The incredible growth potential of Exosomes as minimally invasive liquid biopsies.

All cells release extracellular vesicles (EVs) as part of their normal physiology. We could divide the types of EVs into two wide groups: ectosomes and exosomes. On one hand, ectosomes are EVs are vesicles whose diameter range is between 50nm and 1mm, this category includes and microvesicles, microparticles, and large vesicles. On the other hand, exosomes are EVs whose size is between approximately 40 to 150 nm. The origin of these vesicles also differs, while ectosomes are pinch off the surface of the plasma membrane, exosomes have an endosomal origin.

Exosomes can contain many components of its original cell, including  RNA, lipids, metabolites and cytosolic and cell-surface proteins, which associates them with the immune response, viral pathogenicity, pregnancy, cardiovascular diseases, central nervous system-related diseases and, principally, cancer diagnosis and progression.

▌ Why are exosomes interesting?

Exosomes are thought to provide a means of intercellular communication and of transmission of macromolecules between cells. In the past decades, exosomes have been attributed roles in the spread of elements from the original cell and as contributing factors in the development of several diseases.

As a means of intercellular communication, exosomes may also act as vehicles for spread, as well as miRNAs in the case of cancer, exosomes have been known to contain numerous disease-associated cargos.

Exosomes have been identified in a wide number of human body fluid which include urine, blood, saliva, cerebrospinal fluid, synovial fluid, semen, breast milk, amniotic fluid, aqueous humour (from cadavers) lymph, and bronchoalveolar lavage fluid.  That is one of the reasons why the functions of EVs have been deeply studied over the past years.

On the other hand, exosomes have been proposed to be useful vectors for drugs, given that they are formed of cell membranes instead of synthetic polymers, and therefore they are better tolerated by the host.

▌What is a liquid biopsy?

A liquid biopsy is a blood test widely used to identify cancer cells released from tumours that are circulating in the blood, as well as for fragments of DNA also derived from tumour cells in the blood.

Until recently, in order to diagnose a cancer and to follow its progress, it was necessary to perform a surgery or a puncture in order to extract the sample of tumour tissue to be analysed; such extraction carries some risks and possible complications for the patient, and liquid biopsy makes possible to obtain the same information with a simple blood extraction.

In this sense, a liquid biopsy may be used to help diagnose cancer in early stage, and studies of disseminated tumour cells in bone marrow and circulating tumour cells in peripheral blood have demonstrated to provide important information about cancer biology and progression.

Being able to extract multiple samples of blood easily over time, could definitely help specialist to understand the molecular progression in a tumour, which means that liquid biopsy could be used to help plan treatment or to find out how well the treatment is working.

In this regard, as exosomes are secreted by all cells, they are especially attractive as minimally invasive liquid biopsies with the potential for longitudinal sampling to follow disease progression.

Biogenesis of exosomes allows capturing complex extracellular and intracellular molecular cargo for comprehensive, multi-parametric diagnostic testing.

Recently, advances in the detection and characterization of circulating tumour DNA have finally enabled the introduction of liquid biopsy assays into clinical practice, and multigene assays have been approved in order to detect genetic alterations in plasma cell-free DNA for use as companion diagnostics matched to specific molecularly targeted therapies for cancer.

▌What are the main advantages of liquid biopsies?

► First of all, a liquid biopsy is a non-invasive cancer diagnostic technique which allows to analyse the patient progression taking one or more than one blood sample, enabling to diagnose cancer at early stages. The use of this technique, improve the process for the patient, because it evite unnecessary risks and discomforts for them at the same time that it saves time and resources for the medical professionals.

► The results are obtained from a simple blood analysis, and it can be done as many times as the specialist need, and this makes the monitoring of the evolution of the patient much easier.

► Real-time analysis which helps the early detection of mutations in oncogenes and tumour suppressor genes, as well as the identification of secondary mutations that may lead to resistance.

► On the other hand, there are situations when tissue biopsies are not possible or repeat the biopsy is not really practical, in this sense, liquid biopsies could be more suitable in order to more frequently monitor and evaluate tumours.

Considering the exponential evolution of liquid biopsy in recent decades, traditional solid biopsy shows considerably more limitations and introducing liquid biopsy to clinical practice can be the key to reduce invasive operations and promote more precise medical intervention.

▌ Exosomes for minimally invasive liquid biopsies

Exosomes play a critical role in various physiological and pathological processes, including cancer, pregnancy disorders, cardiovascular diseases, and immune responses. Additionally, the reason why these EVs have so much potential as minimum invasive liquid biopsies is that they can contain a large amount of RNA, both iRNA and mRNA and can be easy analysed.

Furthermore, exosomes become an attractive target for tumour sequencing because DNA and RNA exosomes are not as dispersed as free tumour DNA in sera. In addition, tumour-derived exosomes can also be significantly enriched in various biological fluids which makes exosomal DNA and RNA a reliable sequencing material for tumour liquid biopsy.

Exosomes show a significant advantage over other sources of liquid biopsy, not only because they are present in almost all fluids, and its identification is clear and simple, but also because, they hold high stability encapsulated by lipid bilayers.

This stability helps to reduce the cost of the sample storage and the difficulty of transportation, which reinforce the clinical application of exosomes.

Additionally, exosomes are secreted by living cells and therefore, they contain biological information which make them more representative that de cell-free DNA, which is secreted during necrosis or apoptosis.

Exosomes can represent specific surface proteins from parental cells which can be use even to predict organ-specific metastasis. Over the last years, it has been showed that exosomes express specific proteins such as CD63, ALIX, TS101, and HSP70,20 which are used as markers to effectively distinguish exosomes from other vesicles.

► The most promising uses of exosomes: Remote sensors

Regarding the importance of exosomes in liquid biopsy diagnosis, the most promising use of EVs is as a remote sensor of organs which are inaccessible in routine monitoring, for example, in patients with breast cancer. Recent studies have demonstrated that EVs isolated form the serum contain higher level of glypican 1 1 (GPC1); in patients with early pancreatic cancer; thus, EVs released by the placenta during pregnancies and identified in maternal blood will enable clinicians to routinely monitor the health of placentation and pregnancy throughout gestation without invasive tests.

Later studies have shown that these exosomes can carry the MHC-peptide complexes that are recognized by T-lymphocytes and that secretion of exosomes could promote anti-tumour immune responses.

And it has also been studied that EVs may spread misfolded proteins associated with neurodegenerative diseases, which can be detected in blood, and makes EVs potentially useful in the diagnosis and evaluations of similar neurodegenerative diseases.

▌Exosome-based liquid biopsies for the future

Conventional solid biopsy has gradually shown considerable limitations, whereas liquid biopsies promise to be a great tool for non-invasive diagnosis and prognosis. As we disclosed above, exosomes play a vital rol in various physiological and pathological process which proves them as a potential tool for clinical application.

There are many widely recognize extraction methods to isolate exosomes such ultracentrifugation, and much more under development in order to help the introduction of these for clinical application, including high-performance isolation columns and promising highly sensitive detection kits. However, fundamental questions remain about exosome generation and function, and for this reason is important to keep building research and publications on exosome in order to provide careful and explicit criteria for distinguishing them from other extracellular vesicles to avoid confusing the field and encouraging scepticism.

The rapid and accurate diagnosis of patients with minimally invasive procedures looked difficult in the past, however the discovery of extracellular vesicles and their near ubiquity in body fluids, promises to make EVs and more specifically, exosomes-based liquid biopsies a reality for the future of clinical biopsies.

For this reason, at Immunostep, we work in order to help making the dream come true and we offer our technology and knowledge on exosomes to achieve this objective. Contact us to start a conversarion about your exosome Research!

▌ References

1. Edgar, James R. “Q&A: What Are Exosomes, exactly?” BMC Biology, BioMed Central, 13 June 2016, https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-0268-z.
2. Raghu Kalluri, Valerie S. LeBleu “The biology, function, and biomedical applications of exosomes” Science ,07 Feb 2020, Vol. 367, Issue 6478, eaau6977, DOI: 10.1126/science.aau6977.
3. Ignatiadis, M., Sledge, G.W. & Jeffrey, S.S. Liquid biopsy enters the clinic — implementation issues and future challenges. Nat Rev Clin Oncol 18, 297–312 (2021). https://doi.org/10.1038/s41571-020-00457-x
4. Mining Extracelullar Vesicles for clinically relevant non-invasive diagnostic biomarkers in Cancer.
5. Armstrong, D., & Wildman, D. E. (2018). Extracellular Vesicles and the Promise of Continuous Liquid Biopsies. Journal of pathology and translational medicine, 52(1), 1–8. https://doi.org/10.4132/jptm.2017.05.21
6. Zhou, B., Xu, K., Zheng, X. et al. Application of exosomes as liquid biopsy in clinical diagnosis. Sig Transduct Target Ther 5, 144 (2020). https://doi.org/10.1038/s41392-020-00258-9