. A new marker, A, was validated as a promising exosomal biomarker for PC warranting further testing. Quantitatively pinpointing exosome tumour markers, a clinical field of an unmet need is appealing, yet challenging and requires an attention from health care specialists. The proposed research offers an essentially needed universal platform technology for clinical cancer diagnosis through rapidly and quantitatively monitoring the protein marker of cancer-derived exosomes, in a simple way.
The impact of this work would be remarkable. From basic research to application study, not only will this study contribute to frontier fundamental research of exosomes and cancers, but this robust but simple colorimetric method would also enable feasible application of rapid, reliable, and non-invasive POCT diagnosis of pancreatic cancer at early stages as a routine procedure, thus facilitating possible curative surgical therapy. This simple diagnostic tool will also lead to a new class of rapid and portable testing device, as an alternative to conventional large-sized instruments in clinical laboratories for prognostic applications. The patent application is ongoing in UK at this moment. The outcome of this study would probably end up in a startup company focusing on cancer early diagnosis with innovative technologies. The products, therefore, would be available to GP, end customers in every family, etc. for the rapid screening of cancers, which will largely benefit the society and the economy.
The impact of this work would be remarkable. From basic research to application study, not only will this study contribute to frontier fundamental research of exosomes and cancers, but this robust but simple colorimetric method would also enable feasible application of rapid, reliable, and non-invasive POCT diagnosis of pancreatic cancer at early stages as a routine procedure, thus facilitating possible curative surgical therapy. This simple diagnostic tool will also lead to a new class of rapid and portable testing device, as an alternative to conventional large-sized instruments in clinical laboratories for prognostic applications. The patent application is ongoing in UK at this moment. The outcome of this study would probably end up in a startup company focusing on cancer early diagnosis with innovative technologies. The products, therefore, would be available to GP, end customers in every family, etc. for the rapid screening of cancers, which will largely benefit the society and the economy.