World Cancer Day 2022: Addressing Questions in EV-based Biomarker Development

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On World Cancer Day 2022, we look to progress and challenges in areas critical to the development of extracellular vesicle-based biomarkers for early cancer detection.

Diagnostic information derived from extracellular vesicles (EVs) is expected to have a place in the future of cancer screening, diagnostics, and therapeutic monitoring. Before EV biomarkers can be implemented in the clinic, however, there are a few open questions and challenges which must be addressed.  

Below, we look to recent recommendations for advancing early cancer diagnostics, and share commentary from Ryan Pink, Senior Lecturer in Molecular Biology and Genomics at Oxford Brookes University. Pink is also the lead author of a relevant and recent review article titled ‘Utilising extracellular vesicles for early cancer diagnostics: benefits, challenges and recommendations for the future’.<super-script>1<super-script> But first, an acknowledgement of World Cancer Day 2022:

World Cancer Day 2022: Close the Care Gap

It is hoped that the development of EV-based biomarkers will occur alongside greater health equity, better education about cancer prevention, and continued progress in successful programmes such as the elimination of cervical cancer.<super-script>2<super-script> These goals all align with this year’s World Cancer Day theme of #closethecaregap – a reference to the need to close the gap in cancer care.  

World Cancer Day 2022 (Feb 4th) marks the start of a 3-year campaign, aimed at raising awareness and challenging the inequities of cancer care around the globe. There are many examples highlighted at www.worldcancerday.org, for instance:

  • More than 90% of cervical cancer mortality occurs in low- and middle-income countries<super-script>3<super-script>
  • In New Zealand, Māori are twice as likely to die from cancer as non-Māori<super-script>4<super-script>
  • There are notable differences in cancer-related outcomes for rural and nonrural patients<super-script>5<super-script>
  • Cancer screening among transgender people is lower than in the rest of the population, due in part to discrimination from healthcare practitioners<super-script>6<super-script>

On World Cancer Day, we are encouraged to raise awareness of the cancer care gap, in the hope that governments will be pressured to address the root causes of these inequities – by altering systems that bring everyone up to the same level. The World Cancer Day website is well worth a browse, full of actionable ideas (donate, advocate, inform yourself, learn how to talk about cancer), useful links and resources, and examples of positive change (such as the launch of Oncopadi, a digital tool helping patients in Nigeria to navigate and access earlier cancer diagnosis and treatment).

Progress and challenges in EV-based biomarker development

1. Reference materials required for the quantitative analysis of EV cargo

The reliable detection of cancer-related EV cargo is challenging, given the large number of competing signals, complexity of biofluids, and the technical variation introduced during sample preparation and analysis. In their recent review article, Pink et al. suggest the EV field could learn from other disciplines that have also had to deal with the challenge of extraction, enrichment and signal amplification to gain reliable signals – such as single-cell sequencing.<super-script>1<super-script> Importantly, the development of reference materials for the quantitative analysis of EV cargo is expected to help drive the tuning of biomarkers for clinical settings. Pink elaborates on why the EV field should look to other disciplines for ideas and advances:

“It’s definitely worth keeping an eye on other molecular fields that are receiving huge investments for highly focused technology development – where they are pushing the envelope on greater resolution, and analysing very small sample volumes.

Techniques like single-cell sequencing have exploded in the last few years, and that is partly down to improved assay reliability, improved microfluidics, more specific probes, economies of scale bringing down costs, and the development of informatics to support it.

We can look to the success of single-cell mapping projects like the Human Cell Atlas, which was built to provide an international consortium for the spatial, single-cell multi-omics analysis of every cell in the body. The method itself – spatially resolved transcriptomics – was voted 2020 Method of the Year by Nature Methods.<super-script>7<super-script>

Although lessons do not translate directly, the same technical issues are relevant – like overcoming the challenges of reducing sequencing random read errors at these low concentrations.

Data from such studies also allow the development of tissue-specific biomarkers and tissue-specific normalisation control genes, which are really needed in EV cancer diagnostics to track the tissue of origin in the blood.

The technical drive for multi-omics developments also has knock-on benefits, as seen for example in the Human Protein Atlas project.<super-script>8<super-script> Just in January 2022 we saw the announcement that nucleotide sequencing giant Illumina is forming a partnership with SomaLogic, a proteomic characterisation platform to develop their ‘Plexity Workflow’ – an aptamer-based technology that has already shown success in EVs.”  

2. Standards for EV characterisation: EVs from transfected cells show potential  

The development of reference material and data normalisation approaches is a challenge for many aspects of the EV field and an active area of research. While the use of standardised, synthetic particles is suited to tunable resistive pulse sensing, reference materials with relevant biological characteristics are needed in other settings, such as flow cytometry-based biochemical characterisation. To this end, trackable ‘recombinant EVs’ (rEVs, i.e., EVs from transfected cells) have been proposed as a biological reference material for various applications. Geeurickx et al. (2019) developed rEVs by transiently transfecting cultured HEK293T cells (a known human cell line capable of producing large quantities of recombinant proteins) with DNA encoding gag-EGFP (retroviral gag polyprotein fused to enhanced green fluorescent protein).<super-script>9<super-script>

From these transfected, gag-EGFP-expressing cells, the group obtained fluorescent rEVs with relevant characteristics which appeared to be distinguishable from the sample EVs.  

Ultimately, global access to reliable biological reference materials would be of huge value to the development of EV-based biomarkers for early cancer diagnostics.  

3. Robust studies of different disease stages, including pre-symptomatic clinical samples

To develop diagnostics tests that enable early, pre-symptomatic cancer detection, there is a need for EV profiling of samples from healthy patients who go on to develop cancer. Pink highlights valuable, existing resources that could be utilised for the mapping of cancer progression, such as the UK Biobank. Access to samples from those with high risks of cancer development would also be valuable, therefore clear and robust studies of groups with familiar history or advancing age are also encouraged.  

4. An ongoing need to minimise pre-analytical and EV separation variability

As is the case for all experiments, with EV studies it is good practice to minimise pre-analytical variability, and to comprehensively report as many pre-analytical parameters as are known.<super-script>10<super-script> The need for a deep understanding has been well-documented, with many variables listed across sample collection and storage, and EV separation and EV storage.  

Read more: To Cut Through the Noise in Extracellular Vesicle Analysis, Reproducible Isolation is Essential

5. Extracellular ‘moonshot’ project a possibility

Pink and colleagues are in favour of launching an EV ‘moonshot’ project, i.e., a wide-scale and ambitious project with a cross-disciplinary focus. Pink elaborates:

“Much of the focus is EVs has come from molecular and cell biologists, but I think a wider approach of bringing in biochemists, clinicians and engineers could really accelerate things. Already there are large, population-level multi-omic mapping projects going on, like the UK Biobank. Bringing some of the tools into that space to create the EV omics to map on the phenotype data would push everything forward. Although, I think one of the challenges here is probably around the politics and showing these large projects that the science of EVs is mature enough to justify finances and access to limited and precious samples.”

What will it take to make an EV biomarker successful? Read more: Taking Extracellular Vesicles From Discovery to the Clinic: Requirements, Challenges and General Considerations

References

  1. Pink RC, Beaman E-M, Samuel P, Brooks SA, Carter DRF. Utilising extracellular vesicles for early cancer diagnostics: benefits, challenges and recommendations for the future. British Journal of Cancer. Published online January 10, 2022:1-8. doi:10.1038/s41416-021-01668-4
  2. The Lancet Oncology. Global elimination of cervical cancer is achievable—with commitment. The Lancet Oncology. 2019;20(11):1467. doi:10.1016/s1470-2045(19)30647-3
  3. Hull R, Mbele M, Makhafola T, et al. Cervical cancer in low and middleincome countries (Review). Oncology Letters. 2020;20(3):2058-2074. doi:10.3892/ol.2020.11754
  4. Gurney J, Stanley J, McLeod M, Koea J, Jackson C, Sarfati D. Disparities in Cancer-Specific Survival Between Māori and Non-Māori New Zealanders, 2007-2016. JCO Global Oncology. 2020;6. doi:10.1200/GO.20.00028
  5. Levit LA, Byatt L, Lyss AP, et al. Closing the Rural Cancer Care Gap: Three Institutional Approaches. JCO Oncology Practice. 2020;16(7):422-430. doi:10.1200/op.20.00174
  6. Sterling J, Garcia MM. Cancer screening in the transgender population: a review of current guidelines, best practices, and a proposed care model. Translational Andrology and Urology. 2020;9(6):2771-2785. doi:10.21037/tau-20-954
  7. Marx V. Method of the Year: spatially resolved transcriptomics. Nature Methods. 2021;18(1):9-14. doi:10.1038/s41592-020-01033-y
  8. Paper W. THE HUMAN CELL ATLAS.; 2017. https://arxiv.org/ftp/arxiv/papers/1810/1810.05192.pdf
  9. Geeurickx E, Tulkens J, Dhondt B, et al. The generation and use of recombinant extracellular vesicles as biological reference material. Nature Communications. 2019;10(1):3288. doi:10.1038/s41467-019-11182-0
  10. Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 2018;7(1):1535750. doi:10.1080/20013078.2018.1535750

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