Hunting for the ultimate liquid cancer biopsy - let the TEP dance begin
Feller and Lewitzky Cell Communication and Signaling
Hunting for the ultimate liquid cancer biopsy - let the TEP dance begin
Stephan M. Feller 0
Marc Lewitzky 0
0 Institute of Molecular Medicine, Martin-Luther-University Halle-Wittenberg , Halle (Saale) , Germany
Non-protein coding RNAs in different flavors (miRNAs, piRNAs, snoRNAs, lncRNAs, SHOT-RNAs), exosomes, large oncosomes, exoDNA and now tumor-educated platelets (TEPs) have emerged as crucial signal transmitting, transporting and regulating devices of cells in the last two decades. They are also establishing themselves increasingly in the realm of tumor research. We are currently witnessing a mushrooming of candidate entities for diagnostic and prognostic cancer detection and characterization tests that could have a major impact on how this diverse group of diseases is initially spotted and subsequently treated in the near future. But how do the new kids on the block stand up to the more established circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA)? Without question, much earlier disease detection would be expected to save numerous lives. With all these new players around, will we finally win a major battle in the never-ending war against cancer?
Highly effective early cancer detection could save a huge
number of patients from devastating, marginally effective
therapies that are commonly accompanied by morbidity
and/or followed by early death. Discovering tumors very
early on, ideally before metastasis sets in, has therefore been
on the minds of many cancer researchers and health care
providers alike. After all, it is metastasis that kills the vast
majority of cancer patients .
Cancers typically start from a single cell. However,
with our present day routine methods this single cell
usually will have multiplied into a billion or more cancer
cells and often will have also evolved into several distinct
subclones before the tumor is finally detected.
It is commonly through patient observations and not
specific medical tests that initial cancer signs emerge, for
example in the form of a lump or some sort of pain.
This then sets into motion a series of histological and/or
molecular tests to determine the tumor origin and
possibly even the disease subtype.
Apart from the somewhat disputed successes of large
scale routine mammographies , as well as visual skin
inspections and Pap smears, current medical practice
has fairly little to offer in terms of non- or
minimalinvasive early cancer detection procedures.
Manual prostate inspection and PSA determination are
fairly crude tools that seem to have no substantial impact
on prostate cancer survival rates [3, 4]. Endoscopic
inspections of the aerodigestive tract could probably contribute to
a significant boost in survival rates of some cancer types,
but are by and large ignored as routine screening options
for eligible individuals. They come with a low but
non-negligible risk resulting from mechanical damages
(bleeding, perforation) and anaesthetization
complications (for more details see http://www.bsg.org.uk/
Simple, robust analyses of body fluids like blood, saliva
and urine would therefore be a quantum leap forward in
our probably infinite quest to improve cancer survival
rates. These ‘liquid biopsies’ , would have to detect
cancer cells or their various products with great
reliability to provide a practical, convenient and possibly only
moderately costly expansion of our limited present day
repertoire of cancer detection methods.
Until now, such new test forms are mostly under
development in various research laboratories and not
widely applied in routine medical practice. This might,
however, change in the next years. There are many
candidates in the form of molecules and macromolecular
assemblies to be considered, which could drive these
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Cells and extracellular vesicles
The first category of candidates are the by now ‘classical’
circulating tumor cells (CTCs) [6, 7] and circulating
DNA fragments (ctDNAs) [8, 9]. They are currently
explored in several dozen clinical studies (see e.g.
ClinicalTrials.gov for most recent details). Probably the most
exciting new development in this area is the very recent
finding that ctDNA seems to have a major role to play
as a prognostic marker of surgery success of stage II
colon cancer resections .
In addition, we are witnessing at present the surfacing
of a whole zoo of novel molecule and vesicle classes that
promise to provide information about cancer origins and
subtypes. Some of them will be briefly introduced here
(see also Fig. 1 for selected examples).
Extracellular vesicles come in many forms, sizes,
shapes [11, 12] and their nomenclature and definition is
still somewhat fuzzy, since universal exosomal markers
are still missing [13–15]. Several curated databases like
ExoCarta  and EVpedia  are collecting
information on their molecular components and characteristics.
Exosomes are endocytic secretions that can be generated
by many cell types, at least in vitro, and represent, at
least for now, the most prominent extracellular vesicle
group. They are detectable in many body fluids, form a
diverse bunch of membrane vesicles ranging in size from
ca. 30 to 150 nm and can be isolated by several different
methods, including immunoaffinity capture and
ultracentrifugation [18–20]. Composed of proteins, various
RNAs and lipids, exosomes carry inside and on their
surface a rich load of information that can be deciphered
by molecular readout methods like RNA sequencing and
proteomic approaches. Exosomes have just been
published as suitable leads for the early detection of
pancreatic cancer and the cell surface proteoglycan glypican-1
has been proposed as a cancer exosome marker .
Moreover, exosomes are believed to contain
doublestranded DNA from their generating cells, which has
been christened exoDNA [22, 23], but this finding
requires further validation and exploration before a
possible role of this DNA as biomarker can be considered.
Fairly recently, exosomes have been implicated as major
players in cancer development, for example, in driving
organ-specific metastasis [24, 25]. It is thus to be
expected that exosomes will be a major focus of
biochemical and functional cancer studies in coming years.
Oncosomes, originally described 2008 in gliomas as
vehicles for intercellular transfer of an oncogenic EGFR
variant , are much larger in size (ca. 1–10 μm) and
differ considerably in their composition from exosomes
and other extracellular vehicles [15, 27]. They are
believed to be generated by membrane blebbing in late
stage cancer disease. In prostate cancer cells, oncosome
production was reportedly stimulated by EGFR
activation and elicited in recipient cells an increase in tyrosine
phosphorylation and Akt signalling, thereby altering the
tumor microenvironment and contributing to disease
progression . A new study has further defined the
generation, density, composition and cargo of
oncosomes . It documents the enrichment of cytokeratine
18 (CK18) as a molecular marker of oncosomes and
links enhanced oncosome shedding to silencing of the
cytoskeletal formin protein DIAPH3.
A flourishing superfamily of non-protein coding RNAs
The realization that our genome is not mostly composed
of ‘junk DNA’ and that this term predominantly
reflected our own ignorance, has led to a profound shift
in research activities of scientists focusing on how
genomes and their products are regulated. Non-coding
RNAs have consequently gained a very solid foothold in
most of cell biology research and particularly in human
disease studies [29–31].
MicroRNAs (miRNAs), small ribonucleotide oligomers
typically composed of ca. 22 nucleotides, are the most
Fig. 1 Cells, vesicles and molecules in liquid biopsies that can be queried for information about cancers (selected examples)
intensely studied group of these non-coding RNAs. They
serve as repressors of mRNA translation into proteins 
in a plethora of biological contexts. Many recent reviews
have described their generation and functions in
considerable detail (for two recent examples see [33, 34]). miRNA
involvement in the manifestation and progression of most
diseases, ranging from neurological disorders to
autoimmunity, inflammation, infections, cardiovascular
problems, metabolic pathologies and malignancies is also very
well established [35–40]. Over 1900 miRNAs with critical
functions have been currently described . miRNAs can
act as oncogenes or as tumor suppressors, depending on
the specific tissue and cell type context and miRNA
polymorphisms have been associated with cancer development
risks. Their utility as clinical diagnostics has, however, so
far been severely limited by poor data reproducibility. The
generation of data in the wet lab and the bioinformatics
data processing urgently require optimization and better
Since miRNAs have become a major area of attraction
for researchers at the beginning of this millennium 
over 40,000 publications have investigated the many
facets of these fascinating regulatory systems and
miRNAs are still getting the lion share of attention until
now; but a flurry of additional families of non-protein
coding RNAs are emerging in the more recent literature.
Small nucleolar RNAs (snoRNAs) have long been
viewed as ‘boring’ housekeeping components of the cell.
Hundreds of them have been detected so far. As their
name indicates, they live in the nucleolus, the ribosomal
RNA producing factory of the cell, form complexes with
proteins and aid in the processing of rRNAs .
Interestingly, in the last years various groups have reported
additional functions of snoRNAs, which link them to the
control of cell fates and tumorogenesis . Some
snoRNAs have been detected in plasma and could have
potential as diagnostic as well as prognostic circulating
biomarkers, for example in non-small cell lung cancer
(NSCLC) [45, 46]. Larger studies that confirm and
extend the initial reports are still needed to validate certain
promising snoRNA candidates.
Long non-coding RNAs (lncRNAs), are a highly diverse
group of thousands of RNAs implicated in cancer invasion
and metastasis, as well as many other diseases [47–49]. For
an RNA to be entered into this group, a somewhat arbitrary
size threshold of more than 200 nucleotide in lengths has
been set. At least a few lncRNAs have been detected in
human plasma so far and may have relevance for diagnostic
or prognostic assays [50, 51], but clearly much more work
is needed to gain a robust understanding of the potential
and limits of lncRNAs for cancer detection, classification
PIWI protein-interacting RNAs (piRNAs) are
singlestranded molecules composed of ca. 26–31 nucleotides
and found throughout the animal kingdom . A
major, if not the major, function of piRNAs is the
immobilization of transposons, particularly in the
context of spermatogenesis . Whether piRNAs are really
drivers of cancer development or ‘passengers’ still
remains to be determined . Although they can be
detected as a small fraction of the various RNAs isolatable
from plasma-derived exosomes , it is far from clear
whether they will have any diagnostic value in clinical
Only last year, a new group of RNAs has shot onto the
scene and gained significant attention immediately: sex
hormone-dependent tRNA-derived RNAs
(SHOTRNAs). Generated from mature tRNAs, they are
constitutively present in sex hormone-dependent breast and
prostate cancer cells . The ribonuclease angiogenin
has been implicated in their biogenesis and sex
hormones and their receptors were shown to enhance the
generation of SHOT-RNAs. Their potential as diagnostic
and/or therapeutic targets, if any, remains to be
Developing analyses of circulating macromolecules,
vesicles and cells towards clinical routine applications
CTCs from solid cancers have proven fairly difficult to
work with, for all but a few specialist laboratories. CTCs
are usually exceedingly rare and their vitality at the time
of isolation is often uncertain. Despite nearly two
decades of increasing scrutiny ([57, 58], methods to
actually culture them ex vivo after isolation are just being
developed ). Nevertheless, successful RNA
sequencing from single CTCs of prostate cancer patients has
been reported , implicating non-canonical Wnt
signaling in mediating anti-androgen resistance. This
indicates that it may be possible to omit the ex vivo
amplification step for CTCs, at least for some types of
analyses. CTC plasticity is, however, another significant
worry and clearly needs further elucidation. Along these
lines, EpCAM-positivity cannot be considered a robust
feature of CTCs anymore .
Whole exome and whole genome sequencing
experiments of DNA from tissue biopsies was and is certainly
enormously helpful to gain much deeper knowledge
about the molecular mechanisms and heterogeneity of
virtually all cancer types. These are by now also possible
using single CTCs [62–64]. CTCs could thus, at least in
principle, provide some useful information on the
(sometimes changing) polyclonality of tumors. Multiple
standard biopsies that are otherwise required to analyze
the clonality of tumors, are difficult or impossible to
obtain for certain tumor types, so alternative options would
be most welcome. Until now, however, it is not really
clear how well tumor polyclonality is really reflected in
CTCs and whether the current methods of CTC analysis
are developed far enough to make this a practical
Alternatively, cancer-relevant miRNAs in liquid
biopsies, the so called ‘Oncomirs’, [40, 65] might be analyzed.
But even there, the required technology is just being
developed and still far from being ready for daily routine
usage [66, 67].
In general, it can probably be expected that costly
‘-omics’ approaches will not be the main road forward
for new diagnostic cancer tests, at least when it comes
to broad clinical implementation, even for large
comprehensive cancer centers (CCCs). While ‘-omics’
technologies clearly very important for basic and translational
research, it is highly desirable to generate more focused
analyses, no matter what the material (s) for the analyses
will eventually be. Limited sets of molecular markers,
analyzed by simple, low cost array type (micro-) chips
and automatically read and evaluated by standardized,
automatic machines would seem to be the method of
choice to lead the way into daily routine application.
Tumor educated platelets (TEPs)
It might be at first counterintuitive to assume that highly
chaotic entities like tumor cells could ever be ‘educators’
of anything. Obviously this term is used quite loosely
here and a slightly better one for the recently observed
phenomenon of tumor-influenced platelets [68–70]
might be ‘tumor-conditioned platelets’.
It is, however, also undoubtedly clear that cancer cells
impact very significantly on several other cell types,
ranging from fibroblasts (carcinoma-associated fibroblasts
(CAFs), tumor-associated fibroblasts (TAFs)) and various
immune cells, like tumor- associated macrophages
(TAMs) and tumor-infiltrating lymphocytes (TILs) to
endothelial cells. Molecular studies of the relationships
between different cell types within a tumor mass or a
metastatic lesion are currently under way and provide
evidence for complex, formerly unknown relationships
at a rapid pace [71–73].
By comparison, analyzing effects of tumor cells on
platelets has been somewhat neglected until now. After
all, platelets are merely sheddings of megakaryocytes in
the bone marrow that do not contain a nucleus and
possess a reduced repertoire of proteins, RNAs etc. Their
main normal function is hemostasis, i.e. plugging
vascular holes after physical injuries.
However, cancer progression can be accompanied by
platelet activation and venous thromboembolisms
(VTEs). In addition, platelets can secrete growth factors
that can influence cell growth and angiogenesis. They
are also able to attract other blood cells like monocytes
and granulocytes, which may help to create a
prometastatic microenvironment [74–76].
The latest TEP study by Best and colleagues  is
highly intriguing. Based on extensive platelet RNA
analyses of more than 280 individuals (ca. 22 million reads
per sample) they were able to distinguish healthy
persons from cancer patients with more than 95 % accuracy
and could even provide some information on likely
tumor locations for some major tumor types.
Fivethousand non-coding and protein-coding RNAs were
detectable in pictogram quantities of RNA,
corresponding to platelet numbers that can be found in a single
drop of blood. The global RNA deregulation observed
was massive, with over 2000 changes observed.
Interestingly, prominent tumor-driving mutations
(EGFR, Her-2, c-Met, H-Ras, PI3KCA) were also
accurately reflected in the results obtained from platelet RNA
sequencing and bioinformatics processing of the
resulting data. This finding could have major implications for
future clinical trials and therapy regimens .
While the tumor type was the predominant factor for
the actual platelet conditioning, tumor metastasis did
not significantly impact on them when compared to
samples from patients without metastasis, which is
It should be pointed out that the majority of cancer
patients had metastatic disease (189 of 228), which
usually corresponds to a high tumor mass burden, i.e. many
billions of tumor cells, although patients with localized
disease (albeit unspecified tumor volume) were also
correctly classified. Furthermore, the average age of the
healthy donors was significantly lower than that of the
So are TEPs going to run CTCs out of business?
They are obviously easy to purify, infinitely more
abundant and much less likely to die while moving
through the vascular system. So this may sound like a
done deal, yet it is probably not possible to give a
clearcut answer at present. TEPs will need to be scrutinized
much further until a number of questions have been
Some of the most pressing ones are:
Will other researchers be able to recapitulate the
initially reported results time and time again? In other
words, how robust is this method?
Will it become possible to simplify the analyses to a
degree that becomes feasible in daily routines, for
example by focusing on specific subsets of RNAs
without loosing accuracy?
How much will platelet RNAs from non-cancerous
individuals vary depending on their age or the presence
of various non-malignant diseases?
What is the sensitivity threshold for the cancer patient
studies? Clearly, a single tumor cell should not be able
to ‘educate’ a vast number of platelets. So how many
cancer cells are needed before conditioning effects
become apparent? Many billions?
How does polyclonality factor into this?
Some well-known cancer mutations, mentioned
already above, have been detectable through TEP RNA
analyses. But how many other, rarer mutants and
variants will be detectable robustly?
And what about the highly complex epigenetic
patterns? How will they be reflected in TEPs?
The exciting initial work described by Best and
colleagues is bound to be followed up soon by studies of
larger cohorts of individuals with specific tumor types,
patients with tumors of different sizes etc.
In summary, a new battle has just begun, and there is
certainly some hope for success. We now know our old
enemy cancer much better than only a decade ago,
though it is bound to still have a few more tricks up its
sleeves. Whether it will actually become possible to
snatch a few more mortals away from him for some time
by utilizing liquid biopsies, in the form of TEPs and
other marker vesicles or nucleic acids, remains to be
CTC: Circulating tumor cells; ctDNA: Circulating tumor DNA;
exoDNA: Exosomally-derived DNA; lncRNA: Long non-coding RNA;
miRNA: Micro RNA; piRNA: PIWI protein-interacting RNA; SHOT-RNA: Sex
hormone-dependent tRNA; snoRNA: Small nucleolar RNAs; TAF:
Tumorassociated fibroblast; TAM: Tumor-associated macrophage; TEP: Tumor
educated platelet; TIL: Tumor infiltrating leukocyte; VTE: Venous
We are truly grateful for research support we receive from the institutions
listed below. We apologize to all colleagues who have not been cited with
their important work in this brief summary.
Research funding to our laboratory is provided by Cancer Research UK,
Breast Cancer Campaign (UK, recently renamed to Breast Cancer Now), the
Rosetrees Trust (UK), the Wilhelm-Sander-Stiftung (Munich, Germany), the
VolkswagenStiftung (Hannover, Germany) and the European Regional
Development Fund of the European Commission.
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