We are all too familiar with the statistic that 1 in 2 of us will get cancer in our lifetime. Of that, just under 50% of cancer diagnoses were at stage 3 or 4 in England in 2018, highlighting the urgency for earlier detection.
The earlier a disease is diagnosed, the safer and greater the choice of treatment options available. At present, tissue biopsies remain the gold standard for diagnosis and characterisation of solid tumours. However, the process of obtaining a tissue sample is invasive, unsuitable for some cancer types, and requires repeat hospital visits to follow disease progression. In addition, these samples are often unrepresentative of the heterogeneity of the disease. Thus, there is a pressing need for a more expansive yet less invasive alternative.
Enter the liquid biopsy, where biomarkers identified from a sample of blood, urine, saliva, or even sweat have the potential to comprehensively characterise a disease. Such tumour analytes found in these samples include:
Zooming-in on exosomes
The most abundant biomarker found in a liquid biopsy sample are exosomes. Exosomes are a type of extracellular vesicle around 100 nm in diameter that derive from any and every cell in the body. During their formation, a diverse array of information from the parent cell is packed into the exosome, making for an excellent representation of the parent cell ex situ. For instance, the DNA contained in exosomes is representative of the entire genome and the mutational profile specific to the tumour cell from which it derives, offering the advantage over analysis of ctDNA, for example, which is fragmented and mostly derived from dying cells1. Exosomes, therefore, give a more current overview of the tumour landscape; something that is extremely important in such a rapidly mutating disease.
On the one hand, exosomes present a unique opportunity for multicomponent analyses of cancer biomarkers by high-throughput screening techniques2. Mutagenic DNA, RNA, altered epigenetics, protein and metabolite levels - all contained in exosomes - can be simultaneously analysed to create a comprehensive overview of the cancer. Such approaches have the potential to overhaul the current challenges faced in the field for improving the sensitivity of liquid biopsy analytics.
Alternatively, small-scale analysis can be utilised in cancers where a marker is already known. Such an approach is taken by MetaGuideX, who’s patent-pending technology for the detection of a specific marker on tumour-derived exosomes is reported to potentially prevent unnecessary lymph node removal and subsequent lymphodema in 71% of breast cancer patients who lack the exosome marker that indicates the potential for metastasis.
Exosome Extraction Methods
The method used to isolate and purify tumour analytes is critical to the quality of data obtained from the biomarker they provide, whether that be DNA, RNA, or protein.
The current gold standard method for isolating exosomes is ultracentrifugation (UCF), but this is a specialist technique with a number of drawbacks, notably the co-purification of particles of similar density to liposomes, such as virus and lipoproteins. Ultracentrifuges are expensive pieces of kit that spin at very high speeds and can be dangerous if not handled correctly. Consequently, there is a need for bench-top methods that are easy to use in routine exosome isolation protocols.
An innovative method for isolating exosomes has been developed by New England Peptide. When screening for peptides that bind heat shock proteins (HSPs), they noticed that one particular peptide, Vn96, caused a visible aggregate during pull-down experiments3. Further analysis revealed that this peptide could also bind HSPs found on the surface of exosomes, causing them to precipitate more readily. New England Peptide subsequently developed the METM kit, which uses this property of Vn96 peptides to enable isolation of exosomes in a bench-top centrifuge. Assays can be performed on a ‘miniprep’ scale, requiring much less starting material, however the purity obtained is comparable to that of UCF.
There are many other commercial kits available, as well as microfluidic devices, that have been designed using filtration or immune-affinity capture methods. A number of these methods also avoid UCF; however, purity of the isolate remains a problem.
To combat this, some studies have combined multiple isolation techniques. McKiernan and colleagues used UCF followed by a filtration step to isolate exosomes from the urine of prostate cancer patients. Their methods enabled superior identification of patients with high grade or benign disease, as compared with standard clinical methods4.
Future Perspectives
While other types of tumour analyte have become routinely used in the clinic (with even the NHS rolling out a cfDNA-based test for signs of cancer), this is yet to be seen for exosomes. However, the science is clear: a number of clinical studies have shown that exosomes are packed with diagnostic potential. These have used labour-intensive methods to isolate exosomes, and small patient cohorts. To accomplish routine use of exosomes as a tumour analyte from liquid biopsies, purification methods must be improved and standardised to enable rapid, cost-effective, and reliable isolation of high purity exosomes from a variety of sample types and diseases.
References
- Zhou, B., Xu, K., Zheng, X., Chen, T., Wang, J., Song, Y., Shao, Y. & Zheng, S. Application of Exosomes as Liquid Biopsy in Clinical Diagnosis. Signal Transduction and Targeted Therapy, 5, 144 (2020).
- Valencia, K. & Montuenga, L. M. Exosomes in Liquid Biopsy: The Nanometric World in the Pursuit of Precision Oncology. Cancers,13(9), 2147 (2021).
- Ghosh, A. et al. Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins. PLoS ONE, 9(10), e110443 (2014).
- McKiernan, J. et al. A prospective adaptive utility trial to validate performance of a novel urine exosome gene expression assay to predict high-grade prostate cancer in patients with prostate-specific antigen 2-10ng/ml at initial biopsy. Urol.74(6), 731–738 (2018).
