Background

What are circulating tumour cells and extracellular vesicles?

Circulating tumour cells (CTCs) are cancer cells that have been shed from a primary tumour into the bloodstream or lymphatic system. They are carried around the body and can be the seeds that form metastases. Extracellular vesicles (EVs) are tiny lipid-bilayer bound particles that are shed from most cells. EV composition depends on the type of parent cell that they came from and can include proteins, lipids, nucleic acids and metabolites. They were found to be capable of transferring their cargo from cell to cell, and so wide variety of functions, including transfer of functional proteins, DNA and RNA; molecular recycling; cell signalling; creation of metastatic niches for cancer; and the elimination of waste materials, among other. Hence, their biological significance is also wide ranging, with EVs being implicated in the aging process, chronic inflammation, coagulation, and, of course, disease.

Why study CTCs and EVs?

The detection of CTCs is useful as they can be collected by minimally invasive means of liquid biopsy rather than tissue biopsy, and can be useful for assessing risk of metastasis, prognosis, monitoring therapy effectiveness and aiding treatment decision making. Likewise, the nucleic acid content of EVs can act as a biomarker for disease and be used to identify the original tumour cell type. EVs can also affect the behaviour of other cells – it has been seen that EVs released from colorectal cancer cells encourage the formation of ‘tumour landscapes’ by increasing the migration of fibroblasts. Also, because of their ability to transport and transfer their contents, EVs are also of interest for the delivery of therapeutics to diseased tissue. Additio-nally, it has been found that inhibiting EV release can slow down pancreatic cancer progression in experimental models.

Scan of brain showing glioblastoma (Adobe Stock)

How EVs are usually studied and the challenges involved

EVs are often studied at the population level involving ultracentrifugation and analysis by the usual gamut of techniques, including the nanoscale microscopy techniques, flow cytometry, Western blotting and enzyme-linked immunosorbent assays as well as fluorescence image methods. However, each method has its limitations, not least because the size of the particles ranges from 40 to 2000 nm, with many being less than 300–400 nm and so below the diffraction limit of light. Additionally, the small surface area poses a limit to the number of targets available for staining.

Additionally, the heterogeneous nature of EVs poses an  inherent challenge to the analysis of and characterization of subpopulations that might be responsible for different biological functions and so there has been increasing interest in the analysis of single EVs. Immunofluorescence is an effective way of analysing EV cargoes being of limited cost and having possibilities of multiplexing and automation. The challenge, however, is that signals are very weak and exist only briefly.

Tyramide-based immunofluorescence signal amplification

Tyramide signal amplification (TSA) involves using tyramide reporter probes (tyramide labelled with a fluorescent dye or chromogen) to significantly boost the fluorescent signal compared to conventional techniques. Here, the antigen of interest is bound by the primary antibody, which is itself bound by a horseradish peroxidase (HRP)-conjugated secondary antibody. The HRP then catalyses the conversion of the tyramide into an active form, which then forms a covalent bond with tyrosine residues on proteins or nucleic acids at or near the site of the HRP. This causes a dense deposition of reporter molecules at the site of interest. This technique both improves the signal-to-noise ratio and also reduces the level of non-specific staining. In their recent paper, Cavallero et al. have used this approach to investigate CTCs and EVs from glioblastoma – one of the most common and aggressive forms of malignant brain tumour, which has a very poor 5-year survival rate (less than 5%). After validating the technique on glioblastoma CTCs, they optimized the method for multiplexed staining of single EVs derived from the glioblastoma CTCs, demonstrating both amplified signal intensities and more stable and broader signal ranges compared to conventional methods. The protocol is also shown to be applicable to CTCs and EVs from plasma samples of glioblastoma patients and can also be easily adapted to other cancers of interest. Although mainly performed for proof of principle, the results also gave some clinical observations, showing the presence of glioblastoma CTCs in circulation before disruption of the blood–brain barrier by surgery, and that the presence of EVs positive for tetraspanins (which play a significant role in glioblastoma progression) and other glioblastoma-specific markers generally correlated with CTC counts in most patients analysed. The authors finish by expressing interest in improving the techniques for the isolation of EVs and also for developing the methodology for use with flow cytometry to enhance scalability and sensitivity within a single platform. It is amazing that these particles that are so tiny and so sparse are being encouraged to reveal their secrets. With time, we may see them being used to aid diagnosis, prognosis and treatment decisions.

 Bibliography
1. Cavallaro S, Veiga SI, Ahmad R et al. Signal amplification for fluorescent staining of single particles in liquid biopsies: circulating tumour cells and extracellular vesicles. J Extracell Vesicles 2025;14(10):e70167 (https://doi.org/10.1002/jev2.70167).
2. Chetty VK, Ghanam J, Anchan S et al. Efficient small extracellular vesicles (EV) isolation method and evaluation of EV-associated DNA role in cell-cell communication in cancer. Cancers (Basel) 2022;20;14(9):2068 (https://doi.org/10.3390/cancers14092068).
3. Doyle LM, Wang MZ. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells 2019;8(7):727 (https://doi.org/10.3390/cells8070727).