Extracellular vesicles: platelet dust or diagnostic goldmine?

What are extracellular vesicles?

Extracellular vesicles first gained attention when they were initially identified as ‘platelet dust’ in the 1960s. While the terminology has gone through several iterations since then, the majority of extracellular vesicles are now simply referred to as ‘EVs’.

EVs are essentially a small sac filled with liquid that is secreted from a cell, which contain precious material and information. There are three predominant types of extracellular vesicles found in the body:

1. Exosomes: where the membrane of a cell folds inward and releases a cargo of cellular material
2. Microvesicles: where the membrane of a cell forms a ‘bud’ containing cellular material that eventually detaches from it
3. Apoptotic bodies: where there are sealed sacs containing information from a dying cell

All human cells produce extracellular vesicles, especially tumour cells which have been observed to increase their production of EVs. In 2008, a research group showed that EVs isolated from a brain tumour caused an aggressive increase in development when delivered to other cancer cells. As a result, EVs have gained significant attention for their diagnostic potential as seen in the figure below:

Diagnostic applications of extracellular vesicles

The diagnostic applications of extracellular vesicles in liquid biopsies have generated a lot of excitement in recent years, due to the potential for achieving diagnosis before known symptoms are displayed.

Liquid biopsy is a diagnostic procedure which uses bodily fluids to detect cancers and help healthcare professionals understand more about the disease. The minimally invasive approach usually involves a test done on a blood sample. Other cancer diagnostics, such as tissue biopsies, are much more invasive and can result in damage to the surrounding tissue, potentially leading to an infection.

Tumour growth is notoriously heterogenous, meaning that all the cells within one tumour can be very different. The challenge with this in cancer diagnostics is that if you perform a tissue biopsy, the portion of the cancerous tissue that is sampled might not be representative of the whole tumour.

In comparison, liquid biopsies using bodily fluids can provide a more representative view of the full tumour, since the bodily fluid contains cancer biomarkers (in this case EVs produced by someone with cancer). This can enable a more accurate diagnosis of diseased tissues, something which is not attainable through simple biopsy techniques such as the diagnosis of certain types of lung cancers (for example non-small cell lung cancer).

The increased ease of sampling of liquid biopsies also means diseases can be diagnosed much earlier compared to other methods. This is because the disease’s presence can be detected before patients start expressing evident symptoms. Such early detection offers a significant advantage in healthcare and can lead to a greater probability of successful treatment.

Another key benefit of liquid biopsies is that very small volumes of bodily fluids are required for accurate diagnosis, which helps to reduce the burden on patients and clinicians.

Mass scale testing has already proven successful, with companies such as ExosomeDx delivering test kits for extracellular vesicle biomarkers for prostate cancer. BioFluidica has also created an automated isolation and analysis platform for EV biomarkers for nine different cancers, which isolates EVs from other material in the sample. The NHS is also working alongside The Circulating Tumour Biomarker Network to provide liquid biopsy tests for NHS cancer care, effectively translating research into the clinic.

How does this compare to the non-EV liquid biopsy landscape?

Liquid biopsies are of course not a recent development. There are currently multiple non-extracellular vesicle biomarkers commonly isolated from liquid biopsies for diagnosing diseases, for example, circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs). It’s worth noting that each of these have several associated drawbacks.

ctDNA:

• Is unstable and may be damaged during processing of samples before analysis, for example when centrifugated at the high forces required to separate small particles from a fluid
• Is least prevalent in the early stages of cancer, reducing the chance of early diagnosis and successful treatment
• Is released by dead tumour cells, so it isn’t representative of a live cell
• Is masked by ‘noise’ of other non-tumour originating cell-free DNA

CTCs:

• Are only present during metastasis (when the cancer spreads to other parts of the body), so cannot be used for early localised cancer diagnosis
• Are not abundant, with early metastatic cancers producing less than 3 CTCs per ml in the blood, which can lead to variability in analysis such as false negatives

The benefits of extracellular vesicles compared to alternative biomarkers

As established above, extracellular vesicles contain precious material from their parent cell. This cargo of material, when released outside the cell, can trigger a response by other cells around it. Because EVs communicate in this way, it makes them both a key contributor to the spread of cancer and a key tool for its diagnosis.

Extracellular vesicles release a cargo of material including:

• DNA
• RNA
• Proteins specific to a type of tissue
• Proteins found in the membrane of cells (transmembrane proteins) that are cell-specific

Because EVs contain these materials and can be found systematically in circulation, when they are detected in bodily fluids they can be used to diagnose diseases of distant tissues. For example, a brain tumour could be diagnosed using a blood sample from the arm. This means that accurate diagnosis can be reached for different types of cancers using body fluid samples containing EVs with known biomarkers.

Compared to examples of non-extracellular vesicle biomarkers, EVs can also provide a real-time view of the cancer’s status, as opposed to ctDNA, which is released by dead tumour cells (as explained above).

As EVs are continually released by all living cells, these biomarkers can therefore be found in bodily fluids before the tumour becomes metastatic (unlike CTCs) and starts spreading to other parts of the body. EVs are also very stable and can be used effectively for diagnosis after significant isolation and analytical processing.

Limitations of extracellular vesicles in liquid biopsies

Although tissue-specific, extracellular vesicles can traverse the whole body through systemic transportation networks, meaning they will be more locally concentrated around tissue. For example, EVs acting as biomarkers for neurological diseases are more likely to be concentrated in the cerebrospinal fluid than in urine, and vice versa for prostate-related diseases. However, sampling from the spine can be challenging and increase patient risk. Therefore, to reduce this risk and improve the clinical experience for patients, samples may be taken from blood for the convenience of sampling, though at the cost of a higher yield.

Like other cancer biomarkers, cancerous extracellular vesicles can be detected before metastasis but are least prevalent in the early stages of cancer, when treatment can be most effective. While this is a limitation, it’s worth noting that a screening can still display a positive diagnosis without there being notable cancer symptoms, making this a valuable tool in detecting cancer early on. Specific screening systems are commonly designed for a single type of cancer, which limits their utility since it becomes more costly to perform analysis to detect multiple types of cancer.

Due to the size and diversity of EVs, effective isolation for analysis has been a key focus for the field’s development. Efficient, scalable and cost-effective isolation is yet to be achieved. While extracellular vesicle isolation and pre-analytical processing are not standardised, the variability of these methods will remain an issue when attempting to make the method for EV liquid biopsies reproducible. If there is variability in performance, it can hinder a method’s validation and, in turn, can delay its implementation into the clinic.

How far away are we from using extracellular vesicles in the clinic?

Academia translation to clinic

It is widely accepted that an organised effort is frequently needed to bridge the gap between academia and industry. Initiatives which pair academia and industry, such as the Liquid Biopsy Consortium, may improve the successful translation of extracellular vesicles for clinical diagnosis.

Employing frameworks can improve the quality of research and standardise methodologies. For example, the Biomarker Toolkit, created by Savva KV et al., can be used to display research gaps around EV liquid biopsy, to improve the success rate of clinical translation.

Analytical technologies

The early stages of cancer development are challenging to analyse, leading to many diagnostics reporting poor sensitivity [true positives / (true positives + false negatives)] and specificity [true negatives / (true negatives + false positives)]. The figure below shows how various diagnostic companies perform when detecting early-stage cancer through liquid biopsy, based on data from Connal, S., Cameron, J.M., Sala, A. et al.

This performance worsens as companies aim to detect multi-cancer biomarkers, which leads to the question: how can an effective screening strategy be reached?

Advancements in machine learning will support the accurate identification of biomarkers for multiple cancers, when used alongside spectroscopic detection technologies such as attenuated total reflectance (ATR) and Fourier transform infrared (FTIR). Multi-cancer screening technologies will improve the likelihood of detecting prevalent cancers with great cost-effectiveness, which would help in improving the translation of this approach to low- and mid-income nations.

The future of extracellular vesicle diagnostics

Although extracellular vesicles are not a new discovery, our understanding of these nanoparticles is still maturing, alongside the methods we can employ to efficiently isolate and analyse them to better characterise their therapeutic potential.

The analysis of EVs within liquid biopsies can achieve accurate diagnosis of cancers, even at the early stages of cancerous development (before notable symptoms), thus improving the likelihood of successful treatment. With clinical translation strategies, validated methodologies and investment in multi-cancer screening technologies, the use of EV liquid biopsies will increase.

Extracellular vesicles are far more than the platelet dust we first attributed them. Future developments in isolation and analysis will transform these nanoparticles into a goldmine in the field of diagnostics.