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Flow cytometry, another valuable quantitative analysis of exosome cargo, is used for exosome capture
on streptavidin-coated magnetic beads. It was also demonstrated for the collection of CD63+ exosomes
utilizing biotin-labelled anti-CD63 Ab, starting with fraction number four of exosomes separated by mini-
[40]
SEC . This method demonstrates that the levels of PD-1 and PD-L1 in exosomes obtained from HNSCC
[40]
patients’ plasma is significantly associated with pathological changes such as disease progression .
PD-L1 is a ligand with the potential of inducing immune suppression in activated T cells that express
PD-1. When PD-L1-bearing exosomes were obtained with CD3 and CD69 T lymphocytes, the down-
regulation of CD69 expression was blocked. Anti-PD1 antibodies inactivate this inhibition. Likewise,
when plasma-derived exosomes are obtained with activated CD8 T-cells, fast programmed cell death of T
cells takes place. This reveals that TEX modulate immune cell activity and are able to function as immune
biomarkers [30,40,41] .

Clinical analyses need to be simple, reliable and quick. To meet these criteria, we are currently working on
a new isolation technique using immunoaffinity that allows us to have results in flow cytometry within a
few hours. This will enable a high-throughput analysis of patient samples.

One interesting method described could be the photoacoustic and fluorescence flow cytometry platform, as
[28]
described by Nolan et al. . This is able to perform in vivo flow cytometry on tumor-associated exosomes
in mouse models. Also, more research is needed to bring this technique into human diagnostics, where it
seems to be a challenging but promising path.

Mass spectrometry
MS is one of the most favorable techniques for proteome characterization, where analytes are converted
to gaseous ions and are classified recording to their mass-to-charge ratio. It offers various advantages
such as its specificity, throughput, sensitivity and cost-effective testing. It uses three different proteomic
MS techniques: digestion by trypsin (bottom-up), digestion by a protein with less-frequent cleavage sites
(middle-down), and analysis of native protein (top-down). MS has also been linked with ChIP to quantify
histone modifications, transcription factors and other chromatin-associated proteins.

As already mentioned above, exosomes contain a variety of functional proteins and genetic cargo such
[42]
as DNA, mRNAs, miRNAs and long non-coding RNAs. Tang et al. state: “Sucrose density gradients,
ultrafiltration, high performance liquid chromatography-based protocols and immunoaffinity-capture
methods, singly or combined with the application of ultracentrifugation, can provide high enrichment and
purity of exosomes”. Besides, a number of alternative methods for analysis of exosome RNA extraction
methods have been used: phenol-based techniques (TRIzol) and combined phenol and pure column-based
techniques. The Total Exosome RNA and Protein Isolation kit (TER) has been particularly selected for
RNA isolation .
[42]
Global proteomics is an identification method using as many proteins as possible within one sample. There
are two different strategies: via data dependent acquisition (DDA) or data independent acquisition (DIA),
[43]
with DDA being used more commonly than DIA .

CONCLUSION
Cancer is one of the biggest challenges of medicine, and we still cannot defeat it as a whole. However, liquid
biopsy, the analysis of exosomes, ctDNA and CTCs may give us the possibility for non-invasive diagnosis,
real-time monitoring of disease progression and developing a treatment according to gene profile as well.
The special features of exosomes make them ideal for minimally invasive liquid biopsy. Exosomes are
unique; they express not only “dead” particles such as ctDNA but they also show actual biological activity