cells
ArticleTargeted Phototherapy for Malignant Pleural
Mesothelioma: Near-Infrared Photoimmunotherapy
Targeting Podoplanin
Yuko Nishinaga 1,† , Kazuhide Sato 1,2,3, *,† , Hirotoshi Yasui 1 , Shunichi Taki 1 ,Kazuomi Takahashi 1 , Misae Shimizu 1,2 , Rena Endo 1,2 , Chiaki Koike 1,2 , Noriko Kuramoto 2 ,Shota Nakamura 4 , Takayuki Fukui 4 , Hiroshi Yukawa 2,5,6 , Yoshinobu Baba 5,6 ,Mika K. Kaneko 7 , Toyofumi F. Chen-Yoshikawa 4 , Hisataka Kobayashi 8 , Yukinari Kato 7,9and Yoshinori Hasegawa 10
1
2
3
4
5
6
7
8
9
10

*
†

Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
B3-Unit, Advanced Analytical and Diagnostic Imaging Center (AADIC)/Medical Engineering Unit (MEU),
Nagoya University Institute for Advanced Research, Nagoya 466-8550, Japan
Nagoya University Institute for Advanced Research, S-YLC, Nagoya 464-8601, Japan
Department of Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University,
Nagoya 464-8601, Japan
Department of Biomolecular Engineering, Nagoya University Graduate School of Engineering,
Nagoya 464-8601, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892-1088, USA
New Industry Creation Hatchery Center, Tohoku University, Sendai 980-8575, Japan
National Hospital Organization, Nagoya Medical Center, Nagoya 460-0001, Japan
Correspondence: k-sato@med.nagoya-u.ac.jp; Tel.: +81-52-744-2167; Fax: +81-52-744-2176
These authors equally contributed to this work.

Received: 23 March 2020; Accepted: 16 April 2020; Published: 20 April 2020


Abstract: Malignant pleural mesothelioma (MPM) has extremely limited treatment despite a poor
prognosis. Moreover, molecular targeted therapy for MPM has not yet been implemented; thus, a newtargeted therapy is highly desirable. Near-infrared photoimmunotherapy (NIR-PIT) is a recently
developed cancer therapy that combines the specificity of antibodies for targeting tumors with
toxicity induced by the photoabsorber after exposure to NIR-light. In this study, we developed a new
phototherapy targeting podoplanin (PDPN) for MPM with the use of both NIR-PIT and an anti-PDPN
antibody, NZ-1. An antibody–photosensitizer conjugate consisting of NZ-1 and phthalocyanine
dye was synthesized. In vitro NIR-PIT-induced cytotoxicity was measured with both dead cellstaining and luciferase activity on various MPM cell lines. In vivo NIR-PIT was examined in both theflank tumor and orthotopic mouse model with in vivo real-time imaging. In vitro NIR-PIT-induced
cytotoxicity was NIR-light dose dependent. In vivo NIR-PIT led to significant reduction in both tumor
volume and luciferase activity in a flank model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group).
The PDPN-targeted NIR-PIT resulted in a significant antitumor effect in an MPM orthotopic mousemodel (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). This study suggests that PDPN-targeted
NIR-PIT could be a new promising treatment for MPM.
Keywords: podoplanin (PDPN); near-infrared photoimmunotherapy; malignant pleural mesothelioma

Cells 2020, 9, 1019; doi:10.3390/cells9041019

www.mdpi.com/journal/cells

Cells 2020, 9, 1019

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1. Introduction
Malignant pleural mesothelioma (MPM) is a malignant tumor that originates from mesothelial
cells and has an extremely poor prognosis [1–4]. Although MPM is rarer compared to lung cancer,
the number of patients worldwide is predicted to increase in the next decades [5–7]. Malignant pleural
mesothelioma seldom causes metastasis to other organs but has already progressed widely at the timeof diagnosis, and radical surgery is often impossible. However, the clinically available therapies for
MPM are extremely limited with few regimens [3,4,8]. Therefore, the development of new therapies
for unresectable MPM, especially molecularly targeted ones, is highly desirable.
Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in lymphatic
endothelial cells, type I alveolar epithelial cells, and podocytes of the glomeruli [9]. Podoplanin is also
upregulated in various tumors, including MPM, angiosarcomas, chondrosarcomas, osteosarcomas,
germ-cell tumors, gliomas, glioblastomas, and squamous cell carcinomas (SCCs) of the skin, esophagus,
and head and neck. The antibody (D2-40) for PDPN has been used for a long time as a specificpathological diagnostic marker to determine the development of MPM.
In 2006, NZ-1, a rat anti-human PDPN antibody, was established [10]. Podoplanin has a platelet
aggregation-stimulating (PLAG) domain, of which the disialyl-core-1 structure at Thr52 of PLAG-3
is the center of platelet-aggregating activity. NZ-1 recognizes PLAG-2/3, while D2-40 recognizes
PLAG-1/2 [11]. NZ-1 was shown to inhibit platelet aggregation activity of PDPN by inhibiting its
interaction with platelets. NZ-1 has also been shown to have antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) against MPM in vitro and in vivo [12].
NZ-1 has potent ADCC activity using rat natural killer (NK) cells as effector cells and CDC activity
and mediates in vivo therapeutic antitumor activity against MPM in SCID mice. However, generally,
only ADCC or CDC promotes limited antitumor effects, especially solid tumors, to eradicate tumors.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer photo-target therapy
that combines the specificity of intravenously injected antibodies for targeting tumors with toxicity
induced by photoabsorber (IR700) after exposure to NIR-light at 690 nm [13–17]. Once the NIR-lightirradiates the tumor cells, immediate necrosis develops to eradicate the tumor without normal adjacentcells remaining intact [18]. Recently, the unique mechanism of cell death induced by NIR-PIT has
been clarified, in which photochemical reactions change the hydrophilic status of antibody-IR700 intohydrophobic, resulting in the formation of antigen-antibody complexes aggregated on the membrane
into acute cell rupture [19]. This unique cell death makes NIR-PIT a promising new modality for cancer
therapy [20]. Presently, NIR-PIT is under the international phase III clinical trial against locoregional,
recurrent head and neck SCC (HNSCC) (LUZERA-301, NCT03769506) with FDA fast-track approvaland expected to be clinically licensed in a few years.
Among the organs in the human body, the lung and thoracic cavity can most effectively transmit
NIR-light, because it consists of a large amount of air [21–23]. Thus, MPM is a potentially good target
for NIR-PIT. Moreover, since MPM progresses more locally than distant metastasis to other organs,
NIR-PIT could control local MPM with irradiation of the limited thoracic lesion [1].
In this study, we exploited NIR-PIT to develop a new phototherapy for MPM using NZ-1, targeting
PDPN, and evaluated its clinical efficacy in a pleural disseminated tumor model.
2. Materials and Methods
2.1. Study Design
Our research objective was to establish a new treatment for MPM exploiting NIR-PIT. All in vivo
experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animal
Resources of Nagoya University Animal Care and Use Committee (approval #2017-29438, #2018-30096,#2019-31234, #2020-20104). The ethical board of Nagoya University, Clinical Research Committee,
approved the use of materials (resected specimens) from the patients (approval #2017-0487). Patientswere informed and consented to the use of the materials.

Cells 2020, 9, 1019

3 of 172.2. Immunostaining of Surgical Human Resected MPM Samples
We performed immunostaining of PDPN with resected tumors of patients who underwent
surgery at Nagoya University Hospital from April 2004 to December 2015 and were pathologicallydiagnosed with MPM. Sections of 4 µm thickness were made from formalin-fixed, paraffin-embedded
tumor samples and placed on glass slides. We used NZ-1 (rat monoclonal anti-PDPN antibody,
10 µg/mL) as primary antibodies, and immunohistochemical staining was performed using the
VECTASTAIN Elite ABC Kit (rat IgG) (Vector Laboratories, Burlingame, CA, USA). Antigen retrieval
was not performed. Color development was performed with DAB (ImmPACT DAB Substrate, Vector
Laboratories, Burlingame, CA, USA) and hematoxylin. Podoplanin expression was evaluated at 40×,
100×, and 400× magnification with a bright field of microscope. When >10% of the tumor cells were
stained, it was considered PDPN positive.
Additionally, we also performed immunostaining of PDPN with resected tumors of patients who
were pathologically diagnosed with lung SCC and used NZ-1 and D2-40 (D2-40 mouse monoclonal
antibody, Nichirei Biosciences Inc., Tokyo, Japan) as primary antibodies to compare the results of thestaining. For D2-40, immunohistochemical staining was performed using the HRP-polymer secondary
antibody (EnVision+ System HRP Labelled Polymer Anti-Mouse, Agilent, Santa Clara, CA, USA),and antigen retrieval techniques were performed using pH 6 buffer (Epitope Retrieval Solution pH 6,
Leica Biosystems, Nussloch, Germany) and a pressure cooker.
2.3. Reagents
Water-soluble, silicon-phthalocyanine derivative IRDye 700DX NHS ester was purchased from
LI-COR Biosciences (Lincoln, NE, USA). All other chemicals were of reagent grade.
2.4. Synthesis of IR700-Conjugated NZ-1
The NZ-1 (1 mg, 6.8 nmol) was incubated with IR700 NHS ester (LI-COR Biosciences, Lincoln, NE,
USA) (60.226 µg, 30.8 nmol, 10 mmol/L in DMSO) in 0.1 mol/L Na2 HPO4 (pH 8.6) at room temperaturefor 1 h. The mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ,
USA). The protein concentration was determined using a Coomassie Plus protein assay kit (Thermo
Fisher Scientific, MA, USA) by measuring the absorption at 595 nm with spectroscopy (Novaspec
Plus; GE Healthcare, IL, USA). The IR700 concentration was measured by absorption at 689 nm with
spectroscopy to confirm the number of fluorophore molecules conjugated to mAb. The synthesis
was controlled so that an average of three IR700 molecules were bound to a single antibody which issimilar to that in other studies [24–27]. We performed SDS-PAGE as a quality control for the conjugate
according to previous reports [28]. We used diluted NZ-1 as a nonconjugated control. The fluorescentbands were measured with an Odyssey Imager (LI-COR Biosciences) at a 700 nm fluorescence channel.
To confirm the specific binding of NZ-1 to PDPN, an immunoblot with transient overexpression lysateof PDPN was performed. We abbreviate IR700 conjugated to NZ-1 as NZ-1-IR700. The detection ofbinding of NZ-1-IR700 on PDPN was evaluated with immunoblot according to previous reports [21,29].
2.5. Cell Lines
We examined 12 human MPM cell lines in this study. ACC-MESO-1, ACC-MESO-4, Y-MESO-9,
Y-MESO-12, and Y-MESO-14 were established at the Aichi Cancer Research Center Institute [30,31].
MSTO-211H, NCI-H28, NCI-H226, NCI-H2052, NCI-H2373, and NCI-H2452 were purchased from
American Type Culture Collection (ATCC). The 3T3 (mouse fibroblast) cells were also obtained from
ATCC. The cdk4/hTERT-immortalized normal human bronchial epithelial cell line HBEC3 was obtained
from the Hamon Center (University of Texas Southwestern Medical Center, Dallas, TX, USA).

Cells 2020, 9, 1019

4 of 172.6. Cell Culture
The PDPN, GFP, and luciferase-expressing MSTO-211H cells were established by transfecting
RediFect Red-FLuc lentiviral particles (PerkinElmer, Waltham, MA, USA) and PDPN-GFP lentiviral particles(OriGene, Rockville, MD, USA). Luciferase-expressing H2373 cells were also established by transfecting
RediFect Red-FLuc lentiviral particles (PerkinElmer, Waltham, MA, USA). Stable high PDPN, GFP, andluciferase expression was confirmed after >10 passages. We abbreviate PDPN, GFP, and luciferase-expressing
MSTO-211H as MSTO-211H-PDPN-luc-GFP and luciferase-expressing H2373 as H2373-luc.
Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Rockford, IL, USA)supplemented with 10% fetal bovine serum and penicillin (100 IU/mL)–streptomycin (100 mg/mL)(Thermo Fisher Scientific) in tissue culture flasks in a humidified incubator at 37 ◦ C in an atmosphereof 95% air and 5% carbon dioxide.
2.7. Flow Cytometry
To evaluate PDPN expression in cell lines, IR700 fluorescence from cells after incubation with
NZ-1-IR700 was measured using a flow cytometer (Gallios, Beckman Coulter, Brea, CA, USA) andKaluza software (Beckman Coulter). Cells (1 × 105 ) were seeded on 12 well plates and incubated for
8 h at 37 ◦ C. Thereafter, NZ-1-IR700 was added at a concentration of 10 µg/mL and incubated for
12 h. To validate the specific binding of the conjugated antibody, excess antibody (10 µg) was used
before adding NZ-1-IR700 (Block Study). Both 3T3 and HBEC3 were incubated with CD16 antibody(10 µg/mL; CD16/CD32 monoclonal antibody (93), Thermo Fisher Scientific) for 12 h before the assay toinhibit nonspecific binding with cells and Fc lesion of NZ-1, and almost no signals were observed inthese cell lines. Before flow cytometric analysis, the medium was replaced with PBS, and the cells were
peeled from wells with pipetting or scraping.
2.8. Fluorescence Microscopy
To detect the antigen-specific localization of IR700 conjugates, fluorescence microscopy was
performed (A1Rsi; Nikon Instech, Tokyo, Japan). Ten thousand cells were seeded on glass-bottomed
dishes and incubated for 8 h. Then, NZ-1-IR700 was added to the culture medium at 10 µg/mL andincubated at 37 ◦ C for 12 h. The cells were washed with PBS. Propidium iodide (PI, final concentration
2 µg/mL; Thermo Fisher Scientific) was used to detect ruptured cells, which were added 30 min before theobservation. The cells were exposed to NIR-light (64 J/cm2 ), and serial microscopic images were obtained.
2.9. In Vitro NIR-PIT
One hundred thousand cells were seeded on 12 well plates and incubated with NZ-1-IR700 at
10 µg/mL for 12 h at 37 ◦ C. After the medium was replaced with PBS, the cells were irradiated with an
NIR-light-emitting diode, which emitted light at 670–710 nm wavelength (L690-66-60, Ushio-Epitex,
Kyoto, Japan). The actual power density (mW/cm2 ) was measured with an optical power meter (PM100,
Thorlabs, Newton, NJ, USA).
2.10. Cytotoxicity or Phototoxicity Assay
The cytotoxic effects of NIR-PIT with NZ-1-IR700 were determined by luciferase activity andflow cytometry with PI staining. For luciferase activity, 150 µg/mL of D-luciferin-containing media(GoldBio, St. Louis, MO, USA) was administered to PBS-washed cells 24 h after NIR-PIT and analyzed
with a plate reader for detection of their bioluminescence (Powerscan4, BioTek, Winooski, VT, USA).
We evaluated luciferase activity in vitro after NIR-PIT with a few timepoints (1, 6, and 24 h). With thisestimation, we decided to evaluate luciferase activity in vitro at 24 h after irradiation.
For the flow cytometric assay, the cells were peeled with pipetting or scraping at 1 h after treatment
and washed with PBS. The PI was added to the cell suspension (final 2 µg/mL) and incubated at room

Cells 2020, 9, 1019

5 of 17temperature for 30 min before flow cytometry. The PI fluorescence was evaluated on ten thousandcells with FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA).
2.11. Animal and Tumor Models
All in vivo procedures were conducted in compliance with the local Animal Care and Use
Committee of Nagoya University. Eight-to-twelve-week-old female homozygote athymic nude mice
were purchased from Chubu Kagaku Shizai (Nagoya, Japan). During the procedures, the mice wereanesthetized with isoflurane.
To examine the distribution of NZ-1-IR700 in vivo, we prepared a xenograft tumor mouse modelof MSTO-211H-PDPN-luc-GFP in the right dorsum. Six million MSTO-211H-PDPN-luc-GFP cells were
injected subcutaneously into the right dorsum of the mice. The mice received 100 µg of NZ-1-IR700
intravenously via the tail vein at 14 days after cell implantation and analyzed with a fluorescence
imager (Pearl Imager, LI-COR Biosciences) for detecting IR700 fluorescence.
Six million MSTO-211H-PDPN-luc-GFP and H2373-luc cells were injected subcutaneously into
the dorsum of the mice. The greatest longitudinal diameter (length) and greatest transverse diameter(width) were measured with an external caliper. Tumor volumes based on caliper measurements were
calculated using the following formula: tumor volume = length × width2 × 0.5. Tumors that reachedapproximately 100 mm3 in volume were selected for further experiments. Body weight was checked on ascale. For bioluminescence imaging (BLI), D-luciferin (15 mg/mL, 200 µL) was injected intraperitoneally
into mice 13 days after cell implantation and analyzed with IVIS imaging system (PerkinElmer) for
luciferase activity. Then, mice were selected for further study based on tumor size and bioluminescencesignals. The mice were sacrificed with carbon dioxide when the tumor length was >15 mm.
To create a pleural disseminated MPM model, six million MSTO-211H-PDPN-luc-GFP or one
hundred thousand cells H2373-luc cells in PBS were injected into the thoracic cavity through theright intercostal space using a 30 G needle according to a previous study [14]. To prevent lung injury,
the needle could only be inserted 5 mm (a foam styrol stopper prevented deeper insertion). After 4 days,
BLI was performed after D-luciferin (15 mg/mL, 200 µL) was injected intraperitoneally, and mice with
sufficient luciferase activity were selected for further studies.
2.12. Characterization of Mouse Model
Both the subcutaneous bilateral flank model and disseminated pleural model received 100 µg of
NZ-1-IR700. One day after injection, serial images were obtained with a fluorescence imager (Pearl
Imager, LI-COR Biosciences) for detecting IR700 fluorescence with the IVIS imaging system for BLI.
2.13. In Vivo Local PDPN-Targeted NIR-PIT
The day when the tumor cells were injected into the mice was indicated as day 1. The mice withtumors on both dorsal sides were injected with 100 µg of NZ-1-IR700 on day 13, and larger tumors
were irradiated with NIR-light at 50 J/cm2 on day 14 and 100 J/cm2 on day 15.
For evaluation of NIR-PIT effects in the pleural disseminated MPM mouse model, the mice were
injected with 100 µg of NZ-1-IR700 on day 4 and administered at 30 J/cm2 on day 5. The NIR-light wasapplied 15 J/cm2 from two directions transcutaneously followed by serial fluorescence imaging (FLI) and BLI.
2.14. Statistical Analysis
Data are expressed as mean ± SEM from a minimum of three experiments, unless otherwise
indicated. Statistical analyses were performed using a statistical program (GraphPad Prism, GraphPad
Software, San Diego, CA, USA). For two-group comparisons, the Mann–Whitney test or t-test wasused. p-Value < 0.05 indicated a statistically significant difference.

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2.14. Statistical Analysis

3. Results

Data are expressed as mean ± SEM from a minimum of three experiments, unless otherwise
indicated. Statistical analyses were performed using a statistical program (GraphPad Prism,
GraphPad Software, San Diego, CA, USA). For two-group comparisons, the Mann–Whitney test or t3.1. Immunostainingof Surgical Specimens of Human-Resected MPM
test was used. p-Value < 0.05 indicated a statistically significant difference.

To confirm3. Results
the coincidence of the staining between NZ-1 and D2-40, we first checked bothstaining on the same specimens, and no staining difference was detected (Supplementary Materials
3.1. Immunostaining of Surgical Specimens of Human-Resected MPM
Figure S1). Immunostaining for PDPN with NZ-1 was performed on 18 surgical specimens of MPM in
To confirm the coincidence of the staining between NZ-1 and D2-40, we first checked both
our institution (Figure
Of specimens,
18 patients,
had difference
epithelioid
mesothelioma,
andthree had biphasic
staining on1a).
the sameand no15
staining
was detected(Supplementary
Materials
S1). Immunostaining
for PDPN withNZ-1
was performed
on 18
surgical specimens
MPM staining wasmesothelioma. Figure
Sarcomatoid
mesothelioma
wasexcluded.
The
positive
rate of ofPDPN
our institution (Figure 1a). Of 18 patients, 15 had epithelioid mesothelioma, and three had biphasic
86.7% (13/15) inin
epithelioid
mesothelioma and 66.7% (2/3) in biphasic mesothelioma. The positive rate
mesothelioma. Sarcomatoid mesothelioma was excluded. The positive rate of PDPN staining wasof all cases acrossthe(13/15)
tumor
type was
83.3% (15/18)(FigureThis
result wasThealmost86.7%
in epithelioid
mesothelioma
and 66.7%(2/3) in1b).
biphasic
mesothelioma.
positiveconsistent with
rate of all cases across the tumor type was 83.3% (15/18) (Figure 1b). This result was almost consistent
those of previous
studies [32–34].
with those of previous studies [32–34].

Figure 1. Immunostaining of surgical specimens of human resected MPM (malignant pleural
Figure 1. Immunostainingof surgical specimens of human resected MPM (malignant pleural
mesothelioma). (a) Hematoxylin-eosin (HE) and podoplanin (PDPN) staining of epithelioid andmesothelioma). (a) Hematoxylin-eosin (HE) and podoplanin (PDPN) staining of epithelioid andbiphasic MPM specimens. Representative images of both PDPN-positive and PDPN-negative MPM
specimens are shown. The medium column showed the magnified view of the dot frame in left column.
The right side column showed the magnified view of the dot frame in the medium column. (b) Number
and rate of PDPN-positive specimens in each MPM type.

3.2. Conjugation of NZ-1 with IR700DX
The NZ-1-IR700 was qualified with the 700 nm fluorescence signals by SDS-PAGE (Figure 2a).
The specific binding of NZ-1-IR700 to PDPN was evaluated by immunoblotting with a transiently
overexpressed PDPN cell lysate (Figure 2b). Blocking of signals on MSTO-211H-PDPN with theaddition of excess NZ-1 was confirmed (Figure 2c), suggesting that NZ-1-IR700 bound specificallyon PDPN. The NZ-1-IR700 did not bind to 3T3 cells (mouse fibroblasts) or human normal tracheal
epithelia, HBEC3 (Figure 2e). Collectively, NZ-1-IR700 was successfully produced and specificallybinds to PDPN.

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3 of 192.1. Study Design

Figure 2. Conjugation of NZ-1-IR700 and its binding capacity. (a) Successful confirmation of conjugated
Our research objective was to establish a new treatment for MPM exploiting NIR-PIT. All in vivo
NZ-1-IR700experimentswith SDS-PAGE(left, colloidal blue staining; right, fluorescence at 700 nm channel). Diluted
were conducted in compliance with the Guide for the Care and Use of Laboratory
NZ-1 servedAnimal
as a control.
Immunoblotting
withCare
NZ-1-IR700
in PDPN-overexpressing
Resources (b)of Nagoya
University Animal
and Use Committee(approval #2017-29438, cell lysates.
#2018-30096,#2019-31234,
Thedetected.
ethical board
NagoyaUniversity,with
ClinicalResearch
Specific bindingof NZ-1-IR700
to#2020-20104).
PDPDN was(c)ofPrior
incubation
excessNZ-1 (to block
Committee, approved the use of materials (resected specimens) from the patients (approval #2017PDPN) inhibitedthe bindingof NZ-1-IR700
to to
MSTO-211H-PDPN0487). Patientswere informed
and consented
the use of the materials. cells, indicating that NA-1-IR700
binds specifically to PDPN. (d) PDPN expression in various MPM cell lines. Examination of PDPN
expression in MPM cell lines established from Caucasian and Japanese patients with MPM. Almost
all MPM cell lines, except for Y-MESO-12 and MSTO-211H, expressed PDPN and showed IR700
fluorescence. (e) Flow cytometric analysis of NZ-1-IR700 in HBEC (normal bronchial epithelial cells)
and mouse fibroblast 3T3 cells. Unspecific binding to normal or mouse fibroblasts was not detected.

3.3. PDPN Expression in the MPM Cell Lines
To evaluate PDPN expression in a variety of MPM cell lines, IR700 fluorescence signals weredetected with NZ-1-IR700 on ACC-MESO-1, ACC-MESO-4, Y-MESO-9, Y-MESO-12, and Y-MESO-14(Japanese MPM cell lines) and MSTO-211H, NCI-H28, NCI-H226, NCI-H290, NCI-H2052, NCI-H2373,
and NCI-H2452 (Caucasian MPM cell lines) using a flow cytometer. Most MPM cell lines, except for
Y-MESO-12 and MSTO-211H, expressed PDPN and showed IR700 fluorescence (Figure 2d).
These data suggest that PDPN is widely expressed in MPM cell lines across races.

Cells 2020, 9, 1019

8 of 173.4. In Vitro Microscopic Observation of NIR-PIT with NZ-1-IR700 on MPM Cell Lines
To optically monitor the effect of NIR-PIT, we genetically modified MSTO-211H (PDPN-negative
cells) to express luciferase and PDPN; MSTO-211H-PDPN-luc-GFP and H2373 to express luciferase;
H2373-luc (Supplementary Materials Figure S2).
Serial fluorescence microscopic observation of MSTO-211H-PDPN-luc-GFP and H2373-luc
cells was performed before and after NIR-PIT. After NIR-light (64 J/cm2 ) irradiation of both
MSTO-211H-PDPN-luc-GFP and H2373-luc cells, cellular swelling, bleb formation, and increase in the
fluorescence of dead cell staining were observed (Figure 3a). Time-lapse imaging of MSTO-211H-PDPNluc-GFP showed acute morphological changes and an increase in fluorescence of PI staining within
30 min (Supplementary Materials Video S1). A decrease in IR700 fluorescence was also observed after
NIR-light exposure.
To assess specific cell death induced by NIR-PIT with NZ-1-IR700, co-cultures of H2373 (PDPN
positive) and MSTO-211H (PDPN negative) were set and followed by NIR-PIT with NZ-1-IR700(Figure 3b). The H2373 begins to swell immediately after NIR-light irradiation into cell rupture,in contrast, MSTO-211H did not change at all.
These data showed that NIR-PIT with NZ-1-IR700 could selectively destroy PDPN-expressing
cancer cells without damaging nontarget cells.
3.5. In Vitro NIR-PIT Effects with NZ-1-IR700 in MPM Cell Lines
To quantitate in vitro NIR-PIT with NZ-1-IR700, we examined luciferase activity and PI staining
with flow cytometry for various MPM cell lines (H2373-luc, MSTO-211H-PDPN-luc-GFP, ACC-MESO-1,
Y-MESO-9, NCI-H28, and NCI-H226). Based on the incorporation of PI, the percentage of cell death
increased in a light dose-dependent manner, regardless of the cell lines (Figure 3c). Luciferase activity
showed significant decreases in relative light units (RLUs) in NIR-PIT-treated cells. The BLI also
showed a decrease in luciferase activity in a light dose-dependent manner, regardless of the cell lines.
No significant cytotoxicity was observed with NIR-light exposure alone or NZ-1-IR700 alone in either
luciferase activity or PI staining (Figure 3d).
Collectively, these studies confirmed that NIR-PIT with NZ-1-IR700 can cause necrotic cell death,universally for MPM cancer cells with PDPN expression.
3.6. In Vivo Biodistribution of NZ-1-IR700
After injection of NZ-1-IR700 into the subcutaneous flank model, IR700 fluorescence was observedon the whole mouse body and gradually accumulated at the tumor site 2 h after the injection (Figure 4a).
Fluorescence quantification showed that the highest accumulation at the tumor site at day 1, while thehighest target-to-background ratio (TBR) was observed on days 2 and 3. There was no other specificaccumulation of IR700-fluorescence, except for the liver and bladder, which involved the process of
metabolism and excretion (Figure 4b).

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Figure 3. In vitro effects of NIR-PIT with NZ-1-IR700 on MPM cell lines. (a) Microscopic observations

Figure 3. In vitro effects of NIR-PIT with NZ-1-IR700 on MPM cell lines. (a) Microscopic observations
before and after PDPN-targeted NIR-PIT on H2373-luc and PDPN-overexpressing MSTO-211H cells
before and after PDPN-targeted
NIR-PIT on H2373-luc and PDPN-overexpressing MSTO-211H cellswith NIR-light (16 J/cm2). (b) Co-cultured H2373 (PDPN positive) and MSTO-211H (PDPN negative)2 ). (b) Co-cultured H2373 (PDPN positive) and MSTO-211H (PDPN negative)
withwere
NIR-light(16
J/cm
used to assess the selective effect of NIR-PIT with NIR-light (16 J/cm2). Only PDPN-expressing
2 ). Only PDPN-expressing
wereH2373
used toassessthe selective
effect of(c)NIR-PIT
withdamage
NIR-light(16 J/cm
cells
ruptured
after the therapy.
Membraneinducedby PDPN-targeted
NIR-PIT
H2373
cells
ruptured
after
therapy.
(c) usingMembranedamageby PDPN-targeted
NIR-PITwasmeasuredwiththethedeadcell countPI staining
withinduced
flow cytometer
which increased
in a wasmeasuredwiththe deadusing
PI arestaining
withasflow
cytometera mannermannerdependent
oncellthe countlight dose.
Data
presented
means± SEM (nwhich≥ 4, * pincreased< 0.05, ** pin< 0.01,Student’s
Thedose.
cell deathof PDPN-targeted
NIR-PITwasdemonstrated
acrossdependent
ont-test).
the light
Dataeffectare presented
as means± SEM(n universally≥ 4, * p < 0.05,** p < 0.01,
Student’s
the
MPM
cell
lines.
(d)
Luciferase
activityinH2373-luc
andMSTO-211H-PDPN-luc-GFP
cells
wasMPMt-test). The cell death effect of PDPN-targeted NIR-PIT was universally demonstrated across themeasuredas
relative
light
unit
(RLU)
which
also
decreased
inan
NIR-light
dose-dependent
manner.
cell lines. (d) Luciferase activity in H2373-luc and MSTO-211H-PDPN-luc-GFP cells was measured
Data are presented as means ± SEM (n ≥ 4, *p < 0.05, **p < 0.01, Student’s t-test).
as relative
light unit (RLU) which also decreased in an NIR-light dose-dependent manner. Data arepresented as means ± SEM (n ≥ 4, *p < 0.05, **p < 0.01, Student’s t-test).

Cells 2020, 9, 1019
Cells 2020, 9, x FOR PEER REVIEW

10 of 17
12 of 19

4. In biodistribution
vivo biodistribution
of NZ-1-IR700.(a)(a)Representative
Representative images
beforeandandafter
FigureFigure
4. In vivo
of NZ-1-IR700.
images(FLI)(FLI)
beforeafter
intravenous
injectionof
NZ-1-IR700(fluorescence
imager,
700
nm).
The
yellowarrow
indicates
theintravenous injection of NZ-1-IR700 (fluorescence imager, 700 nm). The yellow arrow indicates the tumor
tumor
site. (b) Fluorescenceintensity measurementof theand liver
The target-tosite. (b)Fluorescenceintensity measurementof the tumor
andtumorliver (left).
The (left).
target-to-backgroundbackground ratio of the tumor and liver are indicated (right). Data are presented as mean ± SEM (n =ratio of the tumor and liver are indicated (right). Data are presented as mean ± SEM (n = 3). Fluorescence3). Fluorescence quantification showed that the highest accumulation at the tumor site was noted atquantification showed that the highest accumulation at the tumor site was noted at day 1, while theday 1, while the highest target-to-background ratio (TBR) was observed on days 2 and 3. (c)highest target-to-background ratio (TBR) was observed on days 2 and 3. (c) Characterization of the
Characterization of the pleural disseminated MPM model (MSTO-211H-PDPN-luc-GFP). Ex vivo
pleuralfluorescencedisseminated
MPMofmodel(MSTO-211H-PDPN-luc-GFP).
vivo
fluorescenceimagingof theimaging
the disseminatedpleural model at 1 dayEx
after
NZ-1-IR700
injection
and BLIdisseminatedmodelsystem
at 1 dayNZ-1-IR700
and BLI
withst,the
IVIS imaging
systemwith thepleural
IVIS imaging(ki,after
kidney;sp, spleen;injection
pa, pancreas;
li, liver;stomach;bl, bladder;(ki, kidney; sp, spleen; pa, pancreas; li, liver; st, stomach; bl, bladder; in, intestine; lu, lung; he, heart).
Intravenous injection of NZ-1-IR700 was detected in disseminated tumors.

Cells 2020, 9, x FOR PEER REVIEW

13 of 19

Cells 2020, 9, 1019
in, intestine; lu, lung; he, heart). Intravenous injection of NZ-1-IR700 was detected in disseminatedtumors.

11 of 17

These results suggested that NZ-1-IR700 was specifically distributed in PDPN-expressing tumors
resultssuggestedwasspecifically distributedin PDPN-expressing
and that These
treatments
1 and
2 daysthat
afterNZ-1-IR700
NZ-1-IR700
administration
were suitable.
tumors and that treatments 1 and 2 days after NZ-1-IR700 administration were suitable.

3.7. In Vivo Antitumor Effect of PDPN-Targeted NIR-PIT
3.7. In Vivo Antitumor Effect of PDPN-Targeted NIR-PITThe NIR-PIT regimen and imaging protocol are depicted in Figure 5a.
The NIR-PIT regimen and imaging protocol are depicted in Figure 5a.

FigureIn vivoantitumorof PDPN-targeted
NIR-PIT.
(a) ThePDPN-targeted
NIR-PIT
Figure5. In5.vivo
antitumor
effecteffectof PDPN-targeted
NIR-PIT.
(a) ThePDPN-targeted
NIR-PITregimenregimen
is
shown
in
a
line.
(b)
In
vivo
FLI
and
BLI
of
subcutaneous
bilateralflank
xenograftedmice
is shown in a line. (b) In vivo FLI and BLI of subcutaneous bilateral flank xenografted micemodel.
model. Arrow showed tumor site. Red Arrow tumor was treated with NIR-PIT. (c) Quantitative RLU
Arrow
showed tumor site. Red Arrow tumor was treated with NIR-PIT. (c) Quantitative RLU
demonstrated a significant decrease in PDPN-targeted NIR-PIT-treated tumors (before NIR-PIT = 1)
demonstrated a significant decrease in PDPN-targeted NIR-PIT-treated tumors (before NIR-PIT = 1)(* p < 0.05 versus control, t-test). PDPN-targeted NIR-PIT also leads to significant reductions in tumor(* p < 0.05 versus control, t-test). PDPN-targeted NIR-PIT also leads to significant reductions in tumor
volume (* p < 0.05 versus control, t-test) in a flank model (n = 4 mice in each group). (d) PDPN-targetedvolume (* p < 0.05 versus control, t-test) in a flank model (n = 4 mice in each group). (d) PDPN-targeted
NIR-PIT in the pleural disseminated model. FLI and BLI of the pleural disseminated model are shownon the left. Quantitative RLUs showed that PDPN-targeted NIR-PIT led to significant reductions inluciferase activity (* p < 0.05 versus control, t-test, n ≥ 3 mice in each group).

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To monitor PDPN-targeted antitumor effects induced by NIR-PIT, BLI and FLI were
examined in a mouse xenograft model with a subcutaneous bilateral flank tumor (H2373-luc,
MTSO-211H-PDPN-luc-GFP) model. The IR700 fluorescence disappeared only at the treated site(right) immediately after NIR-PIT, suggesting that the therapy went well (Figure 5b, Supplementary
Materials Figure S4a). The BLI showed that luminescence inside the treated tumor disappeared 1 day
after NIR-PIT (* p < 0.05 versus no NIR-light irradiation tumor (control; NZ-1-IR700 iv only), t-test),
indicating that the antitumor effect was strong. In contrast, the RLU of non-irradiated tumors increased
along with tumor growth. Antitumor effect evaluated by both tumor volume and luciferase activity
showed a significant decrease (* p < 0.05 versus no NIR-light irradiation (control; NZ-1-IR700 iv only),t-test) (Figure 5c, Supplementary Materials Figure S4b).
Therefore, PDPN-targeted NIR-PIT with NZ-1-IR700 demonstrated a significant antitumor effecton the subcutaneous flank tumor model.
3.8. Characterization of the Pleural Disseminated MPM Model
Next, to evaluate the PDPN-targeted NIR-PIT with NZ-1-IR700 in a more precise model,
an orthotopic thoracic disseminated MPM model was established. To confirm the MPM pleuraldisseminated orthotopic model with both MSTO-211H-PDPN-luc-GFP and H2373-luc cells, BLI and
FLI were performed. The implanted thoracic disseminated tumors with NZ-1-IR700 intravenously
injected via the tail vein 1 day before imaging showed both high IR700 fluorescence and luciferase
activities, indicating that intravenously injected NZ-1-IR700 accumulated on the disseminated pleuraltumor sites (Figure 4c, Supplementary Materials Figure S3).
These data suggested that MPM pleural disseminated orthotopic models were successfully establishedand that intravenous injection of NZ-1-IR700 could reach and accumulate on the disseminated tumors.
3.9. In Vivo PDPN-Targeted NIR-PIT Effect on MPM Pleural Disseminated Orthotopic Model
To evaluate the effect on the MPM pleural disseminated orthotopic model with H2373-luc tumor,
PDPN-targeted NIR-PIT with NZ-1-IR700 was performed. After treatment with NIR-PIT, pleuraldisseminated tumors in the model showed a decrease in both IR700 fluorescence and luciferase
activities. While the RLU did not increase in the NIR-PIT-treated groups, RLU in the control group
showed a gradual increase along with tumor growth (* p < 0.05 versus control, t-test) (Figure 5d).
Collectively, these data suggested that NIR-PIT caused significant antitumor effects even in the MPM
pleural disseminated orthotopic model, and the therapy was feasible.
4. Discussion
This study established a novel molecular targeted therapeutic approach targeting PDPN for
MPM. Immunostaining of the MPM-resected specimens showed that approximately 80% of MPM
specimens were positive for PDPN. We also found that a variety of MPM cell lines express PDPN
across races. In vitro NIR-PIT showed high selective cytotoxicity to the various PDPN-positive MPM
cell lines. We also demonstrated the antitumor effect on orthotopic MPM mice model with the
combination of NZ-1 and NIR-PIT technology. The therapy is feasible and can be performed withrepeated NIR-light irradiation.
The only standard chemotherapy for MPM is cisplatin + pemetrexed (CDDP + PEM) combination
therapy. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor(VEGF), has been shown to prolong the median overall survival (OS) from 16.1 to 18.8 months andprogression-free survival from 7.3 to 9.2 months when used in combination with CDDP + PEM in thephase III MAPS (NCT00651456) [35]. Additional anti-angiogenic drugs, such as cediranib (anti-VEGF
receptor and platelet-derived growth factor (PDGF) receptor inhibitor), nintedanib (anti-VEGFR,PDGFR, and fibroblast growth factor (FGF) receptor inhibitor), axitinib (anti-VEGFR inhibitor),and soferanib (multi-target inhibitor of VEGFR1/2/3, FGFR-1, PDGFR-β, and RAF/cKit pathway) havealso been examined for additive effects; however, they have only limited effects. Recently, the effects

Cells 2020, 9, 1019

13 of 17of immune checkpoint inhibitors (ICIs), including anti-CTLA-4 antibodies and anti-PD-1/PD-L1
antibodies, on various malignant tumors have been reported [36–38]. A combination of ICIs andchemotherapy has been evaluated in several clinical trials. With this progression, patients with MPM
still have few options for therapy.
The development of antibodies targeting PDPN had started decades ago. NZ-1 was developed
as a new anti-human PDPN antibody by immunizing rats with a PLAG domain of PDPN in 2006.
NZ-1 inhibits the platelet aggregation activity of PDPN by suppressing its interaction with platelets.
Many other clones targeting PDPN have also been produced and proved to show some antitumor
effect based on ADCC and CDC. Since anti-human PDPN antibodies including NZ-1 also recognize
normal cells such as lymphatic endothelial cells, a cancer-specific mAb (CasMab) that specificallyrecognizes human PDPN in tumor cells was established in 2014 [39]. When the same glycoprotein isexpressed in cancer and normal cells, CasMab focuses on the difference in the type of glycosylation
and recognizes both peptides and sugar chains simultaneously. With this new technology of antibody
modification, more specific and effective antibodies have emerged.
PDPN has been recently reported to be expressed in not only cancer cells but also cancer-associatedfibroblasts (CAFs) of various malignant tumors [9,40]. PDPN expression in CAFs is thought to becorrelated with poor prognosis and associated with lymph node metastasis and reduced OS in many
malignancies [41,42]. Thus, targeting PDPN has the additional advantage of depleting CAFs which isessential for tumor progression.
NIR-PIT is a selective and novel cancer treatment that is labeled with an antibody and reacts
only at the site irradiated with NIR-light. Thus, the therapy can control spatiotemporal anti-cancer
effects, resulting in an ideal targeted molecular therapy for patients with MPM. The results of a phase
II clinical trial of NIR-PIT in patients with locoregional recurrent HNSCC were reported in American
Society of Clinical Oncology 2019 (55th Annual Meeting of the American Society of Clinical Oncology,
NCT02422979). The overall response rate was 43% of which four had complete response. Based onthese results, a randomized, double-arm, open-label, controlled international phase III clinical trial(LUZERA-301) is now underway. This clinical trial obtained “fast-track” designation from the US FDA
and “SAKIGAKE” designation from Japan Pharmaceuticals and Medical Devices Agency (PMDA),
and this NIR-PIT technology is expected to be in clinical use in a few years.
The detailed cytotoxic mechanism of NIR-PIT has been unclear for several years. A recent study
on the mechanism of NIR-PIT revealed that photochemical ligand reactions trigger rapid necrotic
cell death. This reaction converts the hydrophilic side chains (silanol) of IR700 into hydrophobic
which renders the antibody-IR700 conjugates aggregated on the cell membrane. This photochemical
reaction-induced cell death by NIR-PIT provides therapy uniqueness for acute photonecrosis [19,20].
This new concept of cell death makes NIR-PIT totally different from conventional phototherapies.
There are interesting approaches to treat cancers with antibodies [43]. Firstly, the effect ofradioimmunotherapy (RIT) using the rat-human chimeric antibody NZ-12 has been reported [44].
NZ-12 is applicable for RIT and radiolabeled NZ-12 enhances the antitumor effect. RIT with radiolabeled
NZ-12 significantly suppressed tumor growth and prolonged survival without body weight loss andobvious adverse effects. RIT irradiates β particles (90 Y), thus RIT has to give normal tissue damaged andthe accumulation of the liver caused liver damage. Additionally, linking toxins to antibodies to generate“immunotoxins” has shown some success as an alternative cancer therapy. However, the efficacy isoften dose dependent, as use of immunotoxins has been associated with adverse side effects such as
liver damage [45]. Conversely, the advantage of NIR-PIT is that it is safe; phototherapy is targeted to
the region of interest, and there is no toxicity associated with antibody-IR700 conjugates in the absenceof NIR-light irradiation.
There are some concerns in this study. First, since PDPN is also expressed in lymphatic endothelial
cells, the antibody targeting PDPN also binds to them. To avoid binding to lymphatic endothelial
cells [9], CasMab, which specifically recognizes tumor cells based on the difference in the glycosylationof PDPN between tumor and normal cells, has recently emerged [39]. With the combination of NIR-PIT

Cells 2020, 9, 1019

14 of 17and CasMab technology for PDPN targeting, we could achieve ideal selective cancer cell death targeting
PDPN. Second, it is difficult to irradiate the chest cavity via external light sources. However, in actual
clinical use, we intend to use diffuser light sources to irradiate the entire intrathoracic cavity via thethoracic drainage tube, since we usually do pretreat to discharge the pleural effusion and place thedrainage device (create a hole and place the tube into the cavity) in the chest wall [8]. Thus, we canirradiate the entire thoracic cavity via both the external and internal chest walls. With the device
development in the future, this technology could overcome this difficulty.
MPM is thought to be suitable for NIR-PIT because it tends to locally invade only in the thoracic
cavity and has rare distant metastases [1]. However, in some cases, NIR-PIT has limited therapeutic
effects. In such cases, since the adverse events caused by NIR-PIT are negligible, NIR-PIT could be
repeated or combined with the therapy used with ADCs [46,47], chemotherapy [8], immune checkpoint
inhibitors [48], and anti-regulatory T-cell therapies [49].
Therefore, NIR-PIT targeting PDPN with NZ-1 showed sufficient antitumor effects and wasthought to be feasible. Thus, NIR-PIT targeting PDPN could be a promising new treatment for MPM.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/4/1019/s1, Figure S1:
Immunostaining of surgical specimens of human resected lung squamous cell carcinoma. Figure S2: Confirmation of
the stable expression of luciferase. Figure S3: Characterization of the pleural disseminated MPM model (H2373-luc).
Figure S4: In vivo antitumor effect of PDPN-targeted NIR-PIT on MSTO-211H-PDPN-luc-GFP tumor. Video S1:Time-lapse imaging of MSTO-211H-PDPN-luc-GFP with microscopic observations before and after NIR-PIT for 30 min.
Author Contributions: Y.N. and K.S. mainly conducted all experiments, performed analysis and wrote themanuscript; Y.H., S.T., K.T., M.S., R.E., C.K., N.K., H.Y., Y.B., H.K., and Y.H. conducted analysis; M.K.K. and Y.K.
produced the NZ-1, S.N., T.F., and T.F.C.-Y. conducted surgical operation to gather the specimens; K.S. supervisedand conducted all the project. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partly supported by the Program for Developing Next-generation Researchers (Japan
Science and Technology Agency), KAKEN (18K15923, 17K07299, 19K07705, JSPS), Takeda Science Foundation,
Kowa Life Science Foundation, Aichi Cancer Research Foundation, Toyoaki Scholarship Foundation, Suzuken
Memorial Foundation, Research Grant of the Japan Cancer Society, Research grant of The Nagoya University
Medical Association, Foundation for Promotion of Cancer Research, Project Mirai Cancer Research Grant, Chukyo
Choju Research Grant, Nagoya-Igaku Shinkokai, and AMED under Grant Numbers: JP19am0401013 (Y.K.),
JP19am0101078 (Y.K.), and JP19ae0101028 (Y.K.). Funders only provided funding, and had no role in the study
design, data collection, data analysis, interpretation, and writing of the report.
Acknowledgments: We thank the nano-platform at Nagoya University and core technical staff in Nagoya University
Equipment Sharing System. MPM cell lines were kindly provided by Yoshitaka Sekido (Aichi Cancer Center).
Conflicts of Interest: The authors declare no conflict of interest.

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