Cellular and Molecular Bioengineering ( 2015)

DOI: 10.1007/s12195-015-0418-3

Therapeutic Targeting of Circulating Tumor Cells: An Important Problem

That Deserves Careful Study
In cancer research, the discovery and study of circulating tumor cells (CTCs) have seemed to open a
world of possibilities. We now have the potential to
gain cellular and molecular understanding of individual cases of metastatic cancer without invasive procedures. This area of research is, however, not without
some basic pitfalls. In this commentary, we address
some of these pitfalls by considering two recent
examples in the published literature and discuss ways
to overcome their limitations with the hope of
informing those who may be entering the growing eld
of CTC research. Careful research design should always be followed to prevent incomplete or misleading
studies from entering the literature, and thereby avoid
setting back this burgeoning eld.
Two recent CTC papers appearing in the journal
PLOS ONE can serve as examples of preliminary
studies lacking the necessary controls to yield meaningful results. In the rst, Kim and Gaitas outline a
creative application of photodynamic therapy (PDT)
intended to prevent cancer metastasis, in which tubing
could be attached to a patients vasculature to divert
blood into an external device where it would be exposed to LED illumination to induce cancer cell
death.5 The illuminationcombined with a photosensitizer conjugated to an antibody against CD44, a
cell adhesion protein found on many cancer cellsis
intended to eliminate CTCs in the blood. Their study
attempts to examine the feasibility of this technique
in vitro. Upon rst glance the results of the paper might
seem promising, with the vast majority of prostate
cancer cells spiked into whole blood dying within an
hour after 2 min treatment; however, the authors
overlooked key aspects of treatment in blood that,
when considered, signicantly weaken the overall impact of the paper.
When critically evaluating this proposed therapy,
one must rst look at the feasibility of treating the
entire blood volume in a realistic time frame. Limitations of throughput are a common issue in many
proposed CTC technologies. Most devices test a small
volume of blood and while the information gleaned
from such volumes can be of scientic interest,4,10,13,14
the scale-up of these devices becomes more complicated. The scale-up of the PDT device to treat an adult
blood volume (~5 L) in this case does seem feasible,

however. One may calculate that at a physiological

blood velocity (~30 cm/s) typical of small arteries of
equal diameter as the 1 mm internal diameter tubing
used, 36 meters of tubing would be needed to achieve
their prescribed 2 min of LED light exposure and thus
the entire blood volume could be processed within
about 6 h. This amount of tubing would contain a
blood volume of 28 mL, also a reasonable design
parameter. A single or double layer of this tubing
could, in theory, be arranged in a small bedside or even
portable extracorporeal device. Unfortunately no
analysis of this form was presented in the 2015 paper.5
Importantly, one must also consider the proposed
therapys eect on the normal blood cells, which would
likewise also receive exposure to the photodynamic
sensitizer and light source. This is the most problematic issue with the proposed technology. The authors
chose CD44 as the molecular target for the photosensitizer therapy. While CD44 expression is well documented in cancer,3,6,7,11 it is also a major adhesion
molecule for leukocytes.8,16 CD44 goes by the alternate
name phagocytic glycoprotein-1, and is a receptor for
hyaluronic acid. The implication of this, of course, is
that while the photosensitizer may target CTCs,
leukocytes will almost certainly become bound to the
sensitizer and also killed during light treatment. Furthermore, even if a more appropriate antigen was selected to better avoid off-target binding, the reactive
oxygen species (ROS) generated by the therapy would
still be likely to affect surrounding blood cells and
accumulate within the plasma during the course of
treatment. The authors argue that since the diffusion
distance of ROS is small (~100 nm, reportedly), the
ROS generated at the surface of CTCs should not
reach neighboring blood cells. While this might hold
true under static conditions, under dynamic blood ow
conditions it is the much greater convective length scale
that must be considered when estimating whether ROS
generated in the vicinity of one cell surface may affect
neighboring cells. The chaotic mixing of bulk blood
ow will nearly ensure that all cells within the
peripheral blood owing through the device will be
equally exposed to ROS generated in the extracellular
uid. Remarkably, Kim and Gaitas demonstrated the
ability to kill PC-3 cancer cells within spiked blood
samples, but never tested whether the PDT approach

 2015 Biomedical Engineering Society

J. R. MARSHALL

was also killing blood cells at the same rate, a minimum requirement of any selective therapy designed to
act in the bloodstream.5 In our opinion, this important
control measurement should have been a necessary
precursor to publication in a peer-reviewed research
journal, and the absence of basic measurements such as
these cast the overall conclusions into considerable
doubt. The authors used the loss of uorescence signal
from PC-3 prostate cancer cells labeled with the live
cell dye Calcein AM as their sole measure of cell death,
whereas an additional assay of apoptosis and necrosis
such as Annexin V, TUNEL, or MTT would be recommended, as well as the use of an additional cancer
cell line.
Finally, whenever light is used to induce a cellular
response, one must also be concerned with the eects
of heating. This is particularly important in the blood
since red blood cells will undergo lysis at temperatures
above 45 C.2 Anywhere from 60 to 95% of the energy
of LEDs is converted to heat. This heat is generally
believed to not be given off as infrared heat and
therefore may not affect the sample directly. However,
there have been reports of IR heat production and
resulting sample heating by LEDs, an effect that is
dependent on the power used and the type of heat sink.
It may thus be possible to avoid this issue through
careful engineering, but heating should nevertheless
have been considered and tested in the paper (it
wasnt). All of the PDT experiments of Kim and
Gaitas were performed in a foil-lined Styrofoam
insulated cooler, using sealed, static well plates and
blood-lled tubing sealed on both ends. Despite the
diagram of their Figure 1, no ow experiments were
performed.5
The majority of current CTC research focuses on
the isolation and enumeration of cells captured from
patient blood samples for analysis. While this work
does not directly involve the toxicity of reagents on
either cancer or blood cells, it nevertheless holds its
own pitfalls and issues that, if not properly addressed,
can skew results and thereby lead to misleading conclusions.
One example of this is another paper by Gaitas and
Kim published recently in PLOS ONE.1 The authors
present a method for isolating CTCs in a ow device
coated with antibody against epithelial cell adhesion
molecule (EpCAM). The authors showed that they
were able to capture 85% of EpCAM-expressing cancer cells suspended in buffer, and 44% of the cancer
cells from whole blood spiked with cancer cells. This
straightforward method shares similarities with other
EpCAM-antibody isolation approaches previously
described in the literature,4,12,14,15 and would likely not
encounter major challenges in capturing those CTCs
that highly express EpCAM. However, as with the

AND

M. R. KING

previously discussed paper by Kim and Gaitas, some

much needed control experiments and other considerations were neglected, seriously compromising the
soundness of the study.
First, the device is presented as a dynamic
microuidic device intended to capture cells from the
bloodstream of patients to achieve a therapeutic ltering of the blood, however the only experiments
performed in the study were under static (i.e., no ow)
conditions. It is likely that the addition of shear force
would aect the capture eciency of the device and
this is something that should have been addressed to
support the conclusions of the paper.
Second, the authors again failed to take the blood
cells into consideration. In other CTC isolation studies, the capture purity as well as the capture eciency is
reported.4,9,13 This is important for two reasons. First,
if too many white blood cells adhere to the device, they
will block the capture of further cancer cells, thereby
decreasing the capture efciency. This is especially
important for the device discussed in the Gaitas and
Kim paper, as they are suggesting that this technology
could be used as a blood-ltering device for metastatic
cancer patients. Such a device would process many
more white blood cells than cancer cells (>105-fold),
and over time the device would likely become less and
less efcient due to nonspecic adhesion of leukocytes.
The purity is equally important for post-isolation
processing. Certain techniques that involve DNA and
RNA analysis require relatively pure samples to detect
cancer-associated mutations without excessive contamination from blood cell genetic material. For these
reasons, the achieved purity is always reported for new
CTC isolation devices.
We were quite surprised that Gaitas and Kim only
reported the number of cancer cells captured by their
antibody-coated tubing and did not attempt to assess
the number of leukocytes captured, either qualitatively
with a bright eld or phase contrast micrograph to
accompany the uorescence image showing EpCAM+
capture, or quantitatively using a hemocytometer or
ow cytometer. We are even more surprised that such
a preliminary eort would be accepted for publication
in a reputable journal such as PLOS ONE. Surely a
study whose abstracts rst sentence promises a tube
to selectively capture circulating tumor cells should
show some measure of selectively within its pages?
Perhaps bright eld images showed a degree of indiscriminate cell capture which contradicted the stated
goals of the study. The authors sly addition of the
phrase In this preliminary eort to the second sentence of both abstracts should not justify the publication of such incomplete work.
As the title of this commentary states, the development of new technology to study (and treat) circulating

An Important Problem That Deserves Careful Study

tumor cells is sorely needed, but it must be carried out

with sucient rigor to advance the eld. Authors,
reviewers, and editors should work together to avoid
populating the literature with hastily done, inadequate
work that acts more to mislead and confuse rather
than enlighten. We also hope that our commentary can
serve a more productive purpose than simply singling
out one laboratory that is new to the CTC eld, by
pointing out some of the common pitfalls and necessary considerations for researchers to be aware of.
Overall, we are encouraged that the eld of CTC
research is growing and more innovative ideas are
being proposed, such as blood-ltering devices and
new CTC-targeted therapies. These ideas are vital for
the advancement of cancer metastasis research and
treatment. By addressing common issues such as those
presented in this commentary, we believe that CTC
research can become a more important area of cancer
investigation. We hope those who are just entering the
eld can draw inspiration and advice from this commentary and we look forward to seeing what technologies the next wave of CTC research will bring.

REFERENCES
1

Gaitas, A., and G. Kim. Chemically modied plastic tube

for high volume removal and collection of circulating
tumor cells. PLoS One 10:e0133194, 2015.
2
Gershfeld, N. L., and M. Murayama. Thermal instability
of red blood cell membrane bilayers: temperature dependence of hemolysis. J. Membr. Biol. 101:6772, 1988.
3
Hanley, W. D., M. M. Burdick, K. Konstantopoulos, and
R. Sackstein. CD44 on LS174T colon carcinoma cells
possesses E-selectin ligand activity. Cancer Res. 65:5812
5817, 2005.
4
Hughes, A. D., J. Mattison, L. T. Western, J. D. Powderly,
B. T. Greene, and M. R. King. Microtube device for selectin-mediated capture of viable circulating tumor cells
from blood. Clin. Chem. 58:846853, 2012.
5
Kim, G., and A. Gaitas. Extracorporeal photo-immunotherapy for circulating tumor cells. PLoS One
10:e0127219, 2015.
6
Li, X.-P., X.-W. Zhang, L.-Z. Zheng, and W.-J. Guo.
Expression of CD44 in pancreatic cancer and its signicance. Int. J. Clin. Exp. Pathol. 8:67246731, 2015.

Maeda, S., et al. CD44 and CD133 expressions in primary

tumor cells correlate to survival of pancreatic cancer
patients. Open Surg. Oncol. J. 1:17, 2009.
8
McDonald, B., and P. Kubes. Interactions between CD44
and hyaluronan in leukocyte trafcking. Front. Immunol.
6:68, 2015.
9
Nagrath, S., et al. Isolation of rare circulating tumour cells
in cancer patients by microchip technology. Nature
450:12351239, 2007.
10
Okegawa, T., K. Hayashi, H. Hara, K. Nutahara, and E.
Higashihara. Immunomagnetic quantication of circulating tumor cells in patients with urothelial cancer. Int. J.
Urol. 17:254258, 2010.
11
Ouhtit, A., Z. Y. Abd Elmageed, M. E. Abdraboh, T. F.
Lioe, and M. H. G. Raj. In vivo evidence for the role of
CD44s in promoting breast cancer metastasis to the liver.
Am. J. Pathol. 171:20332039, 2007.
12
Sieuwerts, A. M., et al. Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like
breast tumor cells. J. Natl. Cancer Inst. 101:6166, 2009.
13
Stott, S. L., et al. Isolation of circulating tumor cells using a
microvortex-generating herringbone-chip. Proc. Natl.
Acad. Sci. U. S. A. 107:1839218397, 2010.
14
Stott, S. L., et al. Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer. Sci. Transl. Med. 2:25ra23, 2010.
15
Thege, F. I., et al. Microuidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1
capture: characterization, optimization and downstream
analysis. Lab Chip 14:17751784, 2014.
16
Zuchtriegel, G., et al. Spatiotemporal expression dynamics
of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inammatory
response. Arterioscler. Thromb. Vasc. Biol. 35:899910,
2015.

Jocelyn R. Marshall
Meinig School of Biomedical Engineering
Cornell University, Ithaca, NY, USA

Michael R. King
Meinig School of Biomedical Engineering
Cornell University
Electronic mail: mrk93@[Link]