MOLECULAR CANCER RESEARCH | NEW HORIZONS IN CANCER BIOLOGY

Podoplanin Drives Amoeboid Invasion in Canine andHuman Mucosal Melanoma
Masahiro Shinada1, Daiki Kato1, Tomoki Motegi2, Masaya Tsuboi2, Namiko Ikeda1, Susumu Aoki1,
Takaaki Iguchi1, Toshio Li1, Yuka Kodera1, Ryosuke Ota1, Yuko Hashimoto2, Yosuke Takahashi2,
James Chambers3, Kazuyuki Uchida3, Yukinari Kato4,5, Ryohei Nishimura1, and Takayuki Nakagawa1

ABSTRACT◥Introduction
Human mucosal melanoma is the most aggressive subtype ofmelanoma, and most patients develop an incurable metastatic disease,irrespective of surgical removal (1). There is little effective systemic
therapy for human mucosal melanoma, and patients with mucosal
melanoma have worse prognoses than those with common cutaneous
melanoma, with a 5-year survival rate of 25% and 90%, respectively.
Therefore, it is crucial to develop a therapeutic strategy to restrict themetastasis of human mucosal melanoma (1–4). However, humanmucosal melanoma research on the molecular mechanisms of tumor
progression, therapeutic targets, and biomarkers has not advanced
owing to its rarity, accounting for only 1% to 2% of all melanomas, withan incidence of 2.2 per 1,000,000 persons/year (5–7).

1
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Tokyo, Japan. 2Veterinary Medical Center, The
University of Tokyo, Tokyo, Japan. 3Laboratory of Veterinary Pathology,
Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo, Japan. 4Department of Antibody Drug Development, Tohoku University
Graduate School of Medicine, Miyagi, Japan. 5Department of Molecular
Pharmacology, Tohoku University Graduate School of Medicine, Miyagi, Japan.

Corresponding Author: Daiki Kato, Laboratory of Veterinary Surgery, Graduate
School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-Ku, Tokyo 113-8657, Japan. E-mail: adk@g.ecc.u-tokyo.ac.jp
Mol Cancer Res 2023;21:1205–19
doi: 10.1158/1541-7786.MCR-22-0929
2023 American Association for Cancer Researchamoeboid invasion and naturally occurring canine mucosal
melanoma can be a novel research model for podoplanin expressing human mucosal melanoma.
Implications: Podoplanin could be a new therapeutic target torestrict the metastatic dissemination of canine and human mucosal
melanoma.

Canine melanoma is a frequent tumor in dogs, reaching 100,000
diagnoses per year in the United States (8–10). In contrast to humanmelanoma, approximately 60% to 70% of canine melanomas originate
from the gingiva of oral cavities and are classiﬁed as mucosal melanomas (8). Generally, complete surgical removal is performed to
manage the local tumor; however, similar to human mucosal melanoma, local recurrence and distant metastasis frequently occur (8, 10).
Notably, naturally occurring canine mucosal melanomas share many
similarities with human counterpart, including genetic mutations,
histopathologic features, and clinical behavior (8, 11–13). Therefore,
naturally occurring canine mucosal melanoma could be a novel
research model for human mucosal melanoma (14–16).
Canine mucosal melanoma cells were recently found to express
podoplanin, a small transmembrane mucin-like glycoprotein (17, 18).
Podoplanin-targeting cytotoxic antibody therapy has exhibited antitumor efﬁcacy in dogs with mucosal melanoma (17). Overexpressionof podoplanin has been observed in human epithelial tumors, and theintracellular domain of overexpressed podoplanin binds to the exrin/
radixin/moesin (ERM), which activates RhoA, a Rho-GTPase family
protein (19–22). Podoplanin expression is related to the frequency ofmetastasis and poor prognosis of human patients with squamous cell
carcinoma (SCC; refs. 23, 24). A previous report showed that podoplanin promotes tumor growth and migration of human and murine
cutaneous melanoma cells (25). Furthermore, the extracellular domainof podoplanin has been shown to bind to platelets to promote tumor
growth and pulmonary metastasis of human cutaneous melanoma
cells (26). However, the intercellular signaling of podoplanin in canineand human mucosal melanoma cells has not been explored.
Invasive human cutaneous melanoma cells have high cellular
plasticity, shifting from the mesenchymal to the amoeboid mode of

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Mucosal melanoma metastasizes at an early stage of the diseasein human and dog. We revealed that overexpression of podoplanin in tumor invasion fronts (IF) was related to poor prognosis of dogs with mucosal melanoma. Moreover, podoplanin
expressed in canine mucosal melanoma cells promotes proliferation and aggressive amoeboid invasion by activating Rhoassociated kinase (ROCK)-myosin light chain 2 (MLC2) signaling. PDPN-ROCK-MLC2 signaling plays a role in cell-cycle
arrest and cellular senescence escape as a mechanism for regulating proliferation. Podoplanin induces amoeboid invasion in
the IFs of mouse xenografted tumor tissues, similar to canine
mucosal melanoma clinical samples. We further identiﬁed that
podoplanin expression was related to poor prognosis of humanpatients with mucosal melanoma, and human mucosal melanoma with podoplanin-high expression enriched gene signatures
related to amoeboid invasion, similar to canine mucosal melanoma. Overall, we propose that podoplanin promotes canine andhuman mucosal melanoma metastasis by inducing aggressive

Shinada et al.

Materials and Methods
Cell culture
Canine melanoma cell lines, CMM2 and CMM12, were established in our laboratory, as previously reported (18, 33, 34). CMM2
cells were cultured in RPMI1640 (FUJIFILM Wako) supplemented
with 10% heat-inactivated FBS (Serana Europe GmbH) and 5 mg/L
gentamicin (Sigma-Aldrich Corp.). CMM12 cells were cultured
in DMEM/Ham F-12 medium (FUJIFILM Wako) with 10% FBS,
100 U/mL penicillin, and 100 mg/mL streptomycin (FUJIFILM
Wako). Cells were maintained at 37 C in a humidiﬁed atmosphere
containing 5% CO2. Podoplanin knocked out (PDPN-KO) cells
were generated using CRISPR/Cas9. The CRISPR/Cas9 vector was
the pGuide-it-ZsGreen1 vector (Takara Bio Inc.). Plasmid DNA wastransfected using Xfect transfection reagent (Takara Bio Inc.). The
sequence of the single-guide RNA (sgRNA) targeting canine podoplanin was ACCGGGCAGCCGACGAGATG. To generate podoplanin-overexpressed PDPN-KO cells (PDPN-KO-OE), CMM2
PDPN-KO#2 and CMM12 PDPN-KO#2 cells were transfected withthe lentivirus vector plasmid pCSII-CMV-MCS (RIKEN BRC DNA
Bank) encoding canine podoplanin cDNA (NM_001003220.1).
Transfected cells were selected with puromycin (FUJIFILM Wako).
Inhibitors
ROCK (Fasudil, GSK269962A, and Y-27632) and myosin II
inhibitors [(–)-blebbistatin] were purchased from Selleck Chemicals. Concentrations used unless otherwise stated were: 100 and
200 mmol/L Fasudil, 1 and 2 mmol/L GSK269962A, 100 and 200 mmol/L
Y-27632, and 30 and 60 mmol/L (–)-blebbistatin for CMM2 andCMM12, respectively. Unless otherwise stated, the treatment timewas 24 hours. Information of antibodies and reagents are describedin Supplementary Data.
IHC
IHC was performed as described previously (18) and detailed
methods are described in Supplementary File. The IFs and TBs were
evaluated separately. The IFs were deﬁned as melanoma cells with at

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least 50% contact with the matrix (29). Quantitative scores of 0, 1, 2,
3, 4, or 5 were assigned when 0, 1 to 10, 11 to 30, 31 to 50, 51 to 80, or
81% to 100% of the melanoma cells were podoplanin positive, respectively. The staining intensity of podoplanin was rated on a scale of 0
to 3 where 0 ¼ negative, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong.
Raw data were converted to IHC scores by multiplying the quantity
and staining intensity scores (24). An IHC score above the medianvalue was considered indicative of high podoplanin expression.
Morphologic analysis of tumor tissues was performed using hematoxylin and eosin (H&E)-stained sections as described previously.
Cell shape was graded from 0 to 3 (0 ¼ round, 1 ¼ ovoid, 2 ¼
elongated, and 3 ¼ spindly), and a score was assigned as follows: cell
shape score ¼ [(percentage of cells; % shape 0  0) þ (% shape 11)
þ (% shape 22) þ (% shape 33)], with values ranging from 0(all cells round) to 300 (all cells spindle). For phosphor-MLC2
staining, three different thresholds were applied according to theintensity scores (0, 1, 2, and 3), and a score was assigned as follows:
phosphor-MLC2 score ¼ [percentage of cells; % intensity 00) þ(% intensity 11) þ (% intensity 22) þ (% intensity 33)], withvalues ranging from 0 (no phosphor-MLC2 expression) to 300 (allcells strongly expressed phosphor-MLC2). All evaluations were performed using blinding clinicopathologic features.
Cell proliferation assay
Cells were stained with trypan blue. The cells were manuallycounted, and the live cell count (unstained cells) was determined.
Annexin V/propidium iodide FACS
Detection of apoptosis cells was performed as described previously (18) and detailed methods are described in Supplementary Data.
Cell-cycle analysis
Cell-cycle analysis was performed as previously described (18) anddetailed methods are described in Supplementary Data.
Wound healing assay
A scratch wound was created in the cell monolayer using a 1,000-mL
pipette tip and washed with culture medium to remove ﬂoating cells.
The migrated areas were manually calculated using ImageJ software(https://imagej.nih.gov/ij; ref. 35).
Transwell invasion assay
Transwell invasion assay was performed as described previously (18) and detailed methods are described in Supplementary Data.
mRNA sequencing
Total RNA was extracted using the RNeasy Mini Kit (Qiagen)according to the manufacturer’s protocol. mRNA sequencing(mRNA-seq) was performed using NovaSeq 6000/PE150. Raw
sequence reads were trimmed to remove adaptors and quality control
using fastp (version. 0.2.0; ref. 36). Mapping and transcript abundance
estimating were performed as in a previous report. These gene raw
count data were normalized by transcripts per kilobase million (TPM;ref. 37). Gene expression differences between control and PDPN-KO
cells were calculated using TPM normalized expression scores, andgenes with fold change > 1.2 or < 0.83 (Ctrl KO#1 and KO#2) weredeﬁned as differentially expressed genes (DEG). Genes whose TPM incontrol cells was < 1.0 were excluded from the analyses. Gene ontology(GO) term enrichment and Kyoto encyclopedia of genes and genomes(KEGG) pathway analyses were performed using DAVID (ver.
DAVID Knowledgebase v2022q1).

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motility to aggressively invade the extracellular matrix (ECM), a
process referred to as amoeboid invasion (27–31). Amoeboidinvading cells are often enriched in the tumor invasion fronts (IF)of primary human cutaneous melanoma compared with the tumor
bodies (TB) and possess high proliferative, migratory, invasive,and metastasis colonizing characteristics (27–31). Therefore, amoeboid invasion is considered a leading driver of the many steps required
for the metastatic success of human cutaneous melanoma, including
separating from the primary tumor nests, invasion into bloodveins and regional lymph nodes, and colonization of metastatic
nests (27–31). Previously, podoplanin was reported to promote amoeboid invasion of murine cutaneous melanoma cells (32), thus wehypothesized that podoplanin drives amoeboid invasion in canine andhuman mucosal melanoma.
Our results show that podoplanin overexpressed in canine mucosal
melanoma IFs drives amoeboid invasion via Rho-associated kinase(ROCK)-myosin right chain 2 (MLC2) signaling. Podoplanin expressing amoeboid invading canine mucosal melanoma cells show highly
proliferative and invasive characteristics, and escape from cell-cycle
arrest and cellular senescence by activating ROCK–MLC2 signaling.
Moreover, human mucosal melanoma with podoplanin-high expression enriched gene signatures related to amoeboid invasion andshowed poor prognosis, similar to canine mucosal melanoma.

Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

Western blot analysis
Western blot analysis was performed as described previously (18).
Brieﬂy, cells were lysed in RIPA buffer supplemented with 10 mmol/L
NaF, 2 mmol/L Na3VO4, and a complete Mini Protease Inhibitor
Cocktail (Roche Diagnostics). After blocking with blocking buffer for
1 hour, primary antibodies were incubated overnight at 4 C. The
membranes were then incubated with horseradish peroxidase (HRP)conjugated antibodies. Membranes were developed using the ECL
Prime western blotting Detection System (GE Healthcare), and luminescence was captured and quantiﬁed using an imaging system(ChemiDoc Image Lab, Bio-Rad Laboratories).

Cell culture on thick layers of collagen I
Collagen I mix (Cellmatrix Type I-A, Nitta Gelatin) was preparedaccording to the manufacturer’s protocol with a ﬁnal concentration of
1.0 mg/mL. After polymerization (4 hours), cells were seeded on top ofcollagen in medium containing 10% FBS, allowed to adhere for24 hours and media was changed to 1% FBS with or without inhibitors.
Cell morphology was analyzed using ImageJ software by manuallydrawing around the cells. The roundness index was calculated as 4 
area  p/perimeter2.
Gel contraction assay
Plates were incubated with 1% BSA in PBS for 1 hour. After washing
the plates with PBS, cells were embedded in collagen I mix (Cellmatrix
Type I-A) with a ﬁnal concentration of 1.8 mg/mL. The gel was
incubated at 37 C for 1 hour, and the medium, with or without
inhibitors, was added. A pipette tip was used to loosen the gel fromthe tissue culture vessel and incubated for 48 hours. The plate image
was scanned using ChemiDoc, and the percentage of gel contraction
was calculated using the ImageJ software.
Time-lapse imaging
Cells were seeded on 24-well plates, and images were captured for8 hours at 30 minutes intervals using a microscope (BZ-X810;
Keyence).
Senescence-associated b-galactosidase stain
Senescence-associated beta-galactosidase (SA-b-GAL) staining was
performed using the Senescence b-Galactosidase Staining Kit (#9860,
Cell Signaling Technology) according to the manufacturer’s protocol.
Three random ﬁelds (200) were assessed in each well.
Animal study
BALB/c-nu/nu mice (BALB/cSlc-nu/nu, 6-week-old female, Japan
SLC) and NOD/Shi-scid, IL-2RgKO (NOG) mice (6- to 12-week-old
females, Central Institute for Experimental Animals) were maintained
under speciﬁc pathogen-free conditions at 24  1 C, 40% to 70%
humidity, and a 12-hour light–dark cycle throughout all the experiments. A total of 2.5106 CMM2 and PDPN-KO cells derived from
CMM2 cells were subcutaneously injected into nude mice. A total of

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GO terms enrichment analysis and gene-set enrichment analysis
for public datasets
Information of the public datasets used is described in Supplementary Data. Gene expression differences between high and low podoplanin expression tumors were evaluated using the Student t test.
Signiﬁcantly higher expressed genes in tumors with high podoplanin
expression were deﬁned as DEGs. When the total sample number
was >10, the statistical signiﬁcance criterion was P < 0.01; when the
total sample number was <10, the criterion was P < 0.05.
Gene sets were downloaded and analyzed using the GSEA
software (ver.4.2.2; ref. 38). The melanoma invasion gene set (39)and mesenchymal amoeboid transition gene set (40) were referred
to in previous studies. Gene-set enrichment analysis (GSEA) was
performed using the following settings: permutations, 1,000; permutation type, gene set; and metric for ranking genes, t test.
Mucosal melanoma samples from dogs and humans were divided
into high and low podoplanin expression groups based on medianpodoplanin expression. To perform GSEA using canine samples,
all ensemble gene IDs were converted from Canis familiaris to
Homo sapiens using R software (ver. 4.2.0, R Development Core
Team, 2019) with the “biomaRt” package (ver. 2.44.4). Signiﬁcantly
enriched gene sets in podoplanin -high expression versus podoplanin -low expression samples were considered according to nominal
P < 0.05. Mesenchymal amoeboid transition gene expression inhuman and canine mucosal melanoma tissues was evaluatedaccording to a previous report (40). Genes whose average expression in high- podoplanin–expressing tissues was more than 1.2-foldcompared with those of low podoplanin–expressing tissues wereselected and heat map images were generated.
Statistical analysis
R software was used for statistical analyses, and used packages were
described in Supplementary Data. Student t test and x2 test were used
for two group comparisons. Dunnett and one-way ANOVA with
Tukey–Kramer tests were used for multigroup comparisons. Survival
curves were estimated by the Kaplan–Meier method and differenceswere estimated by the log rank test. In ﬁgure legends, “n” indicates thenumber of biologic replicates. P < 0.05 was deﬁned as signiﬁcant.

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RhoA-GTP pulldown assay
RhoA-GTP pulldown assay was performed using the RhoA PullDown Activation Assay Biochem Kit, according to the manufacturer’s protocol (#BK-036, Cytoskeleton, Inc.). Brieﬂy, cells werelysed RIPA buffer. Proteins (50 mg) were incubated with 15 mg
glutathione S-transferase (GST)-conjugated Rhotekin RBD beads
for 1 hour. RhoA expression was detected using immunoblotting.
HRP-conjugated anti-mouse IgM antibody (Abcam) was used as thesecondary antibody.

1  107 CMM12 and PDPN-KO cells derived from CMM12 cells
were subcutaneously injected into NOG mice. Tumor volumes were
measured every 2 to 3 days. Tumor volume was calculated using thefollowing approximation formula: tumor volume ¼ 1/2  (major
radius)  (minor radius)2. The study protocol was approved by the
University of Tokyo Animal Care and Use Committee (approval
numbers P19–126H03 and P19–124).
BALB/c-nu/nu mice (BALB/cSlc-nu/nu, 11-week-old female, Japan
SLC) were used for lung metastasis assay. A total of 1  106 CMM2 and
PDPN-KO cells derived from CMM2 cells were intravenously injected
into the mice. Then, 25 days after tumor cell injection, the mice wereeuthanized, lung tissues were collected, and the number of metastatic
foci was counted.

Data availability
mRNA-seq data of control and PDPN-KO canine mucosal melanoma cells are available in the Genome Expression Omnibus withaccession number: GSE208269.
Supplementary experimental procedures
Other detailed experimental procedures are described in Supplementary Materials and Methods.

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Results

Podoplanin regulates canine mucosal melanoma proliferation,
survival, migration, and invasion
To evaluate the functions of podoplanin in canine mucosal melanoma, PDPN-KO cells were generated using CRISPR/Cas9 technology
from two canine mucosal melanoma cell lines (CMM2 and CMM12;
Supplementary Fig. S2A and S2B). PDPN-KO cells exhibited slow
growth and an increase in apoptotic cells with downregulation of theantiapoptotic protein; BCL-2 and upregulation of the proapoptotic
protein; BAX compared with control cells (Fig. 2A and B; Supplementary Fig. S2C and S2D). In addition, PDPN-KO cells showed lower
migration across two-dimensional (2D) surfaces and lower invasiveness in a three-dimensional (3D) ECM compared with control cells(Fig. 2C and D; Supplementary Fig. S2E).

Figure 1.
Podoplanin overexpression in the IFs is associated with poor prognosis in dogs with mucosal melanoma. A–B, Podoplanin expression in normal gingival mucosal
tissues and in the TBs and IFs of canine mucosal melanoma tissues. Podoplanin was stained with alkaline phosphatase, and podoplanin-positive cells were stained red.
Scale bars, 200 mm (top left), 500 mm (bottom left) and 100 mm (top right and bottom middle). C, IHC scores of podoplanin expression in normal tissues (n ¼ 6), theTBs, and IFs from canine mucosal melanoma tissues (n ¼ 43). One-way ANOVA with Tukey–Kramer test. D, Kaplan–Meier survival curves for PFS: left and OS: right ofdogs with mucosal melanoma above and below the median IHC score of PDPN expression in the IFs. Log-rank test.  , P < 0.05,;   , P < 0.01.

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PDPN is overexpressed in canine mucosal melanoma cells in thetumor IFs and associated with poor prognosis in dogs
To evaluate the relationship between podoplanin expression andclinicopathologic features of dogs with mucosal melanoma, IHC for
podoplanin was performed in 43 canine oral mucosal melanoma
tissues (Supplementary Table S1) and 6 normal gingival mucosa
tissues. Podoplanin was expressed in 88% (38/43) of the canine
mucosal melanoma tissues, whereas no podoplanin expression was
detected in the normal gingival mucosa epithelium (Fig. 1A–C).
Podoplanin-positive cells were conﬁrmed as canine mucosal melanoma cells by double staining with podoplanin and SOX10—
canine mucosal melanoma markers (ref. 41; Supplementary Fig. S1A).
The positivity and intensity of podoplanin expression were higher
in the IFs of canine mucosal melanoma tissues than in the TBs(mean positivity 42% vs. 29%, mean intensity score 2.0 vs. 0.8,
respectively; Fig. 1A–C). High podoplanin expression in the IFs was
signiﬁcantly associated with reduced progression-free survival(PFS) and overall survival (OS) rates in dogs with mucosal melanoma,

while high podoplanin expression in the TBs was not signiﬁcantly
associated with PFS and OS rates (Fig. 1D; Supplementary Table S2;
Supplementary Fig. S1B). Overall, podoplanin overexpressed incanine mucosal melanoma IFs was associated with a poor prognosis
in dogs.

Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

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Figure 2.
Podoplanin regulates canine mucosal melanoma proliferation, survival, migration, and invasion. A, Number of live cells at 24, 48, and 72 hours after seeding (n ¼ 3).
B, Percentage of annexin V–positive apoptotic cells analyzed by ﬂow cytometry analysis (n ¼ 3). C, Migrated areas at 8 or 16 hours after wounding (n ¼ 3). D, Numberof invaded cells at 24 hours after cell seeding (n ¼ 3). Representative images of wells are shown under the graphs. Graphs show mean  SD. Dunnett test:  , P < 0.05;
, P < 0.01. Ctrl: control, KO#1: PDPN-KO cell line 1, KO#2: PDPN-KO cell line 2. E, Enriched GO terms in DEGs altered by podoplanin-KO. The x axis indicates the log10
P value.

Furthermore, DEGs in response to PDPN-KO were detected using
mRNA-seq. A total of 560 and 1,132 genes were downregulated andupregulated in the PDPN-KO CMM2 cells, respectively. In addition,
1,726 and 1,122 genes were downregulated and upregulated in the

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PDPN-KO CMM12 cells, respectively (Supplementary Fig. S2F). In
control cells, genes related to cell proliferation and cell-cycle wereenriched compared with PDPN-KO cells, whereas genes related to thenegative regulation of cell proliferation, cell cycle, and cell motility

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Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

were enriched in PDPN-KO cells (Fig. 2E). These results suggest that
podoplanin promoted proliferation, survival, migration, and invasionof canine mucosal melanoma cells.

Loss of podoplanin leads to cell-cycle arrest and cellularsenescence
To elucidate the underlying mechanism of PDPN-mediated cellproliferation, a cell-cycle analysis was performed, and the percentage
of cells in the S-phase cell-cycle was signiﬁcantly decreased in
PDPN-KO cells (Fig. 4A). mRNA-seq of PDPN-KO cells showed anincreased gene expression of cyclin-dependent kinase inhibitor(CDKi) compared with control cells (Supplementary Fig. S4A). However, PDPN-KO did not induce a common change in the expression

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Podoplanin regulates amoeboid invasion features via
ROCK-MLC2 signaling
Podoplanin was reported to active ERM and RhoA (20); thus,
we evaluated their activation status. PDPN-KO cells exhibited
lower ERM phosphorylation and RhoA-GTPase expression, an active
form of RhoA, than those of control cells (Fig. 3A). Furthermore,
PDPN-KO cells exhibited lower myosin phosphatase targeting subunit
1 (MYPT1) and MLC2 phosphorylation, which are ROCK-targeting
proteins, than control cells (Fig. 3A). The number of cells withcytoplasmic stress ﬁbers was signiﬁcantly decreased in PDPN-KO
cells (Fig. 3B and C). mRNA-seq also showed an upregulation ofROCK/cytoskeleton-related genes in control cells compared with
PDPN-KO cells (Supplementary Fig. S3A).
We explored the morphologic alterations induced by PDPN-KO(27–31). Under 3D collagen culture conditions, control cells displayed a reduction in cell size and an increase in the number ofrounded cells compared with 2D culture conditions (Fig. 3D–F).
The size of the PDPN-KO cells was signiﬁcantly larger than that of
the control cells, and PDPN-KO decreased the number of rounded
cells under 3D collagen culture conditions (Fig. 3D–F). Controls
cells showed membrane blebs under 3D collagen culture conditions(Fig. 3G). We then evaluated actomyosin contractile function
and found that the contraction rate of embedded PDPN-KO cells
was signiﬁcantly lower than that of control cells (Fig. 3H and I).
Control cells showed lower adhesive capacity compared with
PDPN-KO cells (Supplementary Fig. S3B). In addition, a previous
report showed that podoplanin regulates proliferation of ﬁbroblasts in a mechanically sensitive mechanism (42). The proliferation of control cells cultured on noncollagen coated dishes(stiffness condition) was increased compared with that of cells
cultured on collagen thick layer dish (soft condition; Supplementary Fig. S3C, left), and the changes were dependent on podoplanin
expression (Supplementary Fig. S3C, right). Overall, podoplanin in
canine mucosal melanoma cells activated ROCK-MLC2 signaling
to drive high levels of actomyosin contraction which is necessary
for amoeboid invasion.

of cyclin and cyclin-dependent kinase genes. A signiﬁcant upregulation of CDKi mRNA, and p21 and p27 proteins in PDPN-KO cells was
conﬁrmed (Supplementary Fig. S4B and S4C). These results indicatethat PDPN promotes cell-cycle S-phase entry to support proliferation.
Interestingly, the number of cells with over 4N DNA (G2–M
phase cells) was signiﬁcantly increased in PDPN-KO cells (Fig. 4A).
Furthermore, PDPN-KO cells displayed a signiﬁcant increase in thenumber of multi-nuclei cells that arise due to cytokinesis failure (43); consequently, cytokinesis failure was frequently observed
during the proliferation of PDPN-KO cells (Fig. 4B and C). These
results suggest that the increase of multi-nuclei PDPN-KO cells
was due to cytokinesis failure. In addition, SA-b-GAL stain-positive
cells were signiﬁcantly increased among the PDPN-KO cells(Fig. 4D and E) with upregulated p21 expression in PDPN-KO
cells (Supplementary Fig. S4C). p21 expression was upregulated
in the nuclei of cytokinesis-failed PDPN-KO cells compared with
control cells with a single nucleus (Supplementary Fig. S4D).
SA-b-GAL staining intensity and positivity in cytokinesis-failed
cells were higher in multi-nuclei PDPN-KO cells than in the control
mono nuclear cells (Supplementary Fig. S4E). Genes associated withcellular senescence pathway were enriched in DEGs induced by
PDPN-KO (Supplementary Fig. S4F and S4G). Overall, PDPN-KO
induced cytokinesis failure in canine mucosal melanoma cells,
resulting in cellular senescence.
ROCK inhibition suppresses cell proliferation, migration, andsenescence
Next, we pharmacologically inhibited ROCK-MLC2 signaling
on canine mucosal melanoma cells to conﬁrm that the loss ofROCK-MLC2 signaling caused cell phenotype modiﬁcations in
PDPN-KO cells. In inhibitory experiments on canine mucosal melanoma cells, ROCK and myosin II inhibition resulted in a dosedependent reduction of cell proliferation (Supplementary Fig. S5A
and S5B), an increase of the proportion of apoptotic cells, andsuppressions of migratory and invasive capacities (Supplementary
Fig. S5C–S5F). Moreover, ROCK and myosin II inhibition suppressedstress ﬁber formation, increased cell area, decreased the number ofrounded cells, and reduced gel contractile capacities (Fig. 5A–C;
Supplementary Fig. S6A–S6C). The percentage of cells in the S-phasecell-cycle was signiﬁcantly reduced, and the percentage of cells withover 4N DNA was signiﬁcantly increased by ROCK and myosin II
inhibition (Supplementary Fig. S7A). The proportion of multi-nuclei
cytokinesis-failure cells and the number of SA-bGAL stain-positive
cells increased with the upregulation of p21 expression by ROCK andmyosin II inhibition (Supplementary Fig. S7B–S7E). Moreover, restoring podoplanin expression in PDPN-KO cells (KO#2) rescued the
podoplanin induced features of amoeboid invasion including thedecrease of cell proliferation, p-MLC2 suppression, increase of cellsize, and change to an elongated cell shape (Fig. 5D–H; Supplementary
Fig. S7F). Overall, the inhibition of ROCK-MLC2 signaling in canine

Figure 3.
Podoplanin regulates amoeboid invasion features via ROCK-MLC2 signaling. A, Immunoblots of p-ERM, RhoA-GTP, p-MYPT1, and p-MLC2. Levels of phospho-protein
expression were calculated after correction to total levels of the relevant protein expression and are indicated under the immunoblot images of each total protein.
B, Confocal images of phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining. Arrowheads show stress ﬁbers in the cytoplasmic regions. Scale bar, 10 mm.
C, Percentage of stress ﬁber–positive and-negative cells (n ¼ 3). D, Representative images of cellular morphologies under 2D and 3D collagen culture conditions.
Scale bar, 50 mm. E, Cell areas under 2D or 3D collagen culture conditions (n ¼ 50). F, Roundness index under the 2D or 3D collagen culture conditions (n ¼ 50).
G, Representative confocal images of control cells with membrane blebs under the 3D collagen culture condition. Phalloidin stain was performed. White arrow heads
indicate membrane blebs. Scale bar, 10 mm. H–I, Representative images (G) and quantiﬁcation (H) of gel contraction rate. The yellow dashed line indicates the shapeof the collagen gels, and the red dashed line indicates the well of the culture plate (n ¼ 3). Graphs show mean  SD. Dunnett test:  , P < 0.05;   , P < 0.01. Ctrl: control,
KO#1: podoplanin-KO cell line 1, KO#2: podoplanin-KO cell line 2.

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Figure 4.
Loss of podoplanin leads to cell-cycle arrest and cellular senescence. A, Cell-cycle analysis (n ¼ 3). B, Phalloidin- and DAPI-stained CMM2 cells (top) and thepercentage of multi nuclei cells (bottom; n ¼ 3). White arrowheads indicate nuclei. Scale bar: 50 mm. C, Time-lapse images during the proliferation of control (CMM2)
and PDPN-KO (KO#2 of CMM2) cells. Red arrowheads show cytokinesis failure cells among PDPN-KO cells, and yellow arrowheads show nuclei in control and
PDPN-KO cells. Scale bar, 10 mm. D, Representative images of SA-b-GAL staining. Scale bar, 50 mm. E, Percentage of SA-bGAL stain-positive cells (n ¼ 3). Graphs
show mean  SD. Dunnett test:  , P < 0.05;   , P < 0.01. Ctrl: control, KO#1: PDPN-KO cell line 1, KO#2: PDPN-KO cell line 2.

mucosal melanoma cells showed similar effects as those found in
PDPN-KO cells, and restoring podoplanin expression in the PDPNKO cells showed recovery of those characteristics, suggesting that
podoplanin regulated the features of amoeboid invasion via ROCKMLC2 signaling in canine mucosal melanoma cells.

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Podoplanin promotes amoeboid invasion features inxenografted mouse models and clinical cases
To determine whether abrogation of podoplanin suppresses amoeboid invasion in canine mucosal melanoma cells in vivo, we generated
xenograft murine xenograft models that exhibited similar podoplanin

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Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

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Figure 5.
Podoplanin-ROCK-MLC2 signaling is the regulator of amoeboid invasion of canine mucosal melanoma cells. A, Representative images of cellular morphologies underthe 2D and 3D collagen culture conditions at 24 hours after inhibitors treatment. Scale bar, 50 mm. B, Cell areas at 24 hours after inhibitors treatment (n ¼ 50). Dunnett
test. C, Roundness index of cells at 24 hours after inhibitors treatment (n ¼ 50). Dunnett test. D, Number of live cells of MOCK control and podoplanin overexpressed
PDPN-KO (PDPN-KO-OE) cells (n ¼ 3). The number of cells were counted at 72 hours after cell seeding. E, Immunoblots of p-MLC2. Levels of phosphor-protein
expression were calculated after correction to total levels of the relevant protein expression and are indicated under the immunoblot images of each total protein.
F, Representative images of cellular morphologies of MOCK control and PDPN-KO-OE cells under 3D collagen culture conditions. Scale bar: 50 mm. G, Cell areas of
MOCK control and PDPN-KO-OE cells under 3D collagen culture conditions (n ¼ 65). Box limits show the 25th and 75th percentiles, the horizontal line represents
the median, and dotted plots show the scores of each cell. Dunnett test. H, Roundness index of MOCK control and PDPN-KO-OE cells under 3D collagen culture
conditions (n ¼ 65). Box limits show the 25th and 75th percentiles, the horizontal line represents the median, and dotted plots show the scores of each cell. Dunnett
test.  , P < 0.05;   , P < 0.01.

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Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

Canine and human mucosal melanomas with high podoplanin
expression are enriched with gene signatures associated withamoeboid invasion
We analyzed three cohorts of public datasets for canine oral mucosal
melanoma (44–46). The expression of the podoplanin gene level was
signiﬁcantly higher in tumor tissues than in normal control tissues(Supplementary Fig. S10A). GO term enrichment analysis showed thattumors that exhibited high podoplanin expression were enriched ingenes related to ROCK/cytoskeleton (Supplementary Fig. S10B).
GSEA displayed that tumors with high PDPN expression wereenriched in gene signatures relevant to amoeboid invasion, proliferation, migration, and invasion (Supplementary Figs. S10C, S10D, andS11). These ﬁndings suggest that podoplanin-dependent induction of
proliferative and aggressive amoeboid invasion is a common characteristic of canine mucosal melanoma cells expressing podoplanin.
To assess the applicability of our discoveries from canine to humanmucosal melanoma, we investigated three human mucosal melanoma
public datasets (47, 48). The expression of the podoplanin gene level
was signiﬁcantly higher in human mucosal melanoma tissues than innormal control tissues (Supplementary Fig. S12A). Survival analysis ofpatients with stage 3 or 4 mucosal melanoma (n ¼ 11) showed thatpatients with high podoplanin-expressing tumors had signiﬁcantly
shorter PFS and OS than those with low podoplanin-expressing
tumors (Fig. 7A). Tumors with high podoplanin expression wereenriched in of ROCK/cytoskeleton-related and amoeboid invasionrelated gene signatures (Fig. 7B and C). Tumors with high podoplanin
expression showed enhanced gene expression of collagens associatedwith amoeboid invasion (COL1A1, COL1A2, COL4A1, and COL4A2;
Supplementary Fig. S12B). Tumors with high podoplanin expression
had enriched gene signatures related to proliferation, migration, andinvasion (Fig. 7D; Supplementary Fig. S12C). Moreover, the upregulation pattern of common mesenchymal amoeboid transition-related
genes was similar between human and canine mucosal melanomas(Fig. 7E). These ﬁndings suggest that podoplanin promotes amoeboid
invasion in human mucosal melanoma through mechanisms similar to
those observed in canine mucosal melanoma.

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expression patterns to those of canine mucosal melanoma clinicalsamples (Supplementary Fig. S8A). Tumor growth was signiﬁcantly
suppressed by PDPN-KO (Fig. 6A). These results indicated that
podoplanin promoted proliferative and aggressive amoeboid invasionin xenografted tumor tissues at the IFs.
To assess whether these observations were recapitulated in clinicalsamples, we conducted IHC analysis. In control tumors, the percentageof ki-67–expressing cells at the IFs was signiﬁcantly higher and, that of
p21-expressing cells at the IFs was signiﬁcantly lower, compared withthat of the TBs (Fig. 6B; Supplementary Fig. S8B). Cell roundness andphosphor-MLC2 expression levels at the IFs were signiﬁcantly higher
than those of the TBs in control tumors (Fig. 6C and D). Notably, thepercentage of ki-67, p21-expressing cells, cell roundness, and phospho-MLC2 expression levels at the IFs of PDPN-KO tumors werereversed to levels similar to those in the TBs of PDPN-KO tumors(Fig. 6B–D; Supplementary Fig. S8B). The IFs of canine mucosal
melanoma enriched rounded tumor cells compared with the TBs, andthe number of rounded cells in the IFs was associated with podoplanin
expression levels in the IFs (Fig. 6E). Phospho-MLC2 expression levels
at the IFs were higher than the TBs, and phosphor-MLC2 expression
levels in the IFs were associated with podoplanin expression levels in
the IFs (Fig. 6F). These data suggested that podoplanin induced
aggressive amoeboid invasion in the IFs of canine mucosal melanoma,
which was linked to unfavorable outcomes in dogs.
To explore the relationship between podoplanin and distant colonization of canine mucosal melanoma cells, an in vivo lung metastasis
assay was performed, and the number of lung metastatic foci wassigniﬁcantly reduced by PDPN-KO (Supplementary Fig. S9A and
S9B). This result suggests that PDPN promotes distant colonization
during the late stage of tumor metastasis.

Discussion
Podoplanin has been reported to drive amoeboid invasion inmurine cutaneous melanoma cells (32); however, that relationship incanine and human melanoma had not been revealed. Especially incanine and human mucosal melanoma, the relationship between
podoplanin and tumor malignancies had not been comprehensively
understood. In this study, we demonstrated that podoplanin overexpressed in the tumor IFs was associated with poor prognosis in dogs
with mucosal melanoma and that podoplanin regulated features ofamoeboid invasion in canine mucosal melanoma cells via podoplaninROCK–MLC2 signaling. Podoplanin expression was also related to theenrichment of amoeboid invasion-related gene signatures in canineand human mucosal melanoma tissues. These ﬁndings suggest that
podoplanin drives amoeboid invasion to acquire highly metastaticfeatures in canine and human mucosal melanoma.
Canine mucosal melanoma is one of the most common oralmalignancies in dogs and is a highly aggressive tumor that canmetastasize during the early stages of the disease (8, 10). The 1-year
survival rate of dogs with mucosal melanoma is less than 35% owing toa high metastatic rate of more than 70% (8, 10). In this study, wedemonstrated that podoplanin overexpressed in the IFs of canine
mucosal melanoma was related to reduced PFS and OS rates, indicating that podoplanin overexpressed in the IFs is a key molecule incanine mucosal melanoma metastasis. We showed that podoplanindepletion in canine mucosal melanoma cells downregulated theactivation of ERM, RhoA, and ROCK-MLC2. Moreover, podoplanindepletion and ROCK inhibition suppressed amoeboid invasion features in canine mucosal melanoma cells, and restoring podoplanin
expression reactivated ROCK-MLC2 signaling in podoplanin-KO
cells. Podoplanin directly binds to ERM protein to activate RhoA (20),and ROCK is a major effector molecule of the activated RhoA.
Amoeboid invasion has been reported to be induced by ROCK-MLC2
signaling activation (27–31). Therefore, podoplanin would promoteamoeboid invasion by activating ERM-RhoA–ROCK-MLC2 signaling. During the early step of tumor metastasis, human cutaneous

Figure 6.
Podoplanin promotes amoeboid invasion features in xenografted mouse models and clinical cases. A, Tumor growth curves xenografted in immunodeﬁcient mice(n ¼ 5–7). Graphs show the mean  SEM. Dunnett test. B–D, Representative images of the TBs and IFs of control and podoplanin-KO tumors (top) and quantiﬁcation(bottom) of the percentage of ki-67–positive cells (B), melanoma cell shape score (C), and p-MLC2 IHC score (D) of xenografted tissues from A (n ¼ 5–6). Ki-67 andp-MLC2 were stained with DAB. Scale bar: 200 mm. Dunnett test. E and F, Representative images of canine mucosal melanoma tissues showing podoplanin
expression, H&E staining, and p-MLC2 expression in the TBs and IFs (left) and quantiﬁcation (right) of melanoma cell shape score (E) and p-MLC2 IHC score (F) in the
TBs and IFs of canine mucosal melanoma clinical samples from Fig. 1 (n ¼ 40 and 34, respectively). High or low podoplanin expression samples were divided by the
median podoplanin expression score in the IFs. Podoplanin and p-MLC2 were stained with alkaline phosphatase, and podoplanin- and pMLC2-positive cells were
stained red. Scale bar, 100 mm. One-way ANOVA with Tukey–Kramer test:  , P < 0.05;   , P < 0.01. C, Control, #1: PDPN-KO cell line 1, #2: PDPN-KO cell line 2.

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Podoplanin Drives Amoeboid Invasion in Mucosal Melanoma

tivated YAP and stabilized p53 (61). Overall, our ﬁndings suggest that
podoplanin-RhoA–ROCK-MLC2 signaling in amoeboid invading
canine mucosal melanoma cells supports the escape from cellularsenescence by preventing cytokinesis failure, caused by high proliferative activity, and further research is required to reveal the detailed
mechanism.
A previous study indicated that PDPN senses a stiffness of external
environment and regulates ﬁbroblast proliferation (42). We found thatthe proliferation of control cells increased on the noncollagen dishcompared to the collagen thick-layer dish, and the changes depended
on the PDPN expression in canine mucosal melanoma cells. As tumor
stiffness increased from the tumor core region to the tumor periphery (62), podoplanin might have sensed a stiffer microenvironment ofcanine mucosal melanoma IFs and promoted the proliferation ofamoeboid-invading cells in the IFs.
Human mucosal melanoma is a rare and the most aggressive
subtype, and it is difﬁcult for surgery to achieve radical excision,owing to the lentiginous growth pattern, multifocal nature, and highmetastasis rate of approximately 80% (1). Although adjuvant radiotherapy is performed for the local control of human mucosal
melanoma, it could not reduce the risk of distant metastasis andprolong patients’ survival (63, 64). In addition, patients withmucosal melanoma show lower response rates to immune checkpoint blockades than those with cutaneous melanoma (23 vs. 40.9%;
ref. 65). Therefore, it is crucial to understand the molecularmechanism of human mucosal melanoma metastasis. Our study
revealed that high podoplanin expression in human mucosal melanoma is associated with poor prognosis and the enrichment of
amoeboid invasion-related gene signatures, as observed in canine
mucosal melanoma. Furthermore, we found that the upregulation
pattern of mesenchymal amoeboid transition-related genes was
similar between podoplanin high-expressing human and canine
mucosal melanoma tissues. These ﬁndings suggest that podoplanin
drives amoeboid invasion and metastasis in human mucosal melanoma through similar mechanisms as those acting in canine
mucosal melanoma. Thus, naturally occurring canine mucosal
melanoma could be a novel research model for podoplanin expressing human mucosal melanoma.
We showed that podoplanin was overexpressed in the IFs of canine
mucosal melanoma tissues; however, the induction mechanism of
podoplanin remains unclear. A previous study showed that podoplanin expression is induced by inﬂammatory cytokines such as IFNg,
TGFb, and/or tumor necrosis factor-a in human SCC cells (66).
Tumor IFs are located adjacent tumor stromal regions enriched withinﬂammatory immune cells; thus, tumor cells in the TFs could bestrongly exposed to inﬂammatory cytokines released from inﬂammatory immune cells in stromal regions (67). Therefore, inﬂammatory
cytokine may induce podoplanin expression on canine melanoma cells
in the TIF, leading to aggressive amoeboid invasion.
Our study opens the possibility that podoplanin plays a crucial rolein amoeboid invasion and metastasis of canine and human mucosal
melanoma. podoplanin could be used as a biomarker and a therapeutictarget for the restriction of amoeboid invasion and metastatic dissemination in canine and human mucosal melanoma.

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melanoma cells change their cellular morphology into the aggressive
amoeboid mode by activating ROCK-MLC2 signaling to invade
adjacent tissues (27–31). Amoeboid invasion is a faster, more efﬁcient,and less energy-consuming mode than the mesenchymal mode,
potentiating the ability of cancer cells to migrate and invade theextracellular environment (49). In the late stage of metastasis, amoeboid invading human cutaneous melanoma cells promote distantcolonization by disrupting endothelial junctions and increasing endothelial cell permeability via secretion of IL1a and activation of NF-kB,
promoting extravasation and metastasis colonization (27). In thisstudy, we obtained similar results that abrogation of podoplaninROCK–MLC2 signaling in canine mucosal melanoma cells suppressedamoeboid invasion features in the IFs of primary tumor tissues andtumor colonization in distant organs. Overall, podoplanin expressed in
the IFs of canine mucosal melanoma cells would be a major promoterof the series of their metastatic process.
Amoeboid-invading human cutaneous melanoma cells have a highproliferative capacity (29). In our study, podoplanin deletion andROCK-MLC2 signaling inhibitions suppressed cell proliferation, suggesting that podoplanin promoted the proliferation of amoeboid
invading cells. In podoplanin-KO cells, S-phase cell-cycle entry was
inhibited, and a high expression of CDKi mRNA was observed. Tumor
cells often downregulate CDKi expression to promote rapid cellproliferation because CDKi negatively regulates cell-cycle progression (50, 51). RhoA activation in tumor cells was reported as a possible
mechanism of CDKi suppression. A previous report showed that
RhoA suppression in human gastric cancer cells induced p21 andp27 upregulation and decreased the number of cells in S-phase cellcycle (52). Therefore, our ﬁndings suggest that podoplanin would
promote S-phase cell-cycle entry in canine mucosal melanoma cells by
suppressing CDKi expression probably through RhoA activation andthat podoplanin promotes the proliferation of amoeboid invading cells
in the canine mucosal melanoma IFs.
Furthermore, we found that podoplanin depletion and inhibition ofROCK-MLC2 induced cellular senescence and cytokinesis failure incanine mucosal melanoma cells. Cellular senescence is a state ofpermanent cell-cycle arrest that occurs in response to stress; therefore,
cellular senescence could be a barrier that tumor cells need to evade tomaintain an aberrant proliferation (53, 54). Cytokinesis failure has
been reported to induce cellular senescence in human colon cancer
cells and rat embryo ﬁbroblasts (55, 56). In addition, it was reported
that RhoA and MLC2 inhibition led to cytokinesis failure in murine
embryo ﬁbroblasts; hence, RhoA–ROCK-MLC2 signaling activation is
involved in cytokinesis progression (57, 58). Overall, cytokinesis
failure caused by podoplanin depletion would induce cellular senescence in canine mucosal melanoma cells. Although it was reported thatcytokinesis failure induced cellular senescence, the detailed mechanism has yet to be comprehensively described. Cytokinesis failure wasreported to generate lagging chromosomes and micronuclei leading to
DNA damage response that induces cellular senescence (59). A
previous report showed that inhibition of ROCK induced DNA
damage in human melanoma cells (60). Other report suggested thatcytokinesis failure in RPE-1 cells prevented cell proliferation by
activating the large tumor suppressor kinase 2 (LATS2), which inac-

Figure 7.
Human mucosal melanomas with podoplanin high expression demonstrates enrichment of gene signatures associated with amoeboid invasion. A, Kaplan–Meiersurvival curves for PFS and OS of human patients with stages 3 or 4 mucosal melanoma above or below the median podoplanin gene expression from The Cancer
Genome Atlas (TCGA) dataset (n ¼ 11). Log-rank test. B, Enriched GO terms in high podoplanin-expressing human mucosal melanoma tissues. The x-axis indicates thelog10 P value. C and D, GSEA plots showing enriched gene signatures in high podoplanin expressing human mucosal melanoma tissues. NES: normalized enrichmentscore. E, Heat maps demonstrating the relative expression of mesenchymal amoeboid transition-related genes in human and canine mucosal melanoma tissues. The
color key indicates the fold change (FC) in gene expression from the average expression.

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Authors’ Disclosures

Acknowledgments

D. Kato reports grants from JSPS KAKENHI during the conduct of the study.
Y. Takahashi reports grants from JSPS KAKENHI during the conduct of the study.
R. Nishimura reports grants from JSPS KAKENHI during the conduct of the study.
No disclosures were reported by the other authors.

We thank all the staff of the Veterinary Medical Center of the University ofTokyo, the Laboratory of Veterinary Pathology who processed the samples, andthe dog owners. This work was supported by JSPS KAKENHI (Grant Numbers
19K22361, 20H03141, and 21K20614) and in part by Japan Agency for Medical
Research and Development (AMED; grant Numbers JP22ama121008 andJP22am0401013).

Authors’ Contributions

The publication costs of this article were defrayed in part by the payment ofpublication fees. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.

Note
Supplementary data for this article are available at Molecular Cancer Research Online(http://mcr.aacrjournals.org/).

Received November 19, 2022; revised April 9, 2023; accepted July 13, 2023;
published ﬁrst July 26, 2023.

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M. Shinada: Data curation, formal analysis, investigation, visualization, writingoriginal draft, writing–review and editing. D. Kato: Conceptualization, data curation,formal analysis, supervision, funding acquisition, validation, project administration,
writing–review and editing. T. Motegi: Resources, software, formal analysis, bioinformatics analysis. M. Tsuboi: Resources, visualization, assist in pathological evaluation.
N. Ikeda: Data curation. S. Aoki: Data curation, formal analysis. T. Iguchi: Data
curation, formal analysis. T. Li: Data curation, formal analysis. Y. Kodera: Data curation,formal analysis. R. Ota: Data curation, formal analysis. Y. Hashimoto: Resources.
Y. Takahashi: Resources, funding acquisition. J. Chambers: Resources, provision ofparafﬁn samples. K. Uchida: Resources. Y. Kato: Resources, funding acquisition,
provision of antibodies. R. Nishimura: Conceptualization, supervision, funding acquisition, project administration. T. Nakagawa: Conceptualization, supervision, project
administration.

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