ArticlePDF Available

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

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
© 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
101
Translational lung cancer research, Vol 3, No 2 April 2014
© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
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
103
Translational lung cancer research, Vol 3, No 2 April 2014
© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
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
105
Translational lung cancer research, Vol 3, No 2 April 2014
© Translational lung cancer research. All rights reserved. Transl Lung Cancer Res 2014;3(2):100-106
www.tlcr.org
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.
References
1. Lianidou ES, Markou A. Circulating tumor cells as
emerging tumor biomarkers in breast cancer. Clin Chem
Lab Med 2011;49:1579-90.
2. O’Flaherty JD, Gray S, Richard D, et al. Circulating
tumour cells, their role in metastasis and their clinical
utility in lung cancer. Lung Cancer 2012;76:19-25.
3. Allard WJ, Matera J, Miller MC, et al. Tumor cells
circulate in the peripheral blood of all major carcinomas
but not in healthy subjects or patients with nonmalignant
diseases. Clin Cancer Res 2004;10:6897-904.
4. Hou JM, Krebs M, Ward T, et al. Circulating tumor cells
as a window on metastasis biology in lung cancer. Am J
Pathol 2011;178:989-96.
5. Moldenhauer G, Momburg F, Möller P, et al. Epithelium-
specic surface glycoprotein of Mr 34,000 is a widely
distributed human carcinoma marker. Br J Cancer
1987;56:714-21.
6. Zhang N, Li X, Wu CW, et al. microRNA-7 is a novel
inhibitor of YY1 contributing to colorectal tumorigenesis.
Oncogene 2013;32:5078-88.
7. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating
tumor cells, disease progression, and survival in metastatic
breast cancer. N Engl J Med 2004;351:781-91.
8. Gauler TC, Theegarten D, Parr A, et al. Decrease
of circulating tumor cells associates with response to
platinum-based chemotherapy in patients with non-small
cell lung cancer, but not with small cell lung cancer. J
Thorac Oncol 2011;6:S1114.
9. Alix-Panabières C, Pantel K. Circulating tumor cells:
liquid biopsy of cancer. Clin Chem 2013;59:110-8.
10. Nel I, Baba HA, Ertle J, et al. Individual proling of
circulating tumor cell composition and therapeutic
106 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
outcome in patients with hepatocellular carcinoma. Transl
Oncol 2013;6:420-8.
11. Nel I, Gauler TC, Eberhardt WE, et al. Formation and
repair kinetics of Pt-(GpG) DNA adducts in extracted
circulating tumour cells and response to platinum
treatment. Br J Cancer 2013;109:1223-9.
12. Nel I, Gauler T, Hoffmann AC. Circulating tumor cell
composition and outcome in patients with solid tumors.
Int J Clin Pharmacol Ther 2014;52:74-5.
13. Sobin LH. TNM: evolution and relation to other
prognostic factors. Semin Surg Oncol 2003;21:3-7.
14. Therasse P, Arbuck SG, Eisenhauer EA, New guidelines
to evaluate the response to treatment in solid tumors.
European Organization for Research and Treatment of
Cancer, National Cancer Institute of the United States,
National Cancer Institute of Canada. J Natl Cancer Inst
2000;92:205-16.
15. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response
evaluation criteria in solid tumours: revised RECIST
guideline (version 1.1). Eur J Cancer 2009;45:228-47.
16. Hoffmann AC, Wild P, Leicht C, et al. MDR1 and
ERCC1 expression predict outcome of patients with locally
advanced bladder cancer receiving adjuvant chemotherapy.
Neoplasia 2010;12:628-36.
17. Hoffmann AC, Danenberg KD, Taubert H, et al. A three-
gene signature for outcome in soft tissue sarcoma. Clin
Cancer Res 2009;15:5191-8.
18. Sarkar FH, Li Y, Wang Z, et al. Pancreatic cancer stem
cells and EMT in drug resistance and metastasis. Minerva
Chir 2009;64:489-500.
19. Barr MP, Gray SG, Hoffmann AC, et al. Generation and
characterisation of cisplatin-resistant non-small cell lung
cancer cell lines displaying a stem-like signature. PLoS
One 2013;8:e54193.
20. Yu M, Bardia A, Wittner BS, et al. Circulating breast
tumor cells exhibit dynamic changes in epithelial and
mesenchymal composition. Science 2013;339:580-4.
21. Yu M, Stott S, Toner M, et al. Circulating tumor cells:
approaches to isolation and characterization. J Cell Biol
2011;192:373-82.
22. Joly E, Hudrisier D. What is trogocytosis and what is its
purpose? Nat Immunol 2003;4:815.
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
... CD133 + CTCs were correlated with N-cad + CTCs, while E + CTCs were associated with treatment response. A significant difference was seen in respect to CK + , N-cad + , and CD133 + and later stage of disease [52]. CD133/CK + ratio and M + presence were associated with shorter PFS based on Kaplan-Meier analysis (p = 0.003, HR = 4.43 and p = 0.03, HR = 2.63, respectively) [52]. ...
... A significant difference was seen in respect to CK + , N-cad + , and CD133 + and later stage of disease [52]. CD133/CK + ratio and M + presence were associated with shorter PFS based on Kaplan-Meier analysis (p = 0.003, HR = 4.43 and p = 0.03, HR = 2.63, respectively) [52]. ...
Article
Full-text available
Lung cancer is the leading cause of cancer-related mortality globally. Among the types of lung cancer, non-small-cell lung cancer (NSCLC) is more common, while small-cell lung cancer (SCLC) is less frequent yet more aggressive. Circulating tumor cells (CTCs), albeit rare, have been portrayed as essential players in the progression of lung cancer. CTCs are considered to adopt an epithelial-to-mesenchymal transition (EMT) phenotype and characteristics of cancer stem cells (CSCs). This EMT (or partial) phenotype affords these cells the ability to escape from the primary tumor, travel into the bloodstream, and survive extremely adverse conditions, before colonizing distant foci. Acquisition of CSC features, such as self-renewal, differentiation, and migratory potential, further reflect CTCs’ invasive potential. CSCs have been identified in lung cancer, and expression of EMT markers has previously been correlated with poor clinical outcomes. Thus far, a vast majority of studies have concentrated on CTC detection and enumeration as a prognostic tools of patients’ survival or for monitoring treatment efficacy. In this review, we highlight EMT and CSC markers in CTCs and focus on the clinical significance of these phenotypes in the progression of both non-small- and small-cell lung cancer.
... Besides NANOG, the cell surface molecule PROM1 (also referred to as CD133), a widely accepted CSC marker [47], proved to be prognostic in our study. This finding is in line with Nel et al. who reported an association of mesenchymal CTCs, an increased ratio of CD133+ stem cell-like CTCs to epithelial CTCs, and poor treatment response [48]. ...
Article
Full-text available
Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis. Methods: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests. Results: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02-10.14, p = 0.016; PROM1: HR 4.23, 95% CI 0.65-27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72-24.14, p = 0.025; PROM1: HR 4.77, 95% CI 0.29-78.94, p = 0.032). Conclusions: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.
... October 2021 | Volume 9 | Article 750444 11 especially for bone metastasis (Cheng et al., 2014;Nel et al., 2014). As our research showed, the number of CTCs in patients with bone metastasis was relatively numerous (Supplementary Table S3). ...
Article
Full-text available
Circulating tumor cells (CTCs) play a crucial role in solid tumor metastasis, but obtaining high purity and viability CTCs is a challenging task due to their rarity. Although various works using spiral microchannels to isolate CTCs have been reported, the sorting purity of CTCs has not been significantly improved. Herein, we developed a novel double spiral microchannel for efficient separation and enrichment of intact and high-purity CTCs based on the combined effects of two-stage inertial focusing and particle deflection. Particle deflection relies on the second sheath to produce a deflection of the focused sample flow segment at the end of the first-stage microchannel, allowing larger particles to remain focused and entered the second-stage microchannel while smaller particles moved into the first waste channel. The deflection of the focused sample flow segment was visualized. Testing by a binary mixture of 10.4 and 16.5 μm fluorescent microspheres, it showed 16.5 μm with separation efficiency of 98% and purity of 90% under the second sheath flow rate of 700 μl min−1. In biological experiments, the average purity of spiked CTCs was 74% at a high throughput of 1.5 × 108 cells min−1, and the recovery was more than 91%. Compared to the control group, the viability of separated cells was 99%. Finally, we validated the performance of the double spiral microchannel using clinical cancer blood samples. CTCs with a concentration of 2–28 counts ml−1 were separated from all 12 patients’ peripheral blood. Thus, our device could be a robust and label-free liquid biopsy platform in inertial microfluidics for successful application in clinical trials.
... The GO chip was optimized to isolate CTCs specifically from NSCLC patients using anti-EPCAM, anti-epidermal growth factor receptor (EGFR), and anti-CD133 to increase the capture of lung cancer CTCs. [118][119][120]122,196 Previous studies have correlated CTCs and CTC metrics to timepoints during treatment, or loosely to clinical outcomes, however none have demonstrated the clinical utility of monitoring CTCs in stage III NSCLC patients. 177,193,197,198 In a cohort of stage I-III NSCLC patients, we used the GO chip to demonstrate that the percent of PD-L1+ CTCs increased after RT, raising from 0.7% of CTCs to 24% of CTCs in 1mL of blood. ...
Thesis
This work hypothesizes that the molecular characterization of extracellular vesicles (EVs) and circulating tumor cells (CTCs), biomarkers found in peripheral blood draws, can be used to monitor, and predict, treatment efficacy and patient outcomes in lung cancer. First, microfluidic EV isolation technologies were developed, and EV protein characterization assays were adapted for use with these technologies. Isolation strategies included (1) Annexin V-phosphatidylserine binding to capture tumor-derived EVs on the device surface and (2) isolating natural killer (NK) cell-derived EVs by first capturing NK cells followed by on-chip EV biogenesis. Western blot protein analyses were optimized for these technologies to verify the presence of EV-specific proteins (CD9, FLOT1, HLA-C) along with cell type-specific proteins (CD56). Finally, the DICE device was developed to both isolate EVs and characterize select EV-Proteins (CD9, Vimentin, EGFR) on-chip. Building off the developed EV characterization methods, EGFR mutations were detected longitudinally in both EV-RNA and EV-Protein from 10 metastatic NSCLC patients. For these patients, identifying the presence of sensitizing (exon 19 del, L858R) and resistance (T790M) EGFR mutations informs sensitivity to tyrosine kinase inhibitors. We demonstrated the presence of exon 19 del and L858R mutations within EV-Protein, marking the first study that demonstrates the presence of these mutations in patient-derived EVs. At the EV-RNA level, exon 19 del mutations were detected in 88% (n=7/8) patients and an increase in mutation burden mirrored disease progression and a decrease mirrored stable disease in 100% (n=5/5) of patients and in 86% (n=12/14) samples. We additionally profiled 2 patients for L858R and T790M mutations, however the detection was more modest at 60% (n=6/10) and 30% (n=3/10) samples, respectively. As such, EV-RNA exon 19 del mutation burden has the potential to inform treatment decisions in a subpopulation of metastatic NSCLC patients. In a cohort of 26 stage III NSCLC patients, CTCs were isolated from all 26 patients using the microfluidic graphene oxide (GO) chip at six timepoints during radiation and immunotherapy therapies. Significantly, it was found that having a decrease in CTCs of less than 75% between pre-treatment and week 4 of radiation therapy is predictive of significantly shorter progression free survival time, 7 months vs 21 months stable monitoring time (p=0.005, log-rank test). Additionally, evaluation of PD-L1 expression on the CTCs demonstrated that having a higher proportion of PD-L1 CTCs before starting treatment was a potential indicator of metastatic potential (p=0.057, log-rank test). Finally, microarray mRNA analysis demonstrated that CTCs develop a more aggressive, proliferative phenotype during radiation treatment. Finally, to assess the heterogeneity of EV biogenesis for applications in NK-EV therapeutics, the droplet microfluidic CellMag-CARWash system was adapted to isolate single NK-92MI cells bound with anti-CD56 Dynabeads. It was found that the CellMag-CARWash isolates cells that have 3+ Dynabeads attached, with an overall efficiency of 58% ± 7 (n=4), while calculations indicate that 1.8 beads should be needed to isolate cells. From a mixed cell population, the CellMag-CARWash isolated NK cells with 95% ± 2 (n=4) purity, achieving an isolation efficiency of 42% ± 14 (n=3). Prolonged droplet stability was demonstrated, and cell viability is 50% after 24 hours in droplets. Taken together, these technological advancements represent necessary developments to move liquid biopsies from the lab to the clinic. These novel isolation and characterization strategies will need to continue to be tested in pilot cohorts and validated in larger cohorts.
... By a Gaussian dual beam trap cells are deformed non-invasively and the relative deformation is measured in a step stress creep experiment [24,[28][29][30][31][32][33][34]. The OS works with a low pulling stress at the order of one Pa, stretching the cells by a few percent over the duration of several seconds, similar to the deforming blood flow [35,36]. Cells exhibit passive viscoelastic and active mechanical properties. ...
Article
Full-text available
Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.
... Cases of gastric cancer with CD44 + in CK + epithelial CTCs are significantly more common among patients with distant metastases and associated with shorter survival than that observed in the case of patients with CK + CD44 − CTCs [71]. NSCLC patients with an increased ratio of CD133 + CTCs to pan-CK-positive cell type (stem cell-like to epithelial ratio) and those that have mesenchymal N-cadherin-positive CTCs are significantly associated with shortened PFS [116]. The co-expression of the stem cell marker CD133 in hybrid CTCs (CK + , vimentin + , and N-cadherin + ) has also been detected at a high frequency in metastatic breast cancer [117,118]. ...
Article
Full-text available
Metastasis-related events are the primary cause of cancer-related deaths, and circulating tumor cells (CTCs) have a pivotal role in metastatic relapse. CTCs include a variety of subtypes with different functional characteristics. Interestingly, the epithelial–mesenchymal transition (EMT) markers expressed in CTCs are strongly associated with poor clinical outcome and related to the acquisition of circulating tumor stem cell (CTSC) features. Recent studies have revealed the existence of CTC clusters, also called circulating tumor microemboli (CTM), which have a high metastatic potential. In this review, we present current opinions regarding the clinical significance of CTCs and CTM with a mesenchymal phenotype as clinical surrogate markers, and we summarize the therapeutic strategy according to phenotype characterization of CTCs in various types of cancers for future precision medicine.
... The heterogeneity of cell populations can be detected also among CTSCs that display mesenchymal or epithelial phenotypes: in a cohort of 43 NSCLC patients it was detected the presence of both epithelial and mesenchymal CTSCs subpopulations. Cells with mesenchymal features had a phenotype that correlated with stemness and with a more aggressive behavior, including a reduced progression free survival [39]. On the other hand, the phenotypic heterogeneity of CTSCs could encourage the use of multiple markers in order to allow a better caption of all the stem population [40]. ...
Article
Circulating tumour cells (CTCs) were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer (NSCLC). Octamer-binding transcription factor-4 positive (OCT4+) and negative (OCT4−) CTCs were identified and captured by interphase fluorescence in situ hybridisation (iFISH). Single cell whole exome sequencing (WES) was performed and the corresponding bioinformatics data were analysed. OCT4+ cells were successfully detected in peripheral blood collected from all four Stage I lung cancer patients. Moreover, the tumour mutational burden (TMB) values observed for OCT4+ samples from the same patients were slightly smaller than those of the OCT4− samples; the difference was not statistically significant (P> 0.05). Thirteen and six characteristic mutations were found in negative samples and positive samples, respectively. The findings indicate that this methodology provides a potential diagnostic index for the early detection of NSCLC.
Article
Full-text available
OBJECTIVE: The aim of the study was the quantification of circulating tumour cells (CTCs) in differentiated thyroid cancer (DTC) patients before and 6 weeks after radioiodine therapy (RIT). CONTEXT: Circulating tumour cells (CTCs) were described more recently in cancer patients, mostly correlating with poor outcome and advanced metastases. DESIGN: Peripheral blood for identification and quantification of CTC before RIT or/and 6 weeks after RIT was provided by 55 DTC patients that received RIT for remnant tissue ablation. PATIENTS: 13 follicular thyroid cancer (FTC) patients, 31 papillary thyroid cancer (PTC) patients and 11 patients having the follicular variant PTC (FV-PTC) were included. MEASUREMENTS: Peripheral blood mononuclear cells (PBMCs) were isolated and EpCAM-positive CTCs were counted by immune fluorescent staining. RESULTS: A CTC positivity of 31.8% before RIT could be observed. Six weeks after RIT, the CTC positivity was reduced to 13.6%. Paired data at both time points of blood sampling could be gathered for n = 33 DTC patients. These patients had significantly higher CTC numbers before RIT than 6 weeks afterwards (0.27 ± 0.47 vs 0.05 ± 0.15, P = .0215). Additionally, significantly reduced CTC numbers were also demonstratedin pre-RIT CTC-positive patients (0.88 ± 0.43 vs 0.05 ± 0.16, P = .0039). CONCLUSION: Our results indicate a reducing effect on the number of CTCs by RIT. Therefore, CTC enumeration should be considered as efficient tool for treatmentmonitoring during RIT. KEYWORDS: circulating tumour cells, differentiated thyroid cancer, measurement limitations, papillary thyroid cancer, quantification, radioiodine therapy, treatment monitoring
Article
Background Circulating tumor cells (CTCs) have been shown to be heterogeneous. Focusing on the epithelial–mesenchymal transition and perioperative kinetics, we evaluated CTCs with mesenchymal phenotypes as a potential prognostic biomarker for patients with gastric cancer.Methods Peripheral blood was collected from 54 patients with gastric cancer before surgery and at 1 week and 1 month after surgery. CTCs were enriched using density-gradient centrifugation and magnetic-activated cell sorting (negative selection). Cell suspensions were characterized by multi-immunofluorescence staining against cytokeratin and N-cadherin, and by 4′,6′-diamidino-2-phenyldole staining.ResultsCTCs were detected in five patients (17%) with early cancer and 14 patients (56%) with advanced cancer (p < 0.05). In our system, N-cadherin, but not cytokeratin, was expressed in the CTCs of 90% (19/21) of patients. Postoperative recurrence was detected in 10 patients, all of whom had N-cadherin+/cytokeratin−/CD45− CTCs preoperatively. Regarding perioperative kinetics, we divided patients into three risk groups: a high-risk group, with one or more preoperative CTCs and increased CTCs postoperatively; an intermediate-risk group, with one or more preoperative CTCs and decreased CTCs postoperatively; and a low-risk group, with no preoperative CTCs. Recurrence rates were 57% (4/7), 33% (4/12), and 6% (2/35), respectively. The relapse-free survival rate was lower in patients at high risk versus those at intermediate or low risk, for all patients (p = 0.00024) and in patients with advanced cancer (p = 0.00103).ConclusionsN-cadherin is a highly useful marker to detect CTCs lacking cytokeratin, and the perioperative kinetics of CTC numbers is beneficial in risk stratification for survival in patients with gastric cancer.
Article
Full-text available
PURPOSE: The role of adjuvant chemotherapy in patients with locally advanced bladder cancer still remains to be defined. We hypothesized that assessing the gene expression of the chemotherapy response modifiers multidrug resistance gene 1 (MDR1) and excision repair crosscomplementing 1 (ERCC1) may help identify the group of patients benefiting from cisplatin-based adjuvant chemotherapy. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 108 patients with locally advanced bladder cancer, who had been enrolled in AUO-AB05/95, a phase 3trial randomizing a maximum of three courses of adjuvant cisplatin and methotrexate (CM) versus methotrexate, vinblastine, epirubicin, and cisplatin (M VEC), were included in the study. Tumor cells were retrieved by laser-captured microdissection and analyzed for MDR1 and ERCC1 expression using a quantitative real-time reverse transcription-polymerase chain reaction assay. Gene expression levels were correlated with clinical outcomes by multivariate Cox proportional hazards regression analysis. RESULTS: Expressions of MDR1 and ERCC1 were independently associated with overall progression-free survival (P = .001, relative risk = 2.9 and P = .01, relative risk = 2.24, respectively). The correlation of high MDR1 expression with inferior outcome was stronger in patients receiving M-VEC, whereas ERCC1 analysis performed equally in the CM and M-VEC groups. CONCLUSIONS: High MDR1 and ERCC1 gene expressions are associated with inferior outcome after cisplatin-based adjuvant chemotherapy for locally advanced bladder cancer. Prospective studies are warranted to define a role for MDR1 and ERCC1 analysis in individualizing multimodality treatment in locally advanced bladder cancer.
Article
Full-text available
Background: Pt-(GpG) intrastrand crosslinks are the major DNA adducts induced by platinum-based anticancer drugs. In the cell lines and mouse models, the persistence of these lesions correlates significantly with cell damage. Here we studied Pt-(GpG) DNA adducts in circulating tumour cells (CTC) treated with cisplatin in medium upfront to systemic therapy from patients with advanced non-small-cell lung cancer (NSCLC). Methods: Blood was drawn before systemic treatment and the CD45/CD15-depleted fraction of mononuclear cells was exposed to cisplatin, verified for the presence of CTC by pan-cytokeratin (pCK) staining and immunoanalysed for the level of Pt-(GpG) in DNA. Results: Immunostaining for pCK, CD45 and subsequently for Pt-(GpG) adducts in the cisplatin-exposed cells (ex vivo) at different time points depicted distinct differences for adduct persistence in CTC between responders vs non-responders. Conclusion: Pt-(GpG) adducts can be detected in CTC from NSCLC patients and assessing their kinetics may constitute a clinically feasible biomarker for response prediction and dose individualisation of platinum-based chemotherapy. This functional pre-therapeutic test might represent a more biological approach than measuring protein factors or other molecular markers.
Article
Full-text available
Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially the epithelial-mesenchymal transition. Recently, Yu and colleagues showed that shifts in these cell populations correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical characteristics. Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell profiles were analyzed per patient and treatment. We detected a remarkable variation of cells with epithelial, mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier log-rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95% confidence interval = 0.01-2.75). Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization and analysis of patients in this ongoing study seems to be warranted.
Article
Full-text available
Epithelial-mesenchymal transition (EMT) of adherent epithelial cells to a migratory mesenchymal state has been implicated in tumor metastasis in preclinical models. To investigate its role in human cancer, we characterized EMT in circulating tumor cells (CTCs) from breast cancer patients. Rare primary tumor cells simultaneously expressed mesenchymal and epithelial markers, but mesenchymal cells were highly enriched in CTCs. Serial CTC monitoring in 11 patients suggested an association of mesenchymal CTCs with disease progression. In an index patient, reversible shifts between these cell fates accompanied each cycle of response to therapy and disease progression. Mesenchymal CTCs occurred as both single cells and multicellular clusters, expressing known EMT regulators, including transforming growth factor (TGF)–β pathway components and the FOXC1 transcription factor. These data support a role for EMT in the blood-borne dissemination of human breast cancer.
Article
Full-text available
Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.