Individual profiling of circulating tumor cell composition in patients with non-small cell lung cancer receiving platinum based treatment

Abstract

Circulating tumor cells (CTC) could serve as a "liquid biopsy" for individualizing and monitoring treatment in patients with solid tumors as recently shown by our group. We assessed which non-hematopoietic cell types are identifiable in the peripheral blood of patients with non-small cell lung cancer (NSCLC) and correlated those to clinical characteristics.
Blood from NSCLC patients (n=43) was processed as previously described. For subtype analyses CTC were negatively enriched by hematopoietic cell depletion. The remaining cell suspension included pre-enriched tumor cells and was spun onto glass slides and further characterized by multi-immunofluorescence staining against epithelial markers pan-cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM), mesenchymal marker N-cadherin, stem cell marker CD133, hematopoietic marker CD45 and nuclear counterstain DAPI. Individual cell type profiles were analyzed and correlated to therapeutic outcome.
Among other associations of CTC subtypes with clinical parameters Kaplan-Meier test revealed that an increased CD133-positive to pan-CK-positive cell type ratio (stem cell like to epithelial ratio) and the presence of mesenchymal N-cadherin+ cells, both were significantly associated to shortened PFS (2 vs. 8 months, P=0.003, HR =4.43; 5 vs. 8 months, P=0.03, HR =2.63).
Our data suggest that different CTC populations are identifiable in peripheral blood and that these individual cell type profiles might be used to predict outcome to platinum based systemic therapies in lung cancer patients.

Full text

© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
Introduction
Circulating tumor cells (CTC) could serve as a “liquid
biopsy” for individualizing and monitoring treatment in
patients with solid tumors (1,2). So far, CTC detection
methods consisted of enrichment and subsequent
identification mostly with anti-cytokeratin (CK) or
epithelial cell adhesion molecule (EpCAM) antibodies. CK-
positive cells are thought to be absent or to be present in
the blood of healthy subjects in very low numbers (3). CTC
have extensively been described in breast and lung cancer
and EpCAM positive CTC quantication has been linked
to patient outcome (4-8). Standardized approaches with
currently available enrichment and detection techniques
are based on physical or biological properties of CTC and
challenged by their cellular heterogeneity and plasticity.
Epithelial-to-mesenchymal transition (EMT) can cause
alteration of cellular features and loss of epithelial properties
leading to a partial or complete switch to a mesenchymal
phenotype. Particularly stem cells have the ability to take on
Original Article
Individual profiling of circulating tumor cell composition in
patients with non-small cell lung cancer receiving platinum based
treatment
Ivonne Nel1, Ulrich Jehn1, Thomas Gauler2, Andreas-Claudius Hoffmann1,3
1Molecular Oncology Risk-Profile Evaluation, Department of Medical Oncology, West German Cancer Center, 2Department of Radiotherapy,
3Department of Medical Oncology, West German Cancer Center, University Duisburg-Essen, 45122 Essen, Germany
Correspondence to: Priv.-Doz. Dr. med. habil. Andreas-Claudius Hoffmann, M.D., Assistant Professor of Experimental Oncology. Department of
Medical Oncology, Molecular Oncology Risk-Prole Evaluation (MORE), West German Cancer Center, University Hospital Essen, Hufelandstrasse
55, 45147 Essen, Germany. Email: hoffmann@more-oncology.de.
Background: Circulating tumor cells (CTC) could serve as a “liquid biopsy” for individualizing and
monitoring treatment in patients with solid tumors as recently shown by our group. We assessed which non-
hematopoietic cell types are identiable in the peripheral blood of patients with non-small cell lung cancer
(NSCLC) and correlated those to clinical characteristics.
Methods: Blood from NSCLC patients (n=43) was processed as previously described. For subtype analyses
CTC were negatively enriched by hematopoietic cell depletion. The remaining cell suspension included pre-
enriched tumor cells and was spun onto glass slides and further characterized by multi-immunouorescence
staining against epithelial markers pan-cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM),
mesenchymal marker N-cadherin, stem cell marker CD133, hematopoietic marker CD45 and nuclear
counterstain DAPI. Individual cell type proles were analyzed and correlated to therapeutic outcome.
Results: Among other associations of CTC subtypes with clinical parameters Kaplan-Meier test revealed
that an increased CD133-positive to pan-CK-positive cell type ratio (stem cell like to epithelial ratio) and
the presence of mesenchymal N-cadherin+ cells, both were signicantly associated to shortened PFS (2 vs. 8
months, P=0.003, HR =4.43; 5 vs. 8 months, P=0.03, HR =2.63).
Conclusions: Our data suggest that different CTC populations are identiable in peripheral blood and
that these individual cell type proles might be used to predict outcome to platinum based systemic therapies
in lung cancer patients.
Keywords: Circulating tumor cells (CTC); mesenchymal cells; epithelial cells; stem cell-like
Submitted Mar 05, 2014. Accepted for publication Mar 23, 2014.
doi: 10.3978/j.issn.2218-6751.2014.03.05
View this article at: http://www.tlcr.org/article/view/2265/2889

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© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
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characteristics of other cell types (9).
We recently developed a CTC detection method based
on multi-immunofluorescence staining that includes but
is not solely dependent on epithelial markers such as CK
or EpCAM and also detects cells with mesenchymal and
stem cell-like characteristics (10-12). In this study, we have
addressed the question whether different types of CTC are
identifiable in the peripheral blood of patients with non-
small cell lung cancer (NSCLC) and, if so, whether their
distribution may serve as a predictor of treatment response
or outcome.
Material and methods
Informed consent and study population
Written informed consent was obtained from all patients
before participating in the study. Blood sample collection
and analyses were approved by the Review Board of the
Medical Department, University of Essen-Duisburg;
Germany (12-5047-BO). The clinico-pathological data
of the patients is listed in Table 1. Tumor staging was
performed according to the criteria of the International
Union Against Cancer (13). Revised Response Evaluation
Criteria in Solid Tumors (RECIST 1.1) were used to dene
response or stable disease in patients after receiving two
cycles of systemic cytotoxic chemotherapy (14,15).
Preparation of blood samples and CTC enrichment
A total of 20 mL citrated peripheral venous blood was
drawn from NSCLC patients prior to or up to three weeks
after platinum-based treatment and processed within 24 h
after collection. Blood sample preparation was done as
follows: 20 mL of blood were diluted with 10 mL PBS and
carefully layered into a Leucosep tube containing 16 mL
Ficoll-Paque (GE-Healthcare) below a porous barrier. After
buoyant density gradient centrifugation (1,600 ×g, 20 ℃,
20 min) the interphase consisting of peripheral blood
mononuclear cells (PBMNC) and CTC was removed and
washed. For subtype analyses CTC were negatively enriched
by hematopoietic cell depletion. PBMNC were treated
with 50 µL of a 1:1 mixture of anti-CD45 and anti-CD15
coated immunomagnetic beads (Dynabeads, Invitrogen)
in a magnetic particle processor (King Fisher mL;
Thermo Fisher). The remaining cell suspension included
bead-free pre-enriched tumor cells and was spun onto
two glass slides per sample using the Cell Spin II
centrifuge (Tharmac, Waldsolms, Germany), air dried and
subsequently xated with 96% Ethanol. Slides were stored
at 4 ℃ until subjected to immunocytochemical staining.
Identication of CTC subtypes using multi-uorescence
labeling
Immunofluorescence staining of epithelial, mesenchymal,
stem cell-like and hematopoietic cells was carried out in
the CD45-depleted pre-enriched tumor cell suspension
Table 1 Patient demographics
DemographicPatients (n=43)
No. %
Tumor stage
IIb 2 5
IIIa 14 33
IIIb 5 12
IV 24 56
Histo
Adenocarcinoma 29 67
Squamous epithelium 14 33
NA 2 5
Grading
G2 15 35
G3 15 35
Gx 15 35
Age 45-77
Median, years 61
Range 27
Therapy
Cis/Pem 8 19
Cis/Pac 30 70
Carbo/Pac 4 9
Carbo/Pem 1 2
Response
PR 18 42
SD 21 49
PD 6 14
PFS (months)
Median 6
Range 0-18
Abbreviations: SD, stable disease; PR, partial response; G1,
well differentiated; G2, moderately differentiated; G3, poorly
differentiated; Gx, Grade cannot be assessed; Cis, cisplatin;
Pem, pemetrexed; Pac, paclitaxel; Carbo, carboplatin.

102 Nel et al. Circulating tumor cell proles in NSCLC
© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
as described previously (10). Briefly, the staining method
included xation and permeabilization of the cells with ice-
cold methanol for five min, washing in PBS, blocking of
unspecific antibody reactions by incubation with blocking
solution containing 5% BSA for 30 min, binding of primary
antibodies (final concentration: 5 µg/mL) either pan-CK
guinea pig polyclonal antibody (ABIN126062, antibodies-
online) and N-cadherin (EPR1792Y) rabbit monoclonal
antibody (2019-1, Epitomics) or CD133 rabbit polyclonal
antibody (orb18124, biorbyt) or EpCAM (ab32392,
Abcam) for CTC and anti-CD45 (MEM-28) mouse
monoclonal antibody (ab8216, Abcam) for hematologic
cells overnight at 4 ℃, wash in 0.1% Tween, binding of
secondary antibodies (FITC-conjugated AffiniPure goat
anti-rabbit and Cy3-conjugated AfniPure goat anti-mouse
or AlexaFlour647-conjugated AfniPure F(ab’)2 Fragment
goat anti-guinea pig; Jackson Immuno Research, Hamburg,
Germany) for 30 min at 37 ℃, washing in 0.1% Tween.
Subsequently, cells were stained with 4',6-Diamidino-
2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich,
St. Louis, MO, USA) for 10 min, mounted with anti-
fading medium (Invitrogen) and stored in the dark until
evaluation. Microscopic evaluation was carried out using
the digital Keyence BZ9000 (Biorevo, Osaka, Japan) all-in-
one fluorescence microscope with integrated camera and
BZ-Analyzer Software. We used pseudo colors to depict
cells. Stained slides were manually examined and CTC were
detected within the same areas, each consisting of ten visual
fields using a 20× magnification on both slides. For CTC
quantication previously published cut-offs were applied to
exclude false-positive events (10).
Statistical analysis
Statistical tests were performed according to previously
published studies by our group (10,16,17). The associations
among CTC subtypes, and clinico-pathological parameters
were tested with Spearman test for bivariate correlations.
Man-Whitney test for independent samples was used to
compare differences of various factors in distinct subgroups.
Kaplan-Meier method was used to test correlations of PFS
with cell types. Survival differences between patients with a
high and low cell type ratio were analyzed by the log-rank
test. The level of signicance was set to P<0.05. All P values
were based on two-sided tests. All statistical analyses were
performed using the Software Packages JMP 10.0 Software
(SAS, Cary, NC, USA), SPSS for Windows (Version 19.0;
SPSS Inc., Chicago, IL, USA), and Medcalc, Version 12.3.0
(Mariakerke, Belgium).
Results
Immunouorescence based identication of CTC subtypes
For the investigation of cellular subtypes a multi-staining
method was required in order to detect various epithelial,
mesenchymal, stem cell-like and hematopoietic markers
and to characterize different cells types. Therefore we
used multi-fluorescence staining for CTC-subtype
detection in NSCLC patients. When examining samples
objects that showed a positive nuclear staining with
DAPI a negative staining against CD45 and a positive
staining for pan-CK, N-cadherin, EpCAM or CD133
were captured and considered as tumor cells (Figure 1).
In blood samples we detected cells with mesenchymal
features such as N-cadherin+/CK-/CD45- and cells with
epithelial properties like CK+/N-cadherin-/CD45-; CK+/
EpCAM+/CD45- and cells with both characteristics like
CK+/N-cadherin+/CD45-. We also detected cells showing
stem cell-like features such as CD133+/CK-/CD45- and
CD133+/CK+/CD45- cells. In addition, we found cells
that stained positive for potential markers of CTC and
CD45 such as CK+/CD45+; CK+/EpCAM+/CD45 (low);
N-cadherin+/CK-/CD45 (low) and CK+/N-cadherin+/
CD45 (low) as well as CK+/CD133+/CD45+ cells. We also
found a sub-population of cells staining positive for CK
and CD45 (low) but negative for EpCAM and cells staining
triple positive for CK, EpCAM and CD45 (low). CTC were
enumerated, normalized and profiles of each patient were
examined. We summarized the total amount of N-cadherin-
positive, CK-positive and CD133-positive cells after
negative enrichment using CD45-depletion. We normalized
the enumerated potential CTC against the total number
of leucocytes obtained from the complete blood count and
expressed the number of CTC per 1,000 PBMNC (Table 2).
Association of CTC subtypes with clinical parameters
Spearman’s rank correlation revealed that the number
of stem cell-like CD133-positive CTC/1,000 PBMNC
(P=0.04; r=0.35) cells were significantly correlated
to the amount of N-cadherin-positive CTC/1,000
PBMNC cells (P=0.002; r=0.53). Cells with epithelial
characteristics (EpCAM-positive) were significantly
associated to treatment response (P=0.007; r=0.97). Mann-
Whitney test revealed a significant difference of CK-
positive CTC/1,000 PBMNC in patients with stage

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© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
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Figure 1 CTC isolated from NSCLC patients were stained against DAPI (nucleus; blue), CD45 (hematopoietic; red, pan-CK (epithelial;
yellow), EpCAM (epithelial; green) and N-cadherin (mesenchymal; green) or CD133 (stem cell; green). Cells marked with a white arrow were
considered as CTC. The image displays various CTC subtypes with epithelial, mesenchymal and/or stem cell-like features such as EpCAM+/
CK+/CD45 low, N-cadherin+/CK-/CD45-; N-cadherin-/CK+/CD45 low; CD133+/CK+/CD45 low and CD133+/CK-/CD45- cells. CTC,
circulating tumor cells; NSCLC, non-small cell lung cancer; CK, cytokeratin; EpCAM, epithelial cell adhesion molecule.
Table 2 Quantication of CTC subtypes
Subgroup No. positive samples (n) Amount of cells/1,000 PBMNC after enrichment
Minimum Maximum Mean SD
CK+ 43 0.1 12.3 1.8 2.3
EpCAM+ 5 0.0 0.5 0.14 0.21
N-cadherin+ 38 0.0 5.9 0.3 0.9
CD133+ 36 0.0 6.3 0.25 1.0
Abbreviations: CK+, pan-cytokeratin-positive cells; CD133+, CD13- positive cells; N-cadherin+, N-cadherin-positive cells;
pCAM+, EpCAM-positive cells. CTC, circulating tumor cells; EpCAM, epithelial cell adhesion molecule; CK, cytokeratin; PBMNC,
peripheral blood mononuclear cells.

104 Nel et al. Circulating tumor cell proles in NSCLC
© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
IV compared to stage III NSCLC (P=0.03; Figure 2).
Furthermore, Mann-Whitney test showed that CD133-
positive CTC/1,000 PBMNC were significantly higher
in patients with tumor grade 3 compared to grade 2
(P=0.04) and confirmed a significantly increased number
of CD133-positive CTC/1,000 PBMNC in the presence
of N-cadherin-positive cells (P=0.002). The number of
N-cadherin-positive CTC/1,000 PBMNC was higher
in patients with stage IV compared to stage III NSCLC
(P=0.09) and the amount of CD133-positive cells was
elevated in stage IV patients compared to stage II (P=0.1).
Noteworthy, Kaplan-Meier test revealed that an increased
CD133-positive to pan-CK-positive cell type ratio (stem
cell like to epithelial ratio) and the presence of mesenchymal
N-cadherin+ cells, both were significantly associated
to shortened PFS (2 vs. 8 months, P=0.003, HR =4.43,
Figure 3A; 5 vs. 8 months, P=0.03, HR =2.63, Figure 3B).
Discussion
With this study we wanted to examine the individual CTC
composition in patients with NSCLC receiving platinum
based treatment. Morphological analysis based on multi-
immunofluorescence staining revealed a variety of CTC
subtypes with epithelial, mesenchymal, stem cell-like or
mixed characteristics such as CK+/N-cadherin-/CD45-;
CK+/EpCAM+/CD45-; CK+/N-cadherin+/CD45-;
CD133+/CK-/CD45- and CD133+/CK+/CD45- cells.
Analyses of individual CTC proles indicated that the presence
of mesenchymal CTC and an increased ratio of stem cell-like
to epithelial CTC was associated to poor treatment response.
If CD133+ cells were detectable, N-cadherin+/CK- cells were
likely to be found. Due to technical limitations (staining on
two slides) it is impossible to determine whether the close
association between N-Cadherin+ and CD133+ cells is related
to coexpression on the same cell or to different cells. However,
it seems to indicate a link between cells with mesenchymal
and stem cell-like characteristics implying both as markers of
poor prognosis. Cancer stem cells (CSC) and EMT-type cells
are believed to play critical roles in drug resistance and cancer
Figure 3 (A) Kaplan-Meier test showed that an increased stem cell-like to epithelial cell type ratio (CD133-positive to pan-CK-positive
CTC ratio; >0) was signicantly associated to shortened PFS: 2 vs. 8 months; P=0.003; (B) Kaplan-Meier test revealed that the presence
of mesenchymal N-cadherin-positive CTC was signicantly associated to shortened PFS: 5 vs. 8 months, P=0.03. CK, cytokeratin; CTC,
circulating tumor cells.
Figure 2 Mann-Whitney test showed that the number of epithelial
CK-positive CTC was signicantly increased in patients with stage
IV compared to stage III NSCLC (P=0.03). CK, cytokeratin; CTC,
circulating tumor cells; NSCLC, non-small cell lung cancer.
100
80
60
40
20
0
100
80
60
40
20
0
Survival probability (%)
Survival probability (%)
0 5 10 15 20 0 5 10 15 20
Progression free survival (months)Progression free survival (months)
CD133+/CK+ cell ratio
<0
>0
Presence
N-cadherin + cells
=1
=0
A B
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
P=0.03
Stage IIIa/b Stage IV
CK-postive CTC/1,000 PBMNC

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metastasis. The formation of CSC and the event of EMT is
a dynamic process which is triggered by the interaction of
various cellular signaling pathways such as Hedgehog, Notch,
PDGF, Wnt, TGF-β, Akt, and NF-κB signaling pathways (18).
Our results are in line with a recent study by Barr et al. who
have generated an isogenic model of cisplatin resistance in
a panel of NSCLC cell lines and reported that the presence
and enrichment of stem-cell markers support the presence of
a chemoresistant population of lung cancer cells with a stem-
like signature (19). It has recently been described by Yu and
colleagues that CTC undergo EMT during treatment and that
these changes, and not only the absolute numbers of certain
subgroups, correlate well with response and resistance to
cytotoxic treatment, respectively (20).
In this study, we were able to observe a distinct
proportion of cells that stained positive for pan-CK and
CD45, a phenomenon already described by Yu et al. (21).
The additional CD45+ staining may not be exclusive for
hematopoietic cells, but may hypothetically be acquired
during dormant stay in the bone marrow or through effects
comparable to trogocytosis, i.e., transfer of membrane
proteins (22). Though this hypothesis cannot be scrutinized
by the data at hand it may warrant waiving any depletion of
CD45-positive cells as this approach might lead to a loss of
cells of interest. However, we took only CD133+/CD45-
cells into account during the abovementioned analyses of
CTC proles with stem cell-like characteristics. Moreover,
our method is based on cell type ratios rather than absolute
cell numbers assuming that a loss of potential CTC might
not affect the proportion of their CD45-negative subtypes.
Taken together our data support the hypothesis that
different CTC populations are identiable in the peripheral
blood of patients with NSCLC and that these individual
cell type profiles may have distinct clinical implications.
This method offers an opportunity to detect changes in
the composition of circulating non-hematopoietic cells
in the blood of NSCLC patients and, therefore, to create
an individual profile of each patient which might help
to predict patient outcome and potentially to select the
appropriate treatment. Further studies addressing the
question whether CTC subtype distributions are changing
during platinum based treatment and whether these
cells bear the potential to develop molecular markers to
individualize treatment are currently ongoing.
Acknowledgements
Authors’ contributions: conception and design: Andreas-
Claudius Hoffmann, Ivonne Nel; provision of study
materials or patients: Andreas-Claudius Hoffmann, Thomas
Gauler; collection and assembly of data: Ulrich Jehn,
Thomas Gauler; data analysis and interpretation: Ivonne
Nel, Ulrich Jehn, Andreas-Claudius Hoffmann; manuscript
writing: Ivonne Nel, Andreas-Claudius Hoffmann; final
approval of manuscript: Andreas-Claudius Hoffmann.
Funding: This project was funded by the Dr. Werner
Jackstädt-Stiftung, Wuppertal, Germany. Results of this
study were partly presented at the 10th Congress on Lung
Cancer of the Spanish Lung Cancer Group in Barcelona,
Spain in November 2013.
Disclosure: The authors declare no conict of interest.
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Cite this article as: Nel I, Jehn U, Gauler T, Hoffmann AC.
Individual profiling of circulating tumor cell composition in
patients with non-small cell lung cancer receiving platinum
based treatment. Transl Lung Cancer Res 2014;3(2):100-106.
doi: 10.3978/j.issn.2218-6751.2014.03.05