Article

Detection of Circulating Tumor Cells (CTCs) in
Malignant Pleural Mesothelioma (MPM) with the“Universal” CTC-Chip and An Anti-Podoplanin
Antibody NZ-1.2
Taiji Kuwata 1, Kazue Yoneda 1,*, Masataka Mori 1, Masatoshi Kanayama 1, Koji Kuroda 1, Mika
K. Kaneko 2, Yukinari Kato 2,3 and Fumihiro Tanaka 1
Second Department of Surgery (Chest Surgery), University of Occupational and Environmental Health, 1-1
Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan;
t-kuwata@med.uoeh-u.ac.jp (T.K.); masataka-m@med.uoeh-u.ac.jp (M.M.);
masatoshi-kanayama@med.uoeh-u.ac.jp (M.K.); kuroda-k@med.uoeh-u.ac.jp (K.K.);
ftanaka@med.uoeh-u.ac.jp (F.T.)
2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; k.mika@med.tohoku.ac.jp (M.K.K.);
yukinarikato@med.tohoku.ac.jp (Y.K.)
3 New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi
980-8575, Japan* Correspondence: yoneda@med.uoeh-u.ac.jp; Tel.: +81-93-691-7442
1

Received: 18 February 2020; Accepted: 2 April 2020; Published: 5 April 2020

Abstract: Circulating tumor cell (CTC) is a potentially useful surrogate of micro-metastasis, but
detection of rare tumor cells contaminated in a vast majority of normal hematologic cells remains
technical challenges. To achieve effective detection of a variety of CTCs, we have developed a novel
microfluidic system (CTC-chip) in which any antibody to capture CTCs is easily conjugated. In
previous studies, we employed an antibody (clone E-1) against podoplanin that was strongly
expressed on mesothelioma cells. The CTC-chip coated by the E-1 antibody (E1-chip) provided amodest sensitivity in detection of CTCs in malignant pleural mesothelioma (MPM). Here, to achievea higher sensitivity, we employed a novel anti-podoplanin antibody (clone NZ-1.2). In an
experimental model, MPM cells with high podoplanin expression were effectively captured with
the CTC-chip coated by the NZ-1.2 antibody (NZ1.2-chip). Next, we evaluated CTCs in the
peripheral blood sampled from 22 MPM patients using the NZ1.2-chip and the E1-chip. One or more
CTCs were detected in 15 patients (68.2%) with the NZ1.2-chip, whereas only in 10 patients (45.5%)
with the E1-chip. Of noted, in most (92.3%, 12/13) patients with epithelioid MPM subtype, CTCs
were positive with the NZ1.2-chip. The CTC-count detected with the NZ1.2-chip was significantly
higher than that with the E1-chip (p = 0.034). The clinical implications of CTCs detected with the
NZ1.2-chip will be examined in a future study.
Keywords: circulating tumor cells (CTCs); malignant pleural mesothelioma (MPM); podoplanin

1. Introduction
Circulating tumor cells (CTCs) are tumor cells that shed from solid tumors and circulate in the
peripheral blood. As CTCs play a pivotal role in development of distant metastases, CTCs potentiallyprovide important clinical information such as prognosis and therapeutic effect in a variety ofmalignant tumors [1–4]. However, detection of rare CTCs contaminated in a large number of normal
Cells 2020, 9, 888; doi:10.3390/cells9040888

www.mdpi.com/journal/cells

Cells 2020, 9, 888

2 of 11

hematologic cells remains technical challenges. In fact, a variety of systems for detection of CTCs
have been developed [5–9], but the CellSearch (Merarini Silicon Biosystems, Huntington Valley, PA,USA) is the only approved system for the clinical use. The CellSearch is an automated system inwhich CTCs are immunomagnetically isolated with an antibody against epithelial cell adhesion
molecule (EpCAM) that is abundantly expressed on epithelial tumor cells [10]. Accordingly, CTCs
without EpCAM expression, such as tumor cells undergoing epithelial-mesenchymal transition(EMT) and non-epithelial tumor cells, may not be detected with the CellSearch. In our previous study,
we evaluated CTCs using the CellSearch in patients with malignant pleural mesothelioma (MPM).
CTCs were detected in only one-third (32.7%) patients, which was mainly caused by negative or low
expression of EpCAM on MPM cells [11]. To capture a wide variety of CTCs including tumor cells
without EpCAM expression, we have developed a novel microfluidic system (“universal CTC-chipsystem”) in which any antibody to capture CTCs is easily conjugated [12–16].
Podoplanin, a transmembrane mucin-type sialoglycoprotein, plays an important role in theprenatal development of lymphatic vasculature and lung. In the adulthood, podoplanin is expressedon some normal tissues such as lymphatic vessels and type I alveolar epithelium [17,18]. Podoplanin
is also expressed on a wide variety of malignant tumors [19–25], which may contribute to tumor
progression mainly by promoting aggregation and activation of platelet [26–30]. Podoplanin is a wellknown diagnostic marker of MPM, as mesothelioma cells show high expression of podoplanis
whereas the specific function has yet to be determined [24]. Accordingly, to capture MPM cells, we
coated the CTC-chip with an anti-podoplanin antibody. In our previous studies, we used a common
anti-podoplanin antibody (clone E-1), and MPM cells were effectively captured with the CTC-chip[14–16]. In addition, the CTC-chip provided a superior sensitivity in detection of CTCs in MPM
patients over the CellSearch [16]. In the current study, to achieve a higher sensitivity, we employed anovel and highly-reactive anti-podoplanin antibody (clone NZ-1.2 [23]) to detect CTCs in MPM.
2. Materials and Methods
2.1. Cell Lines
Human epithelioid-subtype MPM cell lines (ACC-MESO-4, ACC-MESO-1, and NCI-H226),
biphasic-subtype MPM cells line (MSTO-211H), and sarcomatoid-subtype MPM cell line (NCI-H28)
were employed. ACC-MESO-4 and ACC-MESO-1 were purchased from the Riken BioResource
Center (Tsukuba, Japan), and the other cell lines were from American Type Culture Collection(ATCC; Manassas, VA, USA). Cells were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc.,
Waltham, MA, USA) at 37 °C and 5% CO2.
2.2. Flow Cytometry for Evaluation of Podoplanin Expression
Cells were incubated with the anti-podoplanin antibody, NZ-1.2 (50 µg/mL) for 60 min at room
temperature. Next, cells were washed with PBS and were incubated with an Alexa Flour 488conjugated goat anti-rat IgG (H + L) (Thermo Fisher Scientific Inc.) diluted at 1:100. Flow cytometricanalysis was performed using the EC800 Cell Analyzer (Sony, Tokyo, Japan) and the FlowJo software(BD Bioscience, Franklin Lakes, NJ, USA). The mean fluorescence intensity (MFI) ratio wasdetermined as the ratio of the mean fluorescein intensity of NZ-1.2 to that of the negative control.
2.3. Preparation of CTC-Chip
The CTC-chip was used after a two-step coating, as described previously [14–16]. Briefly, thechip was first incubated with a base-antibody at the concentration of 200 µg/mL overnight at 4 °C.
Thereafter, the chip was incubated with a capture-antibody (an anti-podoplanin antibody, clone NZ1.2 or clone E-1 [Santa Cruz Biotechnology, Dallas, TX, USA]) for 60 min at room temperature. A
mouse anti-rat IgG antibody (SouthernBiotech, Birmingham, AL, USA) was used as the base antibody
when the chip was coated with the NZ-1.2 antibody (“NZ1.2-chip”), and a goat anti-mouse IgG
antibody (SouthernBiotech) when the chip was coated with the E-1 antibody (“E1-chip”).

Cells 2020, 9, 888

3 of 112.4. Evaluation of Cell-Capture Efficiency
Cell-capture efficiency was evaluated as described previously [14–16]. In brief, 100 tumor cells
labeled with CellTrace CSFE Cell Proliferation kit (Thermo Fisher Scientific Inc.) were suspended in
1 mL of the blood sampled from a healthy volunteer (K.T.). The cell suspension sample was applied
to the CTC-chip, which was monitored and recorded with a fluorescence microscope (CKX41;
Olympus, Tokyo, Japan) and a digital video camera (Sony). The total number of tumor cells applied
to the CTC-chip (N-total) was determined as the number of cells that passed through the inlet of theCTC-chip. The number of captured cells (N-captured) was determined as the number of CFSE-labeledcells remained on the CTC-chip. The cell-capture efficiency was represented as N-captured/N-total.
Experiments were performed in triplicate.
2.5. Clinical Evaluation of CTCs in MPM Patients
The peripheral blood was sampled from patients with pathological diagnosis of MPM, and was
drawn to a sample tube (BD Vacutainer EDTA-2K; Becton, Dickinson and Company, Franklin Lakes,
NJ, USA). A 1 mL of the blood was taken from the tube and was applied to the NZ1.2-chip, andanother 1 mL of the blood taken from the same sample tube was applied to the E1-chip.
Cells captured on the CTC-chip were stained with an anti-cytokeratin (CK) antibody (rabbit
polyclonal; Abcam, Cambridge, MA, USA) followed by incubation with an anti-rabbit IgG antibody
conjugated Alexa Fluor® 594 (Thermo Fisher Scientific Inc.), an anti-CD45 antibody (rat monoclonal,
clone YTH24.5; Abcam) followed by incubation with an anti-rat IgG antibody conjugated Alexa
Fluor® 488 (Thermo Fisher Scientific Inc.), and with Hoechst33342 (Cell Signaling Technology,
Danvers, MA, USA). Each cell with round to oval morphology, a Hoechst33342-positive nucleus,
positive staining for CK in the cytoplasm, and negative staining for CD45 was judged as a CTC [16].
The revised TNM criteria were used to determine clinical stage (c-stage) [16]. This study wasapproved by the Ethics Committee of Medical Research, University of Occupational andEnvironmental Health, Japan (H26-15), and was performed in accordance with the Declaration of
Helsinki.
2.6. Statistical Analysis
The proportions of un-paired categorical data were compared by the chi-square test or the
Fisher’s exact test, and those of paired categorical data were compared by the McNemar’s test.
Continuous data were compared with a non-parametric test (Wilcoxon signed rank test for paired
data, and Mann-Whitney U-test or Kruskal-Wallis test for un-paired data). Differences wereconsidered to be statistically signified for p-value < 0.05. All statistical analysis was performed with
GraphPad PRISM software (GraphPad Software, Inc., La Jolla, CA, USA).
3. Results
3.1. Podoplanin Expression in MPM Cell Lines
All epithelioid MPM cell lines (ACC-MESO-1, ACC-MESO-4 and NCI-H226) showed highpodoplanin expression. In contrast, non-epithelioid MPM cell lines (NCI-H28 and MSTO-211H)
showed no significant podoplanin expression (Figure 1).

Cells 2020, 9, 888

4 of 11

Figure 1. Flow cytometric analysis of podoplanin expression on malignant mesothelioma (MPM) celllines (ACC-MESO-1, ACC-MESO-4, NCI-H226, NCI-H28, and MSTO-211H). The number in the top
right corner in each figure is the mean fluorescein intensity (MFI) ratio.

3.2. Cell-Capture Efficiency of NZ1.2-Chip
First, we examined the optimal concentration of the NZ-1.2 antibody for the CTC-chip using the
ACC-MESO-4 cells with the highest podoplanin expression. The highest capture efficiency wasachieved at the concentration of 5.0 mg/mL (Figure 2), which was adopted as the optimal
concentration of the NZ-1.2 in further experiments.

Figure 2. Cell-capture efficiency of the CTC-chip coated with an anti-podoplanin antibody (NZ-1.2)
at various concentrations in capturing malignant pleural mesothelioma (MPM) cells (ACC-MESO-4).

Next, we examined the efficiency in capturing various MPM cell lines with the NZ1.2-chip.
Epithelioid MPM cell lines (ACC-MESO-1 and NCI-H226) with high podoplanin expression wereeffectively captured, but the cell-capture efficiency for non-epithelioid cell lines (NCI-H28 andMSTO-211H) was low (Figure 3).

Cells 2020, 9, 888

5 of 11

Figure 3. Cell-capture efficiency of the CTC-chip coated with an anti-podoplanin antibody (NZ-1.2)
at the concentration of 5 mg/mL in capturing various malignant pleural mesothelioma (MPM) cells(ACC-MESO-1, NCI-H226, NCI-H28, and MSTO-211H).

3.3. Clinical Evaluation of CTCs in MPM Patients
We sampled the peripheral blood from a total of 22 MPM patients treated between November
2016 and September 2018 (Table 1) for detection of CTCs (Figure 4) at the diagnosis of MPM. One ormore CTCs were detected with either E1-chip or NZ1.2-chip in 19 (86.4%) patients; 6 (27.3%) patientswere positive with E1-chip and positive with NZ1.2-chip, 4 (18.2%) patients were positive with E1chip and negative with NZ1.2-chip, 9 (40.9%) patients were negative with E1-chip and positive with
NZ1.2-chip, and 3 (13.6%) patients were negative with E1-chip and negative with NZ1.2-chip. In total,
CTCs were detected in 10 (45.5%) patients with the E1-chip and in 15 (68.2%) patients with the NZ1.2chip. Of noted, in most (12/13, 92.3%) epithelioid MPM patients, CTCs were detected in the peripheral
blood with the NZ1.2-chip (Table 1).
Table 1. Characteristics of patients and detection of CTCs with the CTC-chip.

Number of patients(percentage)Median(range)Male
Sex
FemaleEpithelioid
Histologic
Biphasic
subtype
Sarcomatoid
I
II
Clinical stage
III
IV
Total of patients
Age

Number of CTC-positive (CTC-count ≥1/mL) patients (percentage)
E1-chip
NZ1.2-chip

68.5 years (54–79)
21 (95.5%)
1 (4.5%)13 (59.1%)
4 (18.2%)
5 (22.7%)
10 (45.5%)
4 (18.2%)
5 (22.7%)
3 (13.6%)
22 (100.0%)

10 (47.6%)
0 (0%)
6 (46.2%)
2 (50.0%)
2 (40.0%)
3 (30.0%)
3 (75.0%)
3 (60.0%)
1 (33.3%)
10 (45.5%)

14 (66.7%)
1 (100%)12 (92.3%)
1 (25.0%)
2 (40.0%)
7 (70.0%)
2 (50.0%)
4 (80.0%)
2 (66.7%)
15 (68.2%)

Cells 2020, 9, 888

6 of 11

Figure 4. Detection of tumor cells (ACC-MESO-4 cells spiked in blood (A) and circulating tumor cells
in patients with malignant pleural mesothelioma (B)) captured on the CTC-chip coated with an antipodoplanin antibody (clone NZ-1.2).

The distribution of CTC-count detected with the NZ1.2-chip and that with the E1-chip in eachsample was shown in Figure 5A. The median CTC-count for the E1-chip and for the NZ1.2-chip were“0” (range, 0–7) and “2” (range, 0–11), respectively, indicating a significantly higher CTC-count wasachieved with the NZ1.2-chip (p = 0.034). The difference in the CTC-count detected with the E1-chipand the NZ1.2-chip was highly significant in epithelioid patients (median CTC-count, 0 and 3,
respectively; p = 0.013) (Figure 5B). In some patients, changes in the CTC-count during treatment weremonitored with the E1-chip and the NZ1.2-chip (Figure 5C). At most points of time, a higher numberof CTCs were detected with the NZ1.2-chip.

(A)

(B)

Cells 2020, 9, 888

7 of 11(C)
Figure 5. (A) The number of circulating tumor cells (CTC-count) detected with the CTC-chip in 1 mLof the peripheral blood sampled from 22 patients with malignant pleural mesothelioma (MPM). The
CTC-chip was coated with an anti-podoplanin antibody, clone E-1 (E1-chip) or clone NZ-1.2 (NZ1.2chip), for capturing tumor cells. A 1 mL of the peripheral blood taken from the same sample tube wasapplied to the E1-chip or the NZ1.2-chip. (B) The number of circulating tumor cells (CTC-count)detected with the CTC-chip in 1 mL of the peripheral blood sampled from 13 patients with epithelioidtype malignant pleural mesothelioma (MPM). (C) Changes in the number of circulating tumor cells(CTC-count) detected with the E1-chip (black lines and dots) or the NZ1.2-chip (red lines and dots)
during treatment in some selected patients with malignant pleural mesothelioma (MPM).

The median CTC-count detected with the NZ1.2-chip was 3 for epithelioid subtype and 0 forbiphasic or sarcomatoid subtype, and the CTC-count was significantly higher in epithelioid MPM
patients than in non-epithelioid patients (Figure 6). The CTC-count was not significantly different
according to clinical stage (Figure 7A), but the CTC-count trended higher in advanced stage (stage
III and IV) among epithelioid patients (p = 0.051) (Figure 7B).

Cells 2020, 9, 888

8 of 11

Figure 6. Distribution of the number of circulating tumor cells (CTC-count) detected with the CTCchip in 1 mL of peripheral blood sampled from patients with malignant pleural mesothelioma (MPM)
according to histologic subtype. The CTC-chip was coated with an anti-podoplanin antibody (clone
NZ-1.2) to capture CTCs.

(A)

(B)

Figure 7. (A) Distribution of the number of circulating tumor cells (CTC-count) detected with the
CTC-chip in 1 mL of peripheral blood sampled form patients with malignant pleural mesothelioma(MPM) according to clinical stage. The CTC-chip was coated with an anti-podoplanin antibody (clone
NZ-1.2) to capture CTCs. (B) Distribution of the number of circulating tumor cells (CTC-count)detected with the CTC-chip in 1 mL of peripheral blood sampled form patients with epithelioid
malignant pleural mesothelioma (MPM) according to clinical stage.

4. Discussion
In this study, we employed a novel antibody (NZ-1.2) with a higher affinity to podoplanin in
capturing MPM cells using the CTC-chip. A number of pathological studies have demonstrated that
podoplanin expression is positive in most cases (80–100%) of epithelioid subtype of MPM [31,32]. In
the current study, epithelioid-type MPM cell lines showed high podoplanin expression (Figure 1),
and were effectively captured on the CTC-chip coated with the NZ-1.2 antibody (cell-capture
efficiency, 62.8% for ACC-MESO-1, 97.9% for ACC-MESO-4, and 97.6% for NCI-H226) (Figure 3). In
previous studies, the cell-capture efficiency achieved by the CTC-chip coated with a common antipodoplanin antibody (E-1) was 84.1% for ACC-MESO-4 and 76.3% for NCI-H226 [16]. Whereas directcomparison of cell-capture efficiency may not be feasible, MPM cells were more effectively captured
on the CTC-chip when coated with the novel anti-podoplanin antibody (NZ-1.2). Podoplanin may bealso expressed on sarcomatoid subtype of MPM, but the positivity seems lower (30–75%) ascompared with epithelioid MPM [31,33,34]. In fact, non-epithelioid MPM cell lines (NCI-H28, andMSTO-211H) showed no significant podoplanin, which were not effectively captured on the NZ1.2chip ( Figure 1; Figure 3).
Next, we showed that the NZ1.2-chip enabled sensitive detection of CTCs in the peripheral
blood of MPM patients. As MPM cells may not express strong EpCAM expression, CTCs cannot beeffectively captured using EpCAM-dependent methods. In fact, the CellSearch provided only amodest sensitivity in detection of CTCs (32.7% [11] and 44.45% [35]). As an EpCAM-independent
detection method, Bobek and coworkers employed a size-based preparation method [36]. They
reported a promising sensitivity (80%, 4/5) in detection of CTCs in MPM patients, but no further
study has been reported. The sensitivity obtained with the NZ1.2-chip (68.2% for all MPM patientsand 93.2% for epithelioid patients) in this study might be promising. In addition, the CTC-countdetected with the NZ1.2-chip was significantly higher than that with the E1-chip, especially for
epithelioid patients (Figure 5A,B). In addition, dynamic changes in the CTC-count evaluated with the
NZ1.2-chip during treatment were documented in some patients (Figure 5C). These results may

Cells 2020, 9, 888

9 of 11indicate that the NZ1.2-chip provides useful clinical information in the diagnosis and decisionmaking of treatment for patients with epithelioid sub-type of MPM.
There are several limitations in this study. First, the number of patients included in thisretrospective study was only 22, which was too small to draw any definitive conclusion, and we areaware that promising results of the CTC-test in the current study are preliminary. We are now
sampling the peripheral blood from newly-diagnosed MPM patients for the CTC-test, which will bepresented in the future. In addition, the CTC-test was performed only once for a single blood sample
in the current study. The reproducibility of the CTC-test should be investigated using multiple
samples obtained from each patient in a future study. To validate the clinical significance of the CTCtest, a multi-institutional prospective study shall be conducted. Second, the clinical impact of the
CTC-test should be examined after a longer follow-up time. To reveal the prognostic and predictive
value of the CTC-count, we are now conducting retrospective and prospective studies of monitoring
the CTC-count in correlation with patient characteristics and clinical course of MPM patients. Third,
the NZ1.2-chip may enable a sensitive detection of CTCs in epithelioid MPM patients, but not in nonepithelioid MPM patients with the sensitivity of 33.3%. In this study, non-epithelioid MPM cells (H28
and 211H) without significant podoplanin expression were not effectively captured with the NZ1.2chip. To overcome the limitation, we are now testing the CTC-chip coated with multiple antibodies
such as anti-EGFR antibody in combination with an anti-podoplanin antibody, as EGFR is highly
overexpressed on MPM including non-epithelioid subtypes [37,38]. Finally, non-malignant cells suchas reactive mesothelial cells may highly express podoplanin, and can be detected in the peripheral
blood. In the current study, we did not validate whether cells captured on the NZ1.2-chip were true
MPM cells. We have just developed immunostaining of cells captured on the CTC-chip, and will
examine loss of nuclear BAP1 expression, which is a useful marker in discrimination between MPM
cells and non-malignant mesothelial cells [39].
In conclusion, the NZ1.2-chip achieved a higher performance in capturing CTCs in MPM. Futurestudies may reveal clinical relevance of CTCs detected with the NZ1.2-chip.
Author Contributions: Conceptualization, K.Y. and F.T.; methodology, T.K.; software, F.T.; validation, K.Y.,
M.M. and M.K.; formal analysis, K.Y.; investigation, K.K.; resources, M.K. and Y.K.; data curation, K.K.;
writing—original draft preparation, T.K.; writing—review and editing, K.Y. and Y.K; visualization, K.Y.;
supervision, F.T.; project administration, K.Y.; and funding acquisition, F.T. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by Grants-in-Aid for Scientific Research from the Japan Society for the
Promotion of Science (16K10697 and 16H01747), the Promotion of Occupational Health for the Post Graduate
Student of the University of Occupational and Environmental Health, Japan, the UOEH Research Grant for
Promotion of Occupational Health, and AMED under Grant Numbers (JP19am0101078, JP19am0101078, andJP19ae0101028).
Acknowledgments: We thank Eri Kawashima for her valuable technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.

2.

3.

Bidard, F.C.; Peeters, D.J.; Fehm, T.; Nolé, F.; Gisbert-Criado, R.; Mavroudis, D.; Grisanti, S.; Generali, D.;
Garcia-Saenz, J.A.; Stebbing, J.; et al. Clinical validity of circulating tumour cells in patients with metastatic
breast cancer: A pooled analysis of individual patient data. Lancet Oncol. 2014, 15, 406–414. doi:
10.1016/S1470-2045(14)70069-5.
Cohen, S.J.; Punt, C.J.; Iannotti, N.; Saidman, B.H.; Sabbath, K.D.; Gabrail, N.Y.; Picus, J.; Morse, M.;
Mitchell, E.; Miller, M.C.; et al. Relationship of circulating tumor cells to tumor response, progression-freesurvival, and overall survival in patients with metastatic colorectal cancer. J. Clin. Oncol. 2008, 26, 3213–
3221. doi: 10.1200/JCO.2007.15.8923. Erratum in: J. Clin. Oncol. 2009, 1923.
de Bono, J.S.; Scher, H.I.; Montgomery, R.B.; Parker, C.; Miller, M.C.; Tissing, H.; Doyle, G.V.; Terstappen,
L.W.; Pienta, K.J.; Raghavan, D. Circulating tumor cells predict survival benefit from treatment inmetastatic castration-resistant prostate cancer. Clin. Cancer Res. 2008, 14, 6302–6309. doi: 10.1158/10780432.CCR-08-0872. Erratum in: Clin. Cancer Res. 2009, 15, 1506.

Cells 2020, 9, 888

4.

5.

6.

7.

8.

9.
10.

11.

12.

13.

14.
15.
16.

17.

18.

19.

20.

21.

10 of 11

Zhang, L.; Riethdorf, S.; Wu, G.; Wang, T.; Yang, K.; Peng, G.; Liu, J.; Pantel, K. Meta-analysis of the
prognostic value of circulating tumor cells in breast cancer. Clin. Cancer Res. 2012, 18, 5701–5710. doi:
10.1158/1078-0432.CCR-12-1587.
Talasaz, A.H.; Powell, A.A.; Huber, D.E.; Berbee, J.G.; Roh, K.H.; Yu, W.; Xiao, W.; Davis, M.M.; Pease, R.F.;
Mindrinos, M.N.; et al. Isolating highly enriched populations of circulating epithelial cells and other rarecells from blood using a magnetic sweeper device. Proc. Natl. Acad. Sci. U S A. 2009, 106, 3970–3975. doi:
10.1073/pnas.0813188106.
Kirby, B.J.; Jodari, M.; Loftus, M.S.; Gakhar, G.; Pratt, E.D.; Chanel-Vos, C.; Gleghorn, J.P.; Santana, S.M.;
Liu, H.; Smith, J.P.; et al. Functional characterization of circulating tumor cells with a prostate-cancerspecific microfluidic device. PLoS ONE. 2012, 7, e35976. doi: 10.1371/journal.pone.0035976.
Gertler, R.; Rosenberg, R.; Fuehrer, K.; Dahm, M.; Nekarda, H.; Siewert, J.R. Detection of circulating tumor
cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res. 2003, 162, 149–
155.
Hofman, V.; Bonnetaud, C.; Ilie, M.I.; Vielh, P.; Vignaud, J.M.; Fléjou, J.F.; Lantuejoul, S.; Piaton, E.; Mourad,N.; Butori, C.; et al. Preoperative circulating tumor cell detection using the isolation by size of epithelial
tumor cell method for patients with lung cancer is a new prognosis biomarker. Clin. Cancer Res. 2011, 17,
827–835. doi: 10.1158/1078-0432.CCR-10-0445.
Park, J.; Kim, B.; Choi, S.K.; Hong, S.; Lee, S.H.; Lee, K.I. An efficient cell separation system using 3Dasymmetric microelectrodes. Lab. Chip. 2005, 5, 1264–1270.
Allard, W.J.; Matera, J.; Miller, M.C.; Repollet, M.; Connelly, M.C.; Rao, C.; Tibbe, A.G.; Uhr, J.W.;
Terstappen, L.W. 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, 6897–6904.
Yoneda, K.; Tanaka, F.; Kondo, N.; Hashimoto, M.; Takuwa, T.; Matsumoto, S.; Okumura, Y.; Tsubota, N.;
Sato, A.; Tsujimura, T.; et al. Circulating tumor cells (CTCs) in malignant pleural mesothelioma (MPM).
Ann. Surg. Oncol. 2014, Suppl 4, S472-480. doi: 10.1245/s10434-013-3399-2.
Ohnaga, T.; Shimada, Y.; Moriyama, M.; Kishi, H.; Obata, T.; Takata, K.; Okumura, T.; Nagata, T.;
Muraguchi, A.; Tsukada, K. Polymeric microfluidic devises exhibiting sufficient capture of cancer cell line
for isolation of circulating tumor cells. Biomed. Microdevices. 2013, 15, 611–616. doi: 10.1007/s10544-013-97757.
Ohnaga, T.; Takei, Y.; Nagata, T.; Shimada, Y. Highly efficient capture of cancer cells expressing EGFR by
microfluidic methods based on antigen-antibody association. Sci. Rep. 2018, 8, 12005. doi: 10.1038/s41598018-30511-9.
Chikaishi, Y.; Yoneda, K.; Ohnaga, T.; Tanaka, F. Capture of mesothelioma cells with a “Universal CTCchip”. Oncol. Rep. 2017, 37, 77–82. doi: 10.3892/or.2016.5235.
Yoneda, K.; Chikaish, Y.; Kuwata, K.; Ohnaga, T.; Tanaka, F. Capture of mesothelioma cells with ‘universal’
CTC-chip. Oncol. Lett. 2018, 15, 2635–2640. doi: 10.3892/ol.2017.7619.
Yoneda, K.; Kuwata, T.; Chikaishi, Y.; Mori, M.; Kanayama, M.; Takenaka, M.; Oka, S.; Hirai, A.; Imanishi,N.; Kuroda, K.; et al. Detection of circulating tumor cells with a novel microfluidic system in malignant
pleural mesothelioma. Cancer Sci. 2019, 110, 726–733. doi: 10.1111/cas.13895.
Schacht, V.; Ramirez, M.I.; Hong, Y.K.; Hirakawa, S.; Feng, D.; Harvey, N.; Williams, M.; Dvorak, A.M.;
Dvorak, H.F.; Oliver, G.; et al. T1alpha/podoplanin deficiency disrupts normal lymphatic vasculatureformation and causes lymphedema. EMBO J. 2003, 22, 3546–3556.
Ramirez, M.I.; Millien, G.; Hinds, A.; Cao, Y.; Seldin, D.C.; Williams, M.C. T1alpha, a lung type I celldifferentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev. Biol.
2003, 256, 61–72.
Kato, Y.; Fujita, N.; Kunita, A.; Sato, S.; Kaneko, M.; Osawa, M.; Tsuruo, T. Molecular identification of
Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors. J. Biol. Chem. 2003,
278, 51599–51605.
Mishima, K.; Kato, Y.; Kaneko, M.K.; Nishikawa, R.; Hirose, T.; Mastutani, M. Increased expression of
podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression. Acta
Neuropathol. 2016, 111, 483–488.
Kato, Y.; Sasagawa, I.; Kaneko, M.; Osawa, M.; Fujita, N.; Tsuruo, T. Aggrus: A diagnostic marker that
distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors. Oncogene 2004, 23, 8552–
8556.

Cells 2020, 9, 888

22.

23.

24.
25.

26.
27.

28.

29.
30.

31.

32.
33.

34.
35.

36.
37.
38.

39.

11 of 11

Kato, Y.; Kaneko, M.; Sata, M.; Fujita, N.; Tsuruo, T.; Osawa, M. Enhanced expression of Aggrus(T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma. Tumour
Biol. 2005, 26, 195–200.
Kato, Y.; Vaidyanathan, G.; Kaneko, M.K.; Mishima, K.; Srivastava, N.; Chandramohan, V.; Pegram, C.;
Keir, S.T.; Kuan, C.T.; Bigner, D.D.; et al. Evaluation of anti-podoplanin rat monocle antibody NZ-1 fortargeting malignant gliomas. Nucl. Med. Biol. 2010, 37, 785–794. doi: 10.1016/j.nucmedbio.2010.03.010.
Kimura, N.; Kimura, I. Podoplanin as a marker for mesothelioma. Pathol. Int. 2005, 55, 83–86.
Abe, S.; Morita, Y.; Kaneko, M.K.; Hanibuchi, M.; Tsujimoto, Y.; Goto, H.; Kakiuchi, S.; Aono, Y.; Huang, J.;
Sato, S.; et al. A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody. J.
Immunol. 2013, 190, 6239–6249. doi: 10.4049/jimmunol.1300448.
Tsuruo, T.; Yamori, T.; Naganuma, K.; Tsukagoshi, S.; Sakurai, Y. Characterization of metastatic clones
derived from a metastatic variant of mouse colon adenocarcinoma 26. Cancer Res. 1983, 43, 5437–5442.
Sugimoto, Y.; Watanabe, M.; Oh-hara, T.; Sato, S.; Isoe, T.; Tsuruo, T. Suppression of experimental lungcolonization of a metastatic variant of murine colon adenocarcinoma 26 by a monoclonal antibody 8F11
inhibiting tumor cell-induced platelet aggregation. Cancer Res. 1991, 51, 921–925.
Wicki, A.; Lehembre, F.; Wick, N.; Hantusch, B.; Kerjaschki, D.; Christofori, G. Tumor invasion in theabsence of epithelial-mesenchymal tran[sition: Podplanin-mediated remodeling of the actin cytoskeleton.
Cancer Cell. 2006, 9, 261–272.
Martin-Villar, E.; Megias, D.; Castel, S.; Yurrita, M.M.; Vilaro, S.; Quintanilla, M. Podoplanin binds ERM
proteins to activate RhoA and promote epithelial-mesenchymeal transition. J. Cell Sci. 2006, 119, 4541–4553.
Kato, Y.; Kaneko, M.K.; Kuno, A.; Uchiyama, N.; Amano, K.; Chiba, Y.; Hasegawa, Y.; Hirabayashi, J.;
Narimatsu, H.; Mishima, K.; et al. Inhibition of tumor cell-induced platelet aggregation using a novel antipodoplanin antibody reacting with its platelet- aggregation-stimulating domain. Biochem. Biophys. Res.
Commun. 2006, 349, 1301–1307.
Husain, A.N.; Colby, T.V.; Ordóñez, N.G.; Allen, T.C.; Attanoos, R.L.; Beasley, M.B.; Butnor, K.J.; Chirieac,
L.R.; Churg, A.M.; Dacic, S.; et al. Guidelines for Pathologic Diagnosis of Malignant Mesothelioma 2017
Update of the Consensus Statement From the International Mesothelioma Interest Group. Arch. Pathol. Lab.
Med. 2018,142, 89-108.
Ordóñez, N.G. D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers ofepithelioid malignant mesothelioma. Hum. Pathol. 2005, 36, 372–380.
Hinterberger, M.; Reineke, T.; Storz, M.; Weder, W.; Vogt, P.; Moch, H. D2-40 and calretinin - a tissue
microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation. Mod.
Pathol. 2007, 20, 248–255.
Padgett, D.M.; Cathro, H.P.; Wick, M.R.; Mills, S.E. Podoplanin is a better immunohistochemical marker
for sarcomatoid mesothelioma than calretinin. Am. J. Surg. Pathol. 2008, 32, 123–127.
Raphael, J.; Massard, C.; Gong, I.Y.; Farace, F.; Margery, J.; Billiot, F.; Hollebecque, A.; Besse, B.; Soria, J.C.;
Planchard, D. Detection of circulating tumour cells in peripheral blood of patients with malignant pleural
mesothelioma. Cancer Biomark. 2015, 15, 151–156.
Bobek, V.; Kacprzak, G.; Rzechonek, A.; Kolostova, K. Detection and cultivation of circulating tumor cells
in malignant pleural mesothelioma. Anticancer Res. 2014, 34, 2565–2569.
Chia, P.L.; Scott, A.M.; John, T. Epidermal growth factor receptor (EGFR)-targeted therapies inmesothelioma. Exp. Opin. Drug Deliv. 2019, 16, 441–451.
Destro, A.; Ceresoli, G.L.; Falleni, M.; Zucali, P.A.; Morenghi, E.; Bianchi, P.; Pellegrini, C.; Cordani, N.;
Vaira, V.; Alloisio, M.; et al. EGFR overexpression in malignant pleural mesothelioma. An
immunohistochemical and molecular study with clinico-pathological correlations. Lung Cancer. 2006, 51,
207–215.
Carbone, M.; Adusumilli, P.S.; Alexander, H.R. Jr.; Baas, P.; Bardelli, F.; Bononi, A.; Bueno, R.; Felley-Bosco,
E.; Galateau-Salle, F.; Jablons, D.; et al. Mesothelioma: Scientific clues for prevention, diagnosis, andtherapy. CA Cancer J. Clin. 2019, 69, 402–429.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).