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Circulating Tumor Cell Composition in Renal Cell Carcinoma

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Purpose: Due to their minimal-invasive yet potentially current character circulating tumor cells (CTC) might be useful as a "liquid biopsy" in solid tumors. However, successful application in metastatic renal cell carcinoma (mRCC) has been very limited so far. High plasticity and heterogeneity of CTC morphology challenges currently available enrichment and detection techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We recently described a method that enables us to identify and characterize non-hematopoietic cells in the peripheral blood stream with varying characteristics and define CTC subgroups that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize feasibility of this approach and its potential usage in clinical studies. Experimental design: Peripheral blood was drawn from 14 consecutive mRCC patients at the West German Cancer Center and CTC profiles were analyzed by Multi-Parameter Immunofluorescence Microscopy (MPIM). Additionally angiogenesis-related genes were measured by quantitative RT-PCR analysis. Results: We detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics at different time-points during anti-angiogenic therapy. The presence and quantity of N-cadherin-positive or CD133-positive CTC was associated with inferior PFS. There was an inverse correlation between high expression of HIF1A, VEGFA, VEGFR and FGFR and the presence of N-cadherin-positive and CD133-positive CTC. Conclusions: Patients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeutic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic and predictive biomarker in mRCC is warranted.
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RESEARCH ARTICLE
Circulating Tumor Cell Composition in Renal
Cell Carcinoma
Ivonne Nel
1,2
, Thomas C. Gauler
1,3
, Kira Bublitz
1
, Lazaros Lazaridis
1
,
André Goergens
4
, Bernd Giebel
4
, Martin Schuler
3,5
, Andreas-Claudius Hoffmann
1,5
*
1Molecular Oncology Risk-Profile Evaluation, Department of Medical Oncology, West German Cancer
Center, University Duisburg-Essen, Essen, Germany, 2ABA GmbH & Co. KG, BMZ2, Dortmund, Germany,
3Department of Radiotherapy, University of Duisburg-Essen, Essen, Germany, 4Institute for Transfusion
Medicine, University Hospital Essen, University Duisburg-Essen, Essen, Germany, 5Department of Medical
Oncology, West German Cancer Center, University Duisburg-Essen, Essen, Germany
These authors contributed equally to this work.
These authors are co-first authors on this work.
*hoffmann@more-oncology.com
Abstract
Purpose
Due to their minimal-invasive yet potentially current character circulating tumor cells (CTC)
might be useful as a liquid biopsyin solid tumors. However, successful application in meta-
static renal cell carcinoma (mRCC) has been very limited so far. High plasticity and hetero-
geneity of CTC morphology challenges currently available enrichment and detection
techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We
recently described a method that enables us to identify and characterize non-hematopoietic
cells in the peripheral blood stream with varying characteristics and define CTC subgroups
that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize
feasibility of this approach and its potential usage in clinical studies.
Experimental Design
Peripheral blood was drawn from 14 consecutive mRCC patients at the West German Can-
cer Center and CTC profiles were analyzed by Multi-Parameter Immunofluorescence
Microscopy (MPIM). Additionally angiogenesis-related genes were measured by quantita-
tive RT-PCR analysis.
Results
We detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics
at different time-points during anti-angiogenic therapy. The presence and quantity of N-cad-
herin-positive or CD133-positive CTC was associated with inferior PFS. There was an
inverse correlation between high expression of HIF1A,VEGFA,VEGFR and FGFR and the
presence of N-cadherin-positive and CD133-positive CTC.
PLOS ONE | DOI:10.1371/journal.pone.0153018 April 21, 2016 1/14
a11111
OPEN ACCESS
Citation: Nel I, Gauler TC, Bublitz K, Lazaridis L,
Goergens A, Giebel B, et al. (2016) Circulating Tumor
Cell Composition in Renal Cell Carcinoma. PLoS
ONE 11(4): e0153018. doi:10.1371/journal.
pone.0153018
Editor: Min-Hsien Wu, Chang Gung University,
TAIWAN
Received: February 2, 2016
Accepted: March 22, 2016
Published: April 21, 2016
Copyright: © 2016 Nel et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The method described in this manuscript is
part of the translational program in two multicenter
trials in mRCC, C-II-008 Inhibitors (TKI) in Metastatic
Renal Cell Carcinoma (mRCC) (BERAT) Trial,
NCT01731158; FLIPPER Phase IV study,
NCT01521715, and is sponsored by a grant from the
Central European Society for Anticancer Drug
Research EWIV (CESAR).
Competing Interests: Dr. Nel, shared first author,
declares that she has no significant competing
Conclusions
Patients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeu-
tic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic
and predictive biomarker in mRCC is warranted.
Introduction
Circulating tumor cells (CTC) are thought to be useful in individualizing and monitoring treat-
ment in patients with solid tumors [1,2]. So far, CTC detection methods consist of enrichment
and subsequent identification mostly with anti-cytokeratin (CK) or epithelial cell adhesion mole-
cule (EpCAM) antibodies [3]. The epithelial-to-mesenchymal transition (EMT) can cause alter-
ation 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 characteristics of
other cell types [4]. Yu and colleagues reported that the process of EMT is reversible during sys-
temic treatment and that each cycle of response and progression associates with switches in the
phenotype of CTC [5]. Since metastatic renal cell carcinoma (mRCC) cells often lack epithelial
differentiation and currently available enrichment and detection techniques are often challenged
by the cellular heterogeneity and plasticity of CTC, only a few reports have been published on the
isolation of CTC in metastatic renal cell carcinoma mRCC [68].
We recently developed a CTC detection method based on multi-parameter immunofluores-
cence microscopy (MPIM) that includes epithelial markers such as CK or EpCAM and cells with
mesenchymal and stem cell-like characteristics. We were able to identify an individual composition
of CTC subtypes as profiles that associate to therapeutic success in hepatocellular carcinoma, non-
small cell lung carcinoma and head and neck squamous carcinoma [913]. In this study, we have
addressed the question whether different types of CTC are identifiable in the peripheral blood of
patients with mRCC and, if so, whether their distribution may serve as a predictor of treatment
response or outcome. Furthermore, we have assessed whether the distribution of these cells corre-
lated to inter-individual differences in the expression of angiogenesis related molecular markers.
In renal cell carcinoma increased understanding of genetics and molecular biology led to suc-
cessful employment of agents targeting the VEGF and mTOR pathways [6]. The resulting plural-
ity of available treatment options is significantly limited by available parameters for a personalized
implementation of these agents. We therefore tested CTC profiles together with gene expression
levels of several candidate markers involved in angiogenesis like vascular endothelial growth factor
A(VEGFA); vascular endothelial growth factor receptor (KDR1 also known as VEGFR); basic
fibroblast growth factor (FGF2); basic fibroblast growth factor receptor 1 (FGFR1); platelet-derived
growth factor alpha (PDGFA) and hypoxia inducible factor 1 alpha (HIF1A) for their association
with response to first-line VEGF-targeted therapy. We then evaluated the association of the indi-
vidual CTC composition with these molecular markers and their combined correlation to treat-
ment outcome. With this approach we wanted to scrutinize whether developing a blood-based
multi-marker panel for personalized treatment of mRCC is warranted.
Materials and Methods
Ethic statement 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
CTC Profiles in Renal Cell Carcinoma
PLOS ONE | DOI:10.1371/journal.pone.0153018 April 21, 2016 2/14
financial, professional or personal interests that might
have influenced the performance or presentation of
the work described in this manuscript. The lab work
was completed and the manuscript was written while
she was employed at M.O.R.E.Molecular Oncology
Risk-Profile Evaluation, Department of Medical
Oncology, West German Cancer Center, University
Duisburg-Essen. Final changes, submission and
revision of the manuscript was processed during her
employment at ABA GmbH & Co. KG, where she is
currently working on a co-operative project. In
accordance with her ethical obligation as a
researcher, she is reporting that she receives salaries
from the company ABA GmbH & Co. KG that may be
affected by the research reported in the enclosed
paper. This does not alter our adherence to PLOS
ONE policies on sharing data and materials.
Department, University of Essen-Duisburg; Germany (12-5047-BO). Prior to application of
systemic anti-angiogenesis treatment 12 out of 14 patients had undergone nephrectomy during
their history of disease. The clinical-pathological characteristics of the patients are listed in
Table 1. Tumor staging was performed according to the criteria of the American Joint Com-
mittee of Cancer (AJCC) [14]. Response Evaluation Criteria in Solid Tumors (RECIST 1.1)
were used to define response or stable disease in patients after receiving 2 cycles of systemic
anti-angiogenesis therapy [15].
Preparation of cell lines
HepG2 cells were purchased from Sigma Aldrich (St. Louis, MO) and used within 6 months
after resuscitation. DNA profile was characterized by the cell bank using Short Tandem Repeat
(STR)-PCR. Gastrointestinal stromal cells (GIST 882) were a kind gift from Sebastian Bauer
(Department of Medical Oncology, University Hospital Essen) and Jonathan A. Fletcher
Table 1. Patient Demographics.
Patients (n = 14)
Demographic No. %
Tumor stage
IV 14 100
Histo
clear cell 12 86
non-clear cell 1 7
papillary 1 7
Grading
G1 2 14
G2 3 21
G3 7 50
Gx 2 14
Age
Median, years 61
Range 3878
Heng Score
good 2 14
intermediate 9 64
poor 3 21
Therapy
Sunitinib 9 64
Pazopanib 3 21
Temsirolimus 2 14
Response
PR 5 36
SD 9 64
PFS (months)
Median 12
Range 332
Abbreviations: SD, stable disease; PR, partial response; G1: well differentiated; G2: moderately
differentiated; G3: poorly differentiated; Gx: Grade cannot be assessed
doi:10.1371/journal.pone.0153018.t001
CTC Profiles in Renal Cell Carcinoma
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(Department of Pathology, Brigham and Women's Hospital/Harvard Medical School, Boston,
MA). GIST882 cells expressed a c-KIT allele with an exon 13 missense mutation, resulting in a
single amino acid substitution, K642E [16] which was frequently tested using DNA
sequencing.
The stably CD133-expressing K562 cell line K562-CD133:IEG was generated by lentiviral-
mediated gene transfer of a CD133/Prominin-1 splice variant s1 (GenBank accession number
AF507034) cDNAIRES-eGFP expression cassette [17]. Briefly, respective lentiviral superna-
tant was produced by cotransfection of HEK293T cells with the plasmids pCL1-CD133-IEG,
pCD/NL-BH and pcoPE [1820] using Jetpei (Polyplus, Illkirch Cedex, France) transfection
reagent. At 16 h post transfection, cells were cultured for 68 h with sodium butyrate (10 mM,
Sigma-Aldrich), and viral supernatant was collected after 48 h. K562 cells were transduced
with lentiviral supernatants. The established cell line was passaged at least 10 times before
experiments. K562 cells originally were obtained from ATCC and regularly tested in NK cell
killing assays and by flowcytometric immunophenotyping. The cell line K562-CD133:IEG was
characterized to express extracellular CD133 by flowcytometric stainings using PE-conjugated
anti-CD133 antibodies (clone AC133; Miltenyi Biotec, Bergisch Gladbach, Germany; S1 Fig).
Preparation of blood samples and CTC enrichment
Duplicates of 20 ml citrated peripheral venous blood were drawn from mRCC patients after
response assessment (2 cycles of therapy) and processed within 24 h after collection. Blood
sample preparation was done as described previously [912]. Briefly, 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
(1600 x g, 20°C, 20 min) the interphase consisting of peripheral blood mononuclear cells
(PBMNC) and CTC was removed and washed. We employed an enrichment strategy consist-
ing of 2 steps (Fig 1). For (i) 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, Waltham, USA). 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 fixated with 96% Eth-
anol. Slides were stored at 4°C until subjected to immunocytochemical staining. For (ii) gene
expression analysis a second sample was prepared in the same manner, but epithelial CTC
were further enriched using anti-EpCAM immunomagnetic beads (Epithelial Enrich, Dyna-
beads, Life Technologies, Carlsbad, USA) resulting in an EpCAM-positive CTC suspension for
molecular analysis.
Identification of CTC subtypes using MPIM
Immunofluorescence staining of epithelial, mesenchymal, stem cell-like and hematopoietic
cells was carried out in the CD45-depleted pre-enriched tumor cell suspension as described
previously [911]. Briefly, the staining method included fixation of the cells in 4.5% parafor-
maldehyde for 15 min, washing in PBS, permeabilization with 1x Perm/Wash Buffer (BD Bio-
sciences, Franklin Lakes, USA) for 10 min, washing in PBS, blocking of unspecific antibody
reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of pri-
mary antibodies (final concentration: 5 μg/ml) either anti-pan-CK guinea pig polyclonal anti-
body (ABIN126062, antibodies-online, Atlanta, GA) and anti-N-cadherin (EPR1792Y) rabbit
monoclonal antibody (20191, Epitomics, Burlingame, CA) or anti-CD133 rabbit polyclonal
antibody (orb18124, biorbyt, Cambridge, UK) for CTC and anti-CD45 (MEM-28) mouse
CTC Profiles in Renal Cell Carcinoma
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monoclonal antibody (ab8216, Abcam, Cambridge, UK) for hematologic cells overnight at 4°C,
wash in 0,1% Tween, binding of secondary antibodies (FITC-conjugated AffiniPure goat anti-
rabbit and Cy3-conjugated AffiniPure goat anti-mouse or AlexaFlour647-conjugated Affini-
Pure F(ab)2 Fragment goat anti-guinea pig; Jackson Immuno Research, Hamburg, Germany)
for 30 min at 37°C, washing in 0,1% Tween. Subsequently, cells were stained with 46-Diami-
dino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO) for 10 min,
mounted with anti-fading medium (Invitrogen) and stored in the dark until evaluation. As
described previously, for each test a control slide with a mixture of PBMNC (CD45-positive,
pan-CK-negative) from a healthy donor spiked with epithelial cells from the hepatocellular car-
cinoma cell line HepG2 (CD45-negative, pan-CK-positive) was treated under the same condi-
tions. GIST 882 cells were used as positive control for the mesenchymal marker N-cadherin.
Stably transduced CD133-expressing K562 cells (K562-CD133:IEG) were used as positive con-
trol for stem cell marker CD133. Microscopic evaluation was carried out using the digital Key-
ence 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 10 visual
fields using a 20x magnification on both slides. Samples from 5 healthy donors were processed
and examined under the same conditions in order to define cut-off values for false-positive
events.
RNA extraction and quantitative RT-PCR
For gene expression analyses epithelial CTC were sequentially enriched by depletion of
hematopoietic cells and subsequent positive selection using anti-EpCAM immunomagnetic
beads as described above [12]. Total RNA was extracted from recovered EpCAM-positive
tumor cells using MagAttract RNA Cell Mini M48 Kits (Qiagen, Hilden, Germany) and King
Fisher mL magnetic particle processor (Thermo Fisher, Waltham, MA) according to the manu-
facturers instructions. Additionally, remaining DNA was removed using RQ1 RNase free
DNase (Promega, Fichtburg, WI). One-step real time RT-PCR (Roche LightCycler 480, Basel,
Fig 1. Basic principle of CTC isolation. 1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC
mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; 8. Anti-
CD45 beads; 9. Anti-EpCAM beads (positive isolation) or anti-CD15 beads (negative isolation); 10. Washing
buffer; 11. EpCAM-bead bound CTC suspension for qRT-PCR; 12i. Depleted bead free cell suspension
containing CTCs for Cellspin and immunofluorescence staining. 12 ii. Depleted bead free cell suspension
containing CD45-/EpCAM- CTC for qRT-PCR.
doi:10.1371/journal.pone.0153018.g001
CTC Profiles in Renal Cell Carcinoma
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Switzerland) was performed using Precision OneStep qRT-PCR Mastermix Kit with SYBR
Green (Primerdesign, Southampton, UK) for gene expression analysis of VEGFA; KDR1
(VEGFR) FGF2; FGFR1; PDGFA and HIF1A (Primerdesign, Southampton, UK). The primers
for reference gene beta-actin (Eurofins MWG, Nantes, France) were as follows: forward: 5-
GAGCGCGGCTACAGCTT-3, reverse: 5-TCCTTAATGTCACGCACGATTT-3. Assays
were performed in triplicates to determine expression levels. Thermal cycling conditions were
10 min at 50°C and 5 min at 95°C for RT and initial denaturation followed by 50 cycles of 95°C
for 10 sec and 60°C for 30 sec. Triplicates of A549-RNA (10ng/μl) were used as internal stan-
dard to control each run. Each primer was validated in a serial dilution of RNA extracted from
the cell line mentioned above.
Statistical analysis
Statistical tests were performed according to previously published studies by our group [9
12,21,22]. Recursive descent partition analysis was used to identify the strongest divisor of all
factors and the most significant split determined by the largest likelihood-ratio chi-square sta-
tistic in relation to clinical response as described previously [22,23]. The associations among
CTC subtypes, gene expression levels and clinical-pathological parameters were tested with
Spearman test for bivariate correlations. Mann-Whitney test for independent samples was
used to compare differences of various factors in distinct subgroups. For gene expression analy-
sis we used Wilcoxon signed rank test to assess whether expression levels differ in cell fractions
after depletion of hematopoietic cells and enrichment of epithelial CTC. To identify potential
independent factors associated with response multivariate regression models along with estab-
lished clinical parameters were used. The Kaplan-Meier method was used to test correlations
of PFS with cell types and gene expressions, respectively. Survival differences between patients
with a high and low cell type ratio were analyzed by the log-rank test. The level of significance
was set to P<0.05. All P values were based on two-sided tests. All statistical analyses were per-
formed using the Software Packages JMP 10.0 Software (SAS, Cary, NC, USA), SPSS for Win-
dows (Version 19.0; SPSS Inc., Chicago, IL) and Medcalc, Version 12.3.0 (Mariakerke,
Belgium).
Results
Immunofluorescence based identification 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 characteristics. There-
fore, we used MPIM for CTC-subtype detection in mRCC patients. Hematopoietic K562 cells
stably transduced with a CD133-IRES-eGFP expression cassette and GIST 882 cells were used
as controls for stem cell (CD133, Fig 2 Row A) and mesenchymal (N-cadherin) marker expres-
sions, respectively. Cells from patient samples that showed a positive nuclear staining with
DAPI, a negative staining for CD45 and a positive staining for pan-CK, N-cadherin or CD133
were captured and considered as tumor cells (Fig 2 Row B). In mRCC blood samples we
detected cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with epi-
thelial properties like CK+/N-cadherin-/CD45- and cells with both characteristics like CK+/N-
cadherin+ (Fig 3). We also detected cells showing both, stem cell-like and epithelial features
such as CD133+/CK+ cells. CTC were enumerated and CTC profiles of each patient were
examined. We scored the total amount of N-cadherin-positive, CK-positive and CD133-posi-
tive cells, and calculated a ratio of mesenchymal to epithelial cells after negative enrichment
using CD45-depletion. We normalized the enumerated potential CTC against the total
PBMNC number detected in the DAPI channel in each visual field and calculated the number
CTC Profiles in Renal Cell Carcinoma
PLOS ONE | DOI:10.1371/journal.pone.0153018 April 21, 2016 6/14
of CTC per 1000 PBMNC. Analysis of samples from healthy donors revealed the following cut-
off values for false positive events per 1000 PBMNC after CD45 depletion: 0.1 CD133+/CK+
cells; 0.1 CD133/CK- cells; 0.03 N-cadherin+/CK+ cells; 0.05 N-cadherin+/CK- cells and a
total of 0.6 CK+ cells (Table 2).
CTC-subtypes and clinical outcome
Recursive descent partition analysis was used to identify associations of CTC subgroups and
outcome related data. The number of mesenchymal N-cadherin+/CK- cells and the amount of
CD133+ cells showed a high correlation with response and progression free survival (PFS).
The Presence of N-cadherin+/CK- cells was significantly, but inversely correlated to PFS
(p = 0.04; r = -0.59). Mann-Whitney test showed that the number of N-cadherin+/CK- was sig-
nificantly lower in responders vs. non-responders (p = 0.05). Kaplan-Meier log-rank test
revealed that an increased number of N-cadherin+/CK- cells (>0.35) significantly correlated to
shortened PFS (7 vs. 15 months; p = 0.03; [HR] = 0.31; CI: 0.061.59; Fig 4A).
The number of CD133+ cells was significantly associated to the presence of N-cadherin
+ cells (p = 0.04), the total amount of N-cadherin+ cells (p = 0.02) and the number of N-cad-
herin+/CK+ cells (p = 0.04). The ratio of N-cadherin+ cells to CK+ cells (mesenchymal to epi-
thelial cell type) increased significantly when CD133+ cells were present (p = 0.05).
Accordingly, we detected a significantly increased number of N-cadherin+ cells in the presence
of CD133+ cells (p = 0.004) and in turn a significantly increased number of CD133+ cells in
the presence of N-cadherin+ cells (p = 0.05, Fig 4B).
Gene expression analysis and correlation to cell subtypes
Gene expression levels of candidate genes were measured in the EpCAM-enriched (CD45-/
EpCAM+) and in the remaining CD45- and EpCAM-depleted (CD45-/EpCAM-) fraction.
Gene expression levels were correlated with clinical parameters, among each other and
Fig 2. CTC detection. A) Positive control consisting of PBMNC mixed with CD133-expressing K562 cells
which were stably transduced with lentiviral vectors endocoding an internal ribosomal entry site (IRES)-
mediated co-expression cassette of CD133 and enhanced green fluorescent protein (eGFP) (K562-CD133:
IEG). Cells were stained with DAPI (nucleus; blue) and for CD133 (pseudo-color red). The antibodies used
for anti-CD133 immunostaining specifically bound to K562-CD133:IEG cells, visualized by Cy3 (red)
counterstaining (40x magnification). B) CTC isolated from mRCC patients stained with DAPI (blue), for pan-
CK (epithelial; red) and for CD45 (hematopoietic; green; 20x magnification). The cell marked with a white
arrow shows a DAPI-positive (blue)/CD45-negative staining and was positive for pan-CK (red) and
subsequently considered as CTC.
doi:10.1371/journal.pone.0153018.g002
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compared between the CD45-/EpCAM- and the CD45-/EpCAM+ fraction to test for genes
that may be independent from CTC subgroups. The FGFR1 mRNA expression was signifi-
cantly increased in the CD45-/EpCAM- fraction in relation to the CD45-/EpCAM+ fraction
(p = 0.03). VEGFA expression was significantly increased in the CD45-/EpCAM+ fraction
compared to the CD45-/EpCAM- fraction (p = 0.004).
There were no significant differences of PDGFA-,VEGFR-,HIF1A -and FGF2 gene expres-
sion levels between the both cell fractions. Expression levels of HIF1A (p = 0.05, r = 0.56),
PDGFA (p = 0.01, r = 0.67) and VEGFR (p = 0.01, r = 0.71) were significantly correlated to
LDH. FGFR1 mRNA expression in the CD45-/EpCAM- fraction was significantly correlated to
response (p = 0.001; r = 0.72). VEGFA was significantly associated to FGFR1,HIF1A,KDR1
(VEGFR),PDGFA and FGF2.Fig 5A depicts the correlation between gene expression levels.
HIF1A and KDR1 (VEGFR) mRNA expression levels were by trend decreased (p = 0.06 and
p = 0.07) in the EpCAM-/CD45-depleted fraction when CD133+ cells were present in the
blood sample (Fig 5B and 5C).
Fig 3. Detection of CTC subtypes. CTC isolated from mRCC patients were stained with DAPI (nucleus;
blue) and for CD45 (hematopoietic; green), pan-CK (epithelial; red) and N-cadherin (mesenchymal; yellow)
on one slide and with DAPI and for CD45, pan-CK and CD133 (stem cell; yellow) on a second slide. 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 N-cadherin+/CK-/CD45-; N-cadherin-/CK
+/CD45-; CD133+/CK+/CD45+ and CD133-/CK+/CD45 (low) cells.
doi:10.1371/journal.pone.0153018.g003
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Discussion
The aim of this feasibility study was to scrutinize whether our previously established method
for detecting CTC subgroups is feasible in mRCC. MPIM-based morphological analysis
revealed a variety of CTC subtypes with epithelial, mesenchymal, stem cell-like or mixed
Table 2. CTC quantification.
Healthy donors
Subgroup No. of positive Amount of cells/1000 PBMNC after enrichment
samples (n) Minimum Maximum Mean SD cut-off
CK+ 12 0.09 0.57 0.19 0.6
CD133+
CD133+/CK+ 6 0.05 0.11 0.02 0.04 0.1
CD133+/CK- 3 0.03 0.11 0.02 0.03 0.1
N-cadherin+
N-cadherin+/CK+ 2 0.02 0.03 0.02 0.01 0.03
N-Cadherin+/CK- 1 0.05 0.05 0.01 0.05
mRCC patients
Subgroup No. of positive Amount of cells/1000 PBMNC after enrichment
samples (n) Minimum Maximum Mean SD
CK+ 9 2.0 23.5 7.2 7.4
CD133+
CD133+/CK+ 5 0.4 6.9 2.7 2.3
CD133+/CK- 4 0.4 2.2 1.2 0.8
N-cadherin+
N-cadherin+/CK+ 4 0.5 7.3 2.9 3.1
N-Cadherin+/CK- 5 0.7 1.2 0.6 0.3
Abbreviations: CK+: Pan-Cytokeratin-positive cells; CD133+: CD13- positive cells; N-cadherin+:N-cadherin-positive cells; CD133+/CK+: CD133-positive
and Pan-Cytokeratin-positive cells; CD133+/CK-: CD133-positive and Pan-Cytokeratin-negative cells; N-cadherin+/CK+: N-cadherin-positive and Pan-
Cytokeratin-positive cells; N-cadherin+/CK-: N-cadherin-positive and Pan-Cytokeratin-negative cells
doi:10.1371/journal.pone.0153018.t002
Fig 4. CTC subtypes were associated to clinical outcome. A) Kaplan-Meier test showed that the number of N-cadherin+/CK- cells was significantly
associated to progression free survival (PFS) of mRCC patients during first-line treatment with anti-angiogenesistherapy (PFS; 7 vs. 15 months; p = 0.03;
[HR] = 0.31; CI: 0.061.59). B) Mann-Whitney test revealed a significantly increased number of CD133+ cells in the presence of N-cadherin+ cells (p = 0.05).
doi:10.1371/journal.pone.0153018.g004
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characteristics such as N-cadherin+/CK-/CD45-; N-cadherin-/CK+/CD45-; CD133+/CK+/
CD45+ and CD133-/CK+/CD45 (low) cells. Analyses of individual CTC profiles indicated that
the presence as well as the number of mesenchymal and stem cell-like CTC was associated to
poor treatment response. The presence of stem cell-like CD133+ cells and the presence of mes-
enchymal N-cadherin+/CK- cells were correlated to a shortened PFS. If CD133+ cells were
detectable, N-cadherin+/CK- cells were likely to be found. Due to technical limitations (stain-
ing on 2 slides) it is impossible to determine whether the close association between N-Cad-
herin+ and CD133+ cells is related to co-expression on the same cell or to different cells.
However, it seems to indicate a link between cells with mesenchymal and stem cell-like charac-
teristics 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 metastasis. The formation
of CSC and the event of EMT is a dynamic process which is triggered by the interaction of vari-
ous cellular signaling pathways such as Hedgehog, Notch, PDGF, Wnt, TGF-β, Akt, and NF-κ
B signaling pathways [24]. Also in other entities such as lung [25] and pancreatic cancer [26]
CSC have been defined by CD133 expression. Recently, Jiang found that some stem cell mark-
ers like CD133 were expressed during EMT of tubular cells in vitro [27]. Mani and colleagues
described a link between EMT and stemness states in breast cancer models [28]. Their results
illustrated a direct connection of less differentiated stem cells with the mesenchymal-appearing
cells generated by EMT. They revealed that cells, that have undergone EMT, behaved similar to
Fig 5. Gene expression analysis. A) Visualization of significant interrelationships between gene expressions. B and C) Mann-Whitney test showed that
mRNA expression levels of HIF1A (B) and KDR1 (VEGFR) (C) were significantly decreased (p = 0.05) in the EpCAM-/CD45- fraction when CD133+ cells were
present in the blood sample.
doi:10.1371/journal.pone.0153018.g005
CTC Profiles in Renal Cell Carcinoma
PLOS ONE | DOI:10.1371/journal.pone.0153018 April 21, 2016 10 / 14
stem cells from normal or neoplastic cell populations. A study by Armstrong and colleagues
showed that more than 80% of CTC in patients with metastatic castration-resistant prostate
cancer co-expressed epithelial proteins such as EpCAM and CK with mesenchymal proteins
including N-cadherin and the stem cell marker CD133 [29]. Furthermore, they found that
more than 75% of CTC from women with metastatic breast cancer co-expressed CK, vimentin
and N-cadherin. Nakajima and colleagues investigated the expression level of EMT markers
such as N-cadherin, E-cadherin and vimentin in pancreatic primary and metastatic tumors.
They reported a correlation of N-cadherin expression with neural invasion and histological
type [30]. Only few reports have been published on CTC in mRCC patients. In a study by Blue-
mke et al. prognostic significance of CTC in mRCC patients was evaluated using density and
immunomagnetic enrichment as well as CK 8 and 18 for CTC detection. They found two dif-
ferent CTC populations. One was CK 8/18-positive the other one was CK-negative and
hematopoietic lineage-negative, but with tumor like morphology. Cell numbers correlated with
the presence of lymph node and distant metastases [7]. Another study using the CellSearch sys-
tem reported the presence of a CTC population with atypical characteristics and a peculiar
gene expression profile, characterized by lack of cytokeratin expression and gain of CD44low
expression [8]. For most epithelial tumors progression towards malignancy is accompanied by
a loss of epithelial differentiation and a shift towards the mesenchymal phenotype, leading to
enhanced cancer cell migration and invasion [31]. According to Gradilone and colleagues, who
characterized CTC for CK and markers of EMT, the gain of mesenchymal markers in CTC is
correlated to patient prognosis [32,33]. Their data showed that the presence of mesenchymal
markers on CTC more accurately predicted a poor prognosis than the expression of CK alone.
Here, we confirmed that the presence of mesenchymal-like cells correlates to survival in mRCC
patients.
Recently, the existence of circulating mesenchymal stem cells (MSC) derived from periph-
eral blood was reported [3436]. It was described that MSC have the ability to migrate from
bone marrow to damaged tissue via the circulating peripheral blood to promote regeneration
[37]. This process may involve hyper stimulation of bone marrow production using granulo-
cyte colony stimulating factor (G-CSF) resulting in the occurrence of a mixture of MSC, hema-
topoetic stem cells and other immature progenitor cells [35,38,39]. Furthermore, the literature
revealed that MSC migrate to and proliferate within tumor sites [40]. In this study we were
able to observe a distinct proportion of cells that stained positive for pan-CK and CD45, a phe-
nomenon already described by Yu and colleagues [41]. The additional CD45+ staining may
not be exclusive for hematopoietic cells, but may hypothetically be acquired during the dor-
mant state in the bone marrow or through effects comparable to trogocytosis, i.e. transfer of
membrane proteins [42]. Even 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 profiles with stem cell-like characteristics.
Interestingly, HIF1A,KDR1 (VEGFR) and VEGFA expression levels were decreased in the
CD45-/EpCAM- fraction in the presence of CD133+ cells with the latter appearing to be a
marker of poor outcome. These findings are in line with a study by Chen and colleagues who
used immunohistochemistry and described that high expression of CD133 was positively asso-
ciated with tumor invasion depth, presence of distant metastasis, advanced TNM stage and
shorter survival in patients with gastric carcinoma [43] Also other markers, such as circulating
entothelial cells (CEC) which were shown to correlate with vascular damage, were investigated,
recently. Gruenwald et al found that in RCC sunitinib treatment was associated with an early
increase of CECs in patients with a prolonged PFS [44]. Due to the fact that CECs express
CD45 and CD133, using our enrichment strategy, they would remain in the CD45-depleted
CTC Profiles in Renal Cell Carcinoma
PLOS ONE | DOI:10.1371/journal.pone.0153018 April 21, 2016 11 / 14
and EpCAM-depleted cell fraction. It is highly likely that mRNA expression was measured not
exclusively in CTC, but also in CEC and other remaining populations such as hematopoietic
cells. To be correct, our analysis should be referred to as profiling of non-hematopoietic cells
rather than CTC. Additional markers and elaborated molecular characterization are required
to ensure that the mRNA expression profiling reflects CTC gene expression instead of host
response. Recently, the development of a novel, OB-cadherin-based method to capture mesen-
chymal CTC was reported [45] and might be relevant for future analysis.
Taken together, our results support the examination of individual CTC profiles during sys-
temic treatment of metastasized renal cell carcinoma to identify new biomarkers for response
and outcome monitoring.
Supporting Information
S1 Fig. Flowcytometric immunophenotyping. Flowcytometric analysis and validation of the
CD133 cell surface expression on K562 cells engineered to express the CD133 splice variant s1
encoded by an IRES-eGFP expression cassette (CD133:IEG). Cells were stained with PE-conju-
gated isotype control or anti-CD133 antibodies.
(TIF)
Author Contributions
Conceived and designed the experiments: AH IN. Performed the experiments: IN LL KB. Ana-
lyzed the data: IN AH TG. Contributed reagents/materials/analysis tools: TG AG BG MS.
Wrote the paper: IN AH.
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CTC Profiles in Renal Cell Carcinoma
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Supplementary resource (1)

... CTC as a potential biomarker in advanced RCC have been evaluated only in a few studies [13,14]. These studies are limited by inclusion of varied patient population or very small sample sizes. ...
... In a pilot study on 14 mRCC patients, Nel et al. showed a significant correlation between baseline CTC numbers before starting antiangiogenic agents and treatment response [13]. CTC detection also had a significant inverse correlation with PFS (P = 0.04, r = À0.59). ...
... a A multivariate Cox regression analysis with a stepwise selection process was used to evaluate the variables predicting progression-free survival with a P value <0.05 to include and exclude variables in the analysis. (P = 0.05) [13]. Although the authors highlighted the prognostic and predictive role of CTC in a small cohort, their study was limited by heterogenous inclusion of both clearcell and nonclear cell RCC. ...
Article
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Purpose: Circulating tumor cells (CTC) have been demonstrated to have prognostic and predictive role in certain human cancers. However, studies exploring their role in metastatic renal cell carcinoma (mRCC) are scarce. We aimed to evaluate the prognostic and predictive role of CTC in mRCC. Materials and methods: In this prospective study, 35 patients with mRCC were analyzed for the presence of CTC before starting tyro-sine kinase inhibitors (TKI). Progression-free and overall survival rates were estimated using the Kaplan-Meier curves and log-rank test. The prediction to TKI therapy was calculated with the response to treatment determined by standard imaging techniques. Results: Outcomes were assessed according to the CTC positivity at baseline, before the patients started TKI for mRCC. At a mean follow up of 12.4 ± 4.1 months, disease progression was noted in 17 patients (48.6%) including 8 deaths (22.9%). CTC positive patients had a significantly lower progression-free survival rate (12.5% vs. 64.1%, respectively; P = 0.009) but not in the overall survival rate (75% vs. 76.3%, respectively; P = 0.88) in the Kaplan−Meier estimation curves. CTC positivity at baseline significantly predicted a poorer response to TKI (87.5% vs. 37.1%, P = 0.01). The multivariate Cox proportional hazards analysis showed that CTC at baseline was the most significant predictor of progression-free survival (hazard ratio 4.17, 95% confidence interval 1.41−11.99, P = 0.01). Conclusions: Baseline CTC detection can be an important prognostic factor of progression-free survival and significant predictor of poor response to TKI in patients with metastatic RCC. 2020 Elsevier Inc. All rights reserved.
... Since the methods are usually conducted with manual pipetting of the gradient layer, they are imprecise, labor-intensive, operatordependent, and often resulting in incomplete CTC extraction and WBC contamination [4]. To overcome the challenges, OncoQuick ® [22,23], RosetteSep TM [24], RareCyte ® [25], Dynabead [26], and other modified studies based on density have been introduced [22]. While they have a critical advantage with marker-and size-independent isolation, they still showed relatively low purity [27] and recovery rate because of the entrapment of CTCs in the leukocyte-RBC complexes [28] as well as manual or partial automation, which makes them difficult as a routine clinical setting. ...
... Since the methods are usually conducted with manual pipetting of the gradient layer, they are imprecise, labor-intensive, operatordependent, and often resulting in incomplete CTC extraction and WBC contamination [4]. To overcome the challenges, OncoQuick ® [22,23], RosetteSep TM [24], RareCyte ® [25], Dynabead [26], and other modified studies based on density have been introduced [22]. While they have a critical advantage with marker-and size-independent isolation, they still showed relatively low purity [27] and recovery rate because of the entrapment of CTCs in the leukocyte-RBC complexes [28] as well as manual or partial automation, which makes them difficult as a routine clinical setting. ...
Article
Understanding cancer heterogeneity is essential to finding diverse genetic mutations in metastatic cancers. Thus, it is critical to isolate all types of CTCs to identify accurate cancer information from patients. Moreover, full automation robustly capturing the full spectrum of CTCs is an urgent need for CTC diagnosis to be routine clinical practice. Methods: Here we report the full capture of heterogeneous CTC populations using fully automated, negative depletion-based continuous centrifugal microfluidics (CCM). Results: The CCM system demonstrated high performance (recovery rates exceeding 90% and WBC depletion rate of 99.9%) across a wide range of phenotypes (EpCAM(+), EpCAM(-), small-, large-sized, and cluster) and cancers (lung, breast, and bladder). Applied in 30 lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations, the system isolated diverse phenotypes of CTCs in marker expression and size, implying the importance of unbiased isolation. Genetic analyses of intra-patient samples comparing cell-free DNA with CCM-isolated CTCs yielded perfect concordance, and CTC enumeration using our technique was correlated with clinical progression as well as response to EGFR inhibitors. Conclusion: Our system also introduces technical advances which assure rapid, reliable, and reproducible results, thus enabling a more comprehensive application of robust CTC analysis in clinical practice.
... In another study, the authors investigated the presence of CTC with epithelial, mesenchymal, stem cell-like, or mixed-cell characteristics at different time points during antiangiogenic therapy [160]. The presence and quantity of N-cadherin-positive or CD133-positive CTC were associated with reduced progression-free survival. ...
... The presence and quantity of N-cadherin-positive or CD133-positive CTC were associated with reduced progression-free survival. In addition, an inverse correlation between high expression of HIF1A, VEGFA, VEGFR, and FGFR and the presence of N-cadherin-positive and CD133-positive CTC was shown [160]. Two CTC subpopulations were identified in the TARIBO trial. ...
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Renal cell carcinoma (RCC) is an increasingly common malignancy that can progress to metastatic renal cell carcinoma (mRCC) in approximately one-third of RCC patients. The 5-year survival rate for mRCC is abysmally low, and, at the present time, there are sparingly few if any effective treatments. Current surgical and pharmacological treatments can have a long-lasting impact on renal function, as well. Thus, there is a compelling unmet need to discover novel biomarkers and surveillance methods to improve patient outcomes with more targeted therapies earlier in the course of the disease. Circulating biomarkers, such as circulating tumor DNA, noncoding RNA, proteins, extracellular vesicles, or cancer cells themselves potentially represent a minimally invasive tool to fill this gap and accelerate both diagnosis and treatment. Here, we discuss the clinical relevance of different circulating biomarkers in metastatic renal cell carcinoma by clarifying their potential role as novel biomarkers of response or resistance to treatments but also by guiding clinicians in novel therapeutic approaches.
... Bluemke et al. [18] discovered that CK-positive CTCs were significantly correlated with inferior overall survival (OS) of patients with renal cancer. Nel et al. [32] found that the presence and quantity of CD133-positive or N-cadherin-positive CTC were associated with poor PFS in 14 patients with renal cancer. In the present study, patients with higher MCTCs, both before and after surgery, were more likely to have a bad clinical outcome during follow-up. ...
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To explore the clinical significance of the perioperative counts of circulating tumor cells (CTCs), mesenchymal CTCs (MCTCs), and CTC- white blood cells (WBCs) in renal cell carcinoma patients. A total of 131 patients with renal cancer who underwent operation excision from our hospital were enrolled. In addition, 20 patients with benign renal diseases were recruited as a control. Blood samples were collected from the 131 patients, before operation and 3 months after surgery. Samples were also obtained simultaneously from the control group. CanPatrol CTC detection technique was used to enrich and identify CTCs, MCTCs, and CTC-WBCs. All enrolled patients were T1-3N0M0. From these, 52 patients with renal cancer underwent radical resection, while other 79 patients underwent nephron-sparing surgery. The positive rate of CTC, MCTC, and CTC-WBC before surgery were 95.4% (125/131), 61.1% (80/131), and 11.5% (15/131), respectively. Preoperative total CTCs, MCTCs, or CTC-WBCs were poorly correlated with patients’ parameters. Preoperative CTC, MCTC, or CTC-WBC showed no association with progression-free survival (PFS). In contrast, postoperative total CTCs (≥6), positive MCTCs, and positive CTC-WBCs significantly correlated with recurrence and metastasis. These results remained independent indicators for worse PFS. In addition, the increased CTC and MCTC count after surgery also correlated with unfavorable PFS. The detection of six or more total CTCs, MCTC, or CTC-WBCs in peripheral blood after surgery might help to identify a subset of patients that have higher recurrent risk than the overall population of patients with at different stages of renal cancer.
... Upregulation of Notch activity in CTCs is associated with breast cancer metastasis to the brain [74]. The presence and quantity of N-cadherin-positive CTCs are linked to inferior PFS of patients with renal cell carcinoma [75]. The molecules and signaling pathways involved in the regulation of EMT are considered attractive therapeutic targets for the prevention of metastasis [76]. ...
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Metastasis is the main cause of cancer death. Metastatic foci are derived from tumor cells that detach from the primary tumor and then enter the circulation. Circulating tumor cells (CTCs) are generally associated with a high probability of distant metastasis and a negative prognosis. Most CTCs die in the bloodstream, and only a few cells form metastases. Such metastatic CTCs have a stem-like and hybrid epithelial-mesenchymal phenotype, can avoid immune surveillance, and show increased therapy resistance. Targeting metastatic CTCs and their progenitors in primary tumors and their descendants, particularly disseminated tumor cells, represents an attractive strategy for metastasis prevention. However, current therapeutic strategies mainly target the primary tumor and only indirectly affect metastasis-initiating cells. Here, we consider potential methods for preventing metastasis based on targeting molecular and cellular features of metastatic CTCs, including CTC clusters. Also, we emphasize current knowledge gaps in CTC biology that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.
... Recent studies have shown that attempts are now being made to use liquid biopsy as an alternative strategy to understand heterogeneity in different kinds of cancer, such as lung [27], breast [28], gastrointestinal [29,30], and colorectal cancers [31]. With the application of liquid biopsy in BC gradually maturing in recent years, numerous studies have shown that liquid biopsy may play an important role in the management of patients with BC at different stages [32][33][34][35]. ...
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Bladder cancer (BC) is a heterogeneous disease that characterized by genomic instability and a high mutation rate. Heterogeneity in tumor may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. A combination of cytology and cystoscopy is the standard methodology for BC diagnosis, prognosis, and disease surveillance. However, genomics analyses of single tumor‐biopsy specimens may underestimate the mutational burden of heterogeneous tumors. Liquid biopsy, as a promising technology, enables analysis of tumor components in the bodily fluids, such as blood and urine, at multiple time points and provides a minimally invasive approach that can track the evolutionary dynamics and monitor tumor heterogeneity. In this review, we describe the multiple faces of BC heterogeneity at the genomic and transcriptional levels and how they affect clinical care and outcomes. We also summarize the outcomes of liquid biopsy in BC, which plays a potential role in revealing tumor heterogeneity. Finally, we discuss the challenges that must be addressed before liquid biopsy can be widely used in clinical treatment.
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Circulating tumor cells (CTCs) have a potential role as the missing renal cell carcinoma (RCC) biomarker. However, the available evidence is limited, and detection methods lack standardization, hindering clinical use. We performed a systematic review on CTC enrichment and detection methods, and its role as a biomarker in RCC. Full-text screening identified 54 studies. Reviewed studies showed wide heterogeneity, low evidence level, and high risk of bias. Various CTC detection platforms and molecular markers have been used, but none has proven to be superior. CTC detection and CTC count seem to correlate with staging and survival outcomes, although evidence is inconsistent. CTC research is still in an exploratory phase, particularly in RCC. Further studies are still necessary to achieve a standardization of techniques, molecular markers, CTC definitions, and terminology. This is essential to ascertain the role of CTCs as a biomarker and guide future liquid biopsy research in RCC.
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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.
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Sensitive detection of circulating tumor cells (CTCs) from patients' peripheral blood facilitates on-demand monitoring of tumor progression. However, clinically significant capture of renal cell carcinoma CTCs (RCC-CTCs) remains elusive due to their heterogenous surface receptor expression. Herein, a novel capture platform is developed to detect RCC-CTCs through integration of dendrimer-mediated multivalent binding, a mixture of antibodies, and biomimetic cell rolling. The nanoscale binding kinetics measured using atomic force microscopy reveal that dendrimer-coated surfaces exhibit an order of magnitude enhancement in off-rate kinetics compared to surface without dendrimers, which translated into cell capture improvements by ∼60%. Selectin-induced cell rolling facilitates surface recruitment of cancer cells, further improving cancer cell capture by up to 1.7-fold. Lastly, an antibody cocktail targeting four RCC-CTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80%. The optimal surface configuration outperforms the conventional assay solely relying on EpCAM, as demonstrated by detecting significantly more CTCs in patients’ samples (9.8 ± 5.1 vs. 1.8 ± 2.0 CTCs mL⁻¹). These results demonstrate that the newly engineered capture platform effectively detects RCC-CTCs for their potential use as tumor biomarkers.
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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.
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Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples. 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed. We were able to detect cells with epithelial properties like CK+/N-cadherin-/CD45- and CK+/CD133-/CD45- as well as cells with mesenchymal features such as N-cadherin+/CK-/CD45- and cells with both characteristics like N-cadherin+/CK+/CD45-. We also observed cells showing stem cell-like features like CD133+/CK-/CD45- and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45-. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1). This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.
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Hematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.
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Circulating tumor cells (CTC) and cancer stem cells (CSC) have been proposed as tools for detection and characterization of disease and individualization of therapy in patients with many solid tumors. Several automated and semi-automated techniques for identification and isolation of these cells from blood have been proposed and reviewed mostly focusing on their feasibility. In this mini review we summarize the recent relevant literature on this topic and discuss the clinical usability of measuring CTC and CSC in peripheral blood in patients with hepatocellular carcinoma (HCC). Besides literature, the basis for this evaluation was the authors’ experience with treating HCC and research experience on CSC and CTC. Few original reports and reviews have been published focusing on CTC and CSC in HCC. Though HCC is one of the five most common malignancies worldwide only recently these cells have come into focus for detection and characterization of this disease that is characterized by high plasticity and malignancy. A focused and prospective validation of the clinical usability of detecting these cells in HCC is still needed, but results seem promising that they may add great benefit for early detection and individualization of therapy.
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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.
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Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and restricted differentiation of hematopoietic progenitors into neutrophils. To clarify the effects of G-CSF on hematopoietic progenitors, we generated transgenic (Tg) mice that had ubiquitous expression of the human G-CSF receptor (hG-CSFR). In clonal cultures of bone marrow and spleen cells obtained from these mice, hG-CSF supported the growth of myelocytic as well as megakaryocytic, mast cell, mixed, and blast cell colonies. Single-cell cultures of lineage-negative (Lin−)c-Kit+Sca-1+ or Sca-1− cells obtained from the Tg mice confirmed the direct effects of hG-CSF on the proliferation and differentiation of various progenitors. hG-CSF also had stimulatory effects on the formation of blast cell colonies in cultures using 5-fluorouracil–resistant hematopoietic progenitors and clone-sorted Lin−c-Kit+Sca-1+ primitive hematopoietic cells. These colonies contained different progenitors in proportions similar to those obtained when mouse interleukin-3 was used in place of hG-CSF. Administration of hG-CSF to Tg mice led to significant increases in spleen colony-forming and mixed/blast cell colony-forming cells in bone marrow and spleen, but did not alter the proportion of myeloid progenitors in total clonogenic cells. These results show that, when functional G-CSFR is present on the cell surface, hG-CSF stimulates the development of primitive multipotential progenitors both in vitro and in vivo, but does not induce exclusive commitment to the myeloid lineage.
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Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood, so called circulating tumor cells (CTCs), for renal cell carcinoma (RCC) patients. 233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained (Bl+) cells with a tumor-like morphology. After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. A significant correlation between the detection of CTCs and positive lymph node status (p < 0.001; Chi-square test) and the presence of synchronous metastases at the time of primary tumor resection (p < 0.02; Chi-square test) was found. In a multivariate Cox's regression hazard model, presence of CK+ CTCs was significantly correlated with poor overall survival for RCC patients (RR=2.3; p < 0.05). The presence of CTCs correlated to lymph node status and presence of synchronous metastases in RCC. It is important to evaluate CK+ and Bl+ tumor cells together to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2011-5215
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Background: Local ablative techniques such as selective internal radiation therapy (SIRT) have become the mainstay of treating hepatocellular carcinoma (HCC) in the bridging-to-transplant and palliative setting. We recently demonstrated that epithelial circulating tumor cells (CTCs) correlate to an unfavorable outcome. We wanted to scrutinize whether molecular markers detected in this specific CTC subgroup may also have clinical implications. Materials & methods: Mononuclear cells and CTCs were isolated from peripheral blood samples using density gradient centrifugation followed by depletion of hematopoietic and enrichment of epithelial (EpCAM(+)) cells employing immunomagnetic beads. The mRNA expression of candidate markers was correlated with response to SIRT in 25 patients using quantitative real-time reverse-transcription PCR. Results: IGFBP1 mRNA expression levels were significantly correlated with time to progression in a Kaplan-Meier log rank test (p = 0.04; 0 vs 4 months) and receiver operating characteristic analysis demonstrated a potential use to predict patients with shortened time to progression (area under the curve: 0.8; 95% CI: 0.44-0.98; p = 0.03). Conclusion: The EpCAM fraction of CTCs may be useful to detect novel molecular markers to individualize treatment decision in patients with HCC.