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OPEN

Exploration of serum biomarkers
in dogs with malignant melanoma
receiving anti‑PD‑L1 therapy
and potential of COX‑2 inhibition
for combination therapy
Naoya Maekawa1, Satoru Konnai1,2*, Yumie Asano2, Yamato Sajiki2, Tatsuya Deguchi3,
Tomohiro Okagawa1, Kei Watari2, Hiroto Takeuchi2, Satoshi Takagi3,4, Kenji Hosoya3,
Sangho Kim3, Hiroshi Ohta3, Yukinari Kato5,6, Yasuhiko Suzuki1,7,8, Shiro Murata1,2 &Kazuhiko Ohashi1,2Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat humancancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies.
However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore
biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1
antibody c4G12 (n = 27), serum concentrations of prostaglandin E2 ­(PGE2), cytokines, chemokines,
and growth factors were measured prior to treatment initiation. Among 12 factors tested, ­PGE2,
interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) werehigher in OMM dogs compared to healthy dogs (n = 8). Further, lower baseline serum ­PGE2, MCP-1,
and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF
concentrations predicted prolonged overall survival. These observations suggest that ­PGE2 confers
resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for
combination therapy. Indeed, ­PGE2 suppressed IL-2 and interferon (IFN)-γ production by stimulated
canine peripheral blood mononuclear cells (PBMCs), while inhibition of ­PGE2 biosynthesis using theCOX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs.
Thus, serum ­PGE2 may be predictive of c4G12 treatment response, and concomitant use of COX-2
inhibitors may enhance ICI antitumor efficacy.
Cancer has become a major cause of death in domesticated dogs due to gains in lifespan. Like human cancers,
canine cancers are usually treated by surgical excision, radiation, chemotherapy, or a combination of these measures. In addition, new treatment modalities are being developed to provide better veterinary care for canines.
Immunotherapy is one promising strategy because the therapeutic effect is expected to be systemic but stillcancer-specific. In humans, antibodies that inhibit immune checkpoint molecules, such as programmed cell death
1 (PD-1) and PD-ligand 1 (PD-L1), have demonstrated robust antitumor efficacies with acceptable safety profiles
for various cancer ­types1–3. PD-1 is an inhibitory receptor that suppresses the effector functions of activated T
cells. Its ligand, PD-L1, is often overexpressed in tumor cells, suggesting that the PD-1/PD-L1 pathway is a majormechanism for immune evasion by t­ umors4,5. In dogs, PD-1 expression is upregulated in lymphocytes infiltrating
1

Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,
Japan. 2Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,
Japan. 3Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,
Japan. 4Department of Veterinary Surgery 1, School of Veterinary Medicine, Azabu University, Sagamihara,
Japan. 5Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Japan. 6Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai,
Japan. 7International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan. 8Global Station for
Zoonosis Control, Global Institution for Collaborative Research and Education (GI‑CoRE), Hokkaido University,
Sapporo, Japan. *email: konnai@vetmed.hokudai.ac.jp
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oral malignant melanoma (OMM), and PD-L1 is detected in tumor cells of various malignant cancers including
OMM and ­osteosarcoma6–16. A recently developed canine chimeric anti-PD-L1 antibody (c4G12) demonstrated
promising antitumor activity in dogs with OMM and the survival benefit was strongly s­ uggested11,16. However,
the majority of dogs with OMM did not respond to ­c4G1216, suggesting that other factors may limit ICI efficacy.
These findings emphasize the need for further study on predictive biomarkers that can distinguish the subpopulation most likely to benefit from ICI therapy. In addition, studies are required to elucidate mechanisms limiting
ICI antitumor activity and thereby identify possible targets for combination therapy.
Peripheral blood or serum/plasma biomarkers are considered preferable because sampling is easy and lessinvasive. To date, several serum factors have been identified that are predictive of ICI benefit among humancancer patients, including C reactive protein (CRP), interleukin (IL)-6, soluble PD-L1, and various cytokines and­chemokines17–21. We previously reported that high baseline CRP in plasma was associated with poorer overall
survival (OS) in dogs with pulmonary metastatic OMM receiving c4G12 ­treatment16; however, the predictive
values of cytokines, chemokines, and other factors have not been investigated in canine cancer. Hence, to explore
additional predictive biomarkers for ICI efficacy in dogs with OMM, we first measured serum concentrations ofmultiple immune modulators [prostaglandin E2 ­(PGE2), the cytokines interferon-γ (IFN-γ), IL-2, IL-6, IL-10,
IL-12p40, and tumor necrosis factor-α (TNF-α), the chemokines IL-8 and monocyte chemotactic protein 1(MCP-1), and the growth factors nerve growth factor-β (NGF-β), stem cell factor (SCF), and vascular endothelial growth factor-A (VEGF-A)] in healthy controls and dogs with pulmonary metastatic OMM prior to c4G12
treatment. The associations of serum concentrations with OS were assessed by univariate analysis. Next, we
examined the clinical relevance and functions of P
­ GE2 in canine cancers because ­PGE2 is a known suppressorof T cell responses in humans through binding to E prostanoid 2 (EP2) and EP4 ­receptors22 and inhibitors of its
biosynthetic enzyme cyclooxygenase-2 (COX-2) are available for canine diseases with well-known efficacy andsafety profiles. Overexpression of COX-2 has been reported in various canine cancers including transitional cell
carcinoma, squamous cell carcinoma, and mammary ­cancer23, and the selective COX-2 inhibitor ­piroxicam24
has demonstrated clinical benefit against these canine ­cancers25–27. Recent preclinical studies using mouse cancer
models have demonstrated that COX inhibition enhances immunotherapeutic efficacy of anti-PD-1 ­therapy28,29,
however, suppressive functions of P
­ GE2 in the canine immune system and contributions to cancer immune
evasion remain unclear. Therefore, to assess the potential of COX-2 inhibition plus anti-PD-L1 antibody as
combination therapy, we first confirmed COX2 mRNA expression and P­ GE2 upregulation in canine cancers,
and then examined the immunosuppressive effects of ­PGE2 in cultures of canine peripheral blood mononuclear
cells (PBMCs). Finally, the combinational effect of the selective COX-2 inhibitor meloxicam plus anti-PD-L1
antibody was investigated in PBMCs for possible applicability to canine cancer treatment.

Materials and methods

Canine samples. Animal care and use protocols were approved by the Institutional Animal Care and Use
Committee of Hokkaido University (Approval number: 15–0149). All experiments were performed in accordance with relevant guidelines and regulations of the Faculty of Veterinary Medicine, Hokkaido University, which
is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
The use of animals throughout the clinical study was approved by the ethics committee, Faculty of Veterinary
Medicine, Hokkaido University. The reporting of the animal experiment in this study follows the recommendations in the ARRIVE guidelines. Serum samples from pulmonary metastatic OMM dogs (stage IV as defined by
TNM-based ­staging30, n = 27) treated with the canine chimeric anti-PD-L1 antibody ­c4G1211 at the Veterinary
Teaching Hospital of Hokkaido University were collected at baseline (on the first day of immunotherapy). The
detailed dosage and duration of c4G12 therapy and the baseline characteristics of treated dogs were described­elsewhere16. Plasma samples were obtained from tumor-bearing dogs (various tumor types, n = 21) examinedat the Veterinary Teaching Hospital of Hokkaido University. For healthy dog samples, serum, plasma, and heparinized whole blood were collected from clinically healthy beagles housed at the Experimental Animal Facility,
Faculty of Veterinary Medicine, Hokkaido University. Blood sampling was performed without anesthesia. Before
sample collection, informed consent was obtained from the dogs’ owners.
Multiplex immunoassay and enzyme‑linked immunosorbent assay (ELISA). Serum concentra-

tions of IFN-γ, IL-2, IL-6, IL-10, IL-12/IL-23p40, TNF-α, IL-8, MCP-1, NGF-β, SCF, and VEGF-A were quantified by bead-based multiplex immunoassays using Cytokine/Chemokine/Growth Factor 11-Plex Canine ProcartaPlex Panel 1 (Thermo Fisher Scientific, Waltham, MA) and the Luminex 200 System (Luminex, Austin,
Texas). Data were analyzed using Bio-Plex Manager version 6.1 (Bio-Rad, Hercules, CA). P­ GE2 concentrations
in plasma, serum and culture supernatant were quantified using the Prostaglandin E2 Express ELISA Kit (Cayman Chemical, Ann Arbor, MI). IL-2 concentrations in culture supernatant were quantified using the Canine
IL-2 DuoSet ELISA kit, and IFN-γ concentrations using the Canine IFN-γ DuoSet ELISA kit (both from R&D
Systems, Minneapolis, MN). The optical density was measured using the MTP-900 microplate reader (Corona
Electric, Ibaraki, Japan). A heat map showing relative serum levels of each factor was generated using Heatmapper, a web-based tool previously described by Babicki et al. (http://​www.​heatm​apper.​ca/)31. Values below thelower limit of quantification (LLOQ) are marked as missing data.

Cell culture. The canine melanoma cell lines CMeC, LMeC, CMM-1, and CMM-232,33, were cultured as
described ­previously6. The canine osteosarcoma cell lines ­POS34 and H­ MPOS35 were cultured in RPMI 1640
medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine,
200 μg/mL streptomycin, and 200 U/mL penicillin (Thermo Fisher Scientific) at 37 °C under a 5% C
­ O2 atmosphere. Canine PBMCs were purified from heparinized blood obtained from healthy beagles by density gradient
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centrifugation on Percoll (GE Healthcare UK, Buckinghamshire, UK) and cultured as described p­ reviously6.
PBMCs were stimulated with 5 μg/mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich) and 1 μg/mL anticanine CD28 antibody (eBioscience, San Diego, CA). In some experiments, cells were co-treated with 5 μM
meloxicam (Sigma-Aldrich), 2.5 μM Prostaglandin E2 (Cayman Chemical), and/or 20 μg/mL canine chimericanti-PD-L1 antibody c­ 4G1211 as indicated. The same concentrations of DMSO (Nacalai Tesque, Kyoto, Japan)
and dog IgG (Jackson ImmunoResearch, West Grove, PA) were used as negative controls.

Reverse transcription quantitative polymerase chain reaction (RT‑qPCR).

Total RNA wasextracted from canine cancer cell lines using TRI reagent (Molecular Research Center, Cincinnati, OH) and
residual genomic DNA was digested by DNase I (Thermo Fisher Scientific) treatment. cDNA was synthesizedfrom 1 μg of RNA using PrimeScript RTase (TaKaRa, Otsu, Japan) and oligo-dT primer in the presence of 20
U RNase inhibitor (Promega, Madison, WI). For estimation of COX2 expression (GenBank accession number NM_001003354.1), qPCR was conducted in a reaction mixture including the primers 5′-AAG CTT CGA
TTG ACC AGA GCA G-3′ and 5′-TCA CCA TAA AGG GCC TCC AAC-3′, 1 μL cDNA, and SYBR Premix
DimerEraser (TaKaRa) using the LightCycler480 System II (Roche Diagnostics, Mannheim, Germany). HPRT1(GenBank accession number AY283372.1) was used as an internal control g­ ene13. Relative COX2 mRNA expression level was calculated relative to HPRT1 expression.

Statistical analysis. Paired data were compared by Wilcoxon signed rank test, unpaired data by Mann–
Whitney U test, and > 2 group data by Steel–Dwass test. OS was compared between groups stratified according tothe optimal cutoff value for each measured factor (high group > cutoff and low group ≤ cutoff) by Kaplan–Meieranalysis. The optimal cutoff value for each factor was defined as that yielding the most significant split by logrank test as described by Budczies et al. (Cutoff Finder)36. OS was defined as time in days from the first dose of
c4G12 until death. All deaths except for one were considered tumor-related and euthanasia was performed in
one dog due to disease ­progression16 (Supplementary Table 1). Receiver operating characteristic (ROC) curves
for each serum factor were generated in relation to longer survival (OS > median) or tumor response as evidenced by diagnostic i­maging16. Youden’s index was used to determine the cutoff value for calculation of sensitivity and specificity. Statistical analysis was performed using Fisher’s exact test. All tests were performed using
EZR statistical ­software37, and P < 0.05 (two-tailed) was considered statistically significant.

Results

Multiple serum factors are upregulated in dogs with pulmonary metastatic OMM. To examine
whether serum levels of ­PGE2, various cytokines, chemokines, and growth factors are of clinical relevance incanine OMM, we first compared serum concentrations between healthy controls (n = 8) and dogs with pulmonary metastatic OMM (n = 27 among a cohort of 29 ­dogs16) before the first dose of c4G12 using ELISA and abead-based immunoassay panel (Fig. 1a). The baseline characteristics of c4G12-treated dogs, treatment outcome, and serum concentration of each factor are shown in Supplementary Tables 1 and 2. Among these 12 factors tested, P­ GE2, IL-12p40, IL-8, MCP-1, and SCF were quantifiable in most dogs and the concentrations weresignificantly higher in OMM dogs than healthy control dogs (Fig. 1b). In addition, VEGF-A was measurable in
22 OMM dogs (81.5%) and five healthy dogs (62.5%), and serum concentration tended to be higher in OMM
dogs (Fig. 1b). The serum concentrations of the other six measured factors were below the LLOQ in too many
animals to allow statistical comparison.
Baseline concentrations of serum factors are associated with overall survival in dogs withpulmonary metastatic OMM receiving anti‑PD‑L1 antibody therapy. To explore serum biomark-ers predictive of clinical outcome among dogs with OMM receiving anti-PD-L1 therapy, the OMM group wasdichotomized according to serum concentration of each factor and the OS was compared between high (> cutoff) and low (≤ cutoff) groups by Kaplan–Meier analysis. Higher serum levels of ­PGE2, MCP-1, and VEGF-A
were associated with worse OS in animals receiving c4G12 therapy (P = 0.038, 5.7 × ­10−5, and 0.014, respectively;
Fig. 2). In contrast, higher serum IL-2, IL-12p40, and SCF were correlated with improved OS (P = 0.045, 0.034,
and 0.012, respectively; Fig. 2). There were no significant associations between other serum factors (IFN-γ,
IL-6, IL-10, TNF-α, IL-8, and NGF-β) and OS (P > 0.05, Fig. 2). To help interpret the predictive power of each
serum factor, ROC analysis was performed in relation to longer survival (OS > median) or tumor response. Area
under the curve (AUC), sensitivity, and specificity for each factor are shown in Tables 1 and 2. Partially consistent with the Kaplan–Meier analysis, P­ GE2 and IL-6 were significant predictors of longer survival (P = 0.033 and0.031, respectively), whereas IL-2 and IL-6 predicted tumor response to c4G12 treatment (P = 0.030 and 0.013,
respectively).

The COX‑2/PGE2 axis is a potential immune evasion mechanism in canine cancers. Based on

the significant association between elevated serum P­ GE2 and shorter OS in OMM dogs receiving c4G12 therapy, we speculated that the COX-2/PGE2 pathway suppresses antitumor immunity and thus confers resistanceagainst anti-PD-L1 therapy. Consistent with previous studies demonstrating high COX-2 expression in canine
cancers including ­melanoma23, all canine melanoma cell lines (CMeC, LMeC, CMM-1, and CMM-2) and osteosarcoma cell lines (POS and HMPOS) examined had detectable COX2 mRNA expression (Fig. 3a). To verify thatperipheral blood ­PGE2 levels are elevated in dogs with tumor, plasma concentration of ­PGE2 was measured inanother cohort of dogs with various tumor (n = 21; see Supplementary Table 3 for details). Indeed, plasma P­ GE2
concentration was higher in tumor dogs compared to a healthy control group (n = 8; Fig. 3b), suggesting that
­PGE2 is generally upregulated in canine tumor, possibly through aberrant expression of COX-2 in tumor tissues.
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Figure 1.  Serum concentrations of several immune modulators are elevated in dogs with pulmonary metastaticoral malignant melanoma (OMM). (a) Heat map depicting the serum concentrations of each measured factor(as Z-Scores) in dogs with OMM prior to treatment (n = 27) and in healthy dogs (n = 8). Missing values (below
the lower limit of quantitation [LLOQ]) are shown in dark green. (b) Comparison of serum concentration
between OMM and healthy dogs. Red bars indicate the median values. Values below LLOQ are shown as open
circles. Statistical analysis was performed using Mann–Whitney U test.
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Figure 2.  Baseline serum concentrations of several immune modulators are associated with overall survival (OS) in dogs withpulmonary metastatic OMM receiving c4G12 therapy. Dogs were dichotomized into subgroups based on the indicated cutoff valuefor (a) Prostaglandin E2 (­ PGE2), (b) interferon-γ (IFN-γ), (c) interleukin (IL)-2, (d) IL-6, (e) IL-10, (f) IL-12p40, (g) tumor necrosis
factor-α (TNF-α), (h) IL-8, (i) monocyte chemotactic protein 1 (MCP-1), (j) nerve growth factor-β (NGF-β), (k) stem cell factor (SCF),
and (l) vascular endothelial growth factor-A (VEGF-A), and Kaplan–Meier curves were constructed comparing each corresponding
subgroup (high group > cutoff vs. low group ≤ cutoff). Statistical analysis was performed using log-rank test.

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P value**

AUC (95% CI)

Cutoff*Sensitivity

Specificity

PGE2

0.727 (0.501–0.954)

6.5

0.636

0.846

0.033

IFN-γ

0.566 (0.416–0.717)< LLOQ

0.231

0.909

0.596

IL-2

0.626 (0.398–0.854)

17.7

0.545

0.769

0.206

IL-6

0.535 (0.299–0.771)

142.4

0.364

1.000

0.031

IL-10

0.622 (0.412–0.833)

18.4

0.455

0.846

0.182

IL-12p40

0.587 (0.341–0.834)

388.5

1.000

0.308

0.098

TNF-α

0.521 (0.362–0.680)

4.7

0.182

0.923

0.576

IL-8

0.573 (0.329–0.818)

1980.8

0.727

0.538

0.240

MCP-1

0.664 (0.440–0.889)

48.7

0.273

1.000

0.082

NGF-β

0.521 (0.362–0.680)

28.0

0.182

0.923

0.576

SCF

0.741 (0.531–0.951)

87.7

0.727

0.692

0.100

VEGF-A

0.594 (0.353–0.835)

22.1

0.909

0.462

0.211

Table 1.  ROC analysis of each serum factor in relation to longer survival. The sensitivity and specificity ofeach factor to predict longer survival (OS > median) are shown. AUC, area under the curve; CI, confidence
interval; LLOQ, lower limit of quantification. *Cutoffs were determined by calculating the Youden’s index.
**Fisher’s exact test.

P value**

AUC (95% CI)

Cutoff*Sensitivity

Specificity

PGE2

0.618 (0.285–0.951)

3.1

0.400

0.955

0.628

IFN-γ

0.614 (0.524–0.703)< LLOQ

1.000

0.227

0.547

IL-2

0.818 (0.560–1.000)

33.0

0.800

0.864

0.030

IL-6

0.727 (0.414–1.000)

142.4

0.600

0.955

0.013

IL-10

0.691 (0.391–0.991)

84.6

0.400

1.000

0.136

IL-12p40

0.727 (0.470–0.985)

651.9

1.000

0.455

0.561

TNF-α

0.664 (0.407–0.920)

4.7

0.400

0.955

0.079

IL-8

0.618 (0.317–0.920)

2007.0

0.800

0.591

0.326

MCP-1

0.718 (0.524–0.913)

115.2

1.000

0.591

0.474

NGF-β

0.641 (0.380–0.902)

28.0

0.400

0.909

0.144

SCF

0.773 (0.520–1.000)

125.0

0.800

0.818

0.165

VEGF-A

0.500 (0.228–0.772)

17.9

0.600

0.636

1.000

Table 2.  ROC analysis of each serum factor in relation to tumor response. The sensitivity and specificity ofeach factor to predict tumor response are shown. AUC, area under the curve; CI, confidence interval; LLOQ,
lower limit of quantification. *Cutoffs were determined by calculating the Youden’s index. **Fisher’s exact test.

To investigate the immunosuppressive potential of P
­ GE2 in dogs, PBMCs from healthy dogs were cultured
with the nonspecific T cell stimulators SEB and anti-CD28 antibody for 3 d in the presence of ­PGE2. ­PGE2 treatment significantly reduced IL-2 and IFN-γ production by stimulated canine PBMCs (Fig. 3c,d), suggesting that
­PGE2 is a potent suppressor of canine T cell responses.

Combined COX‑2 inhibition and anti‑PD‑L1 antibody treatment enhances cytokine produc‑tion from canine PBMCs. The selective COX-2 inhibitor meloxicam is used routinely for anti-inflamma-tion and analgesia in veterinary practice. To assess whether ­PGE2 production from canine cancer cells is COX-2
dependent, the CMM-1 and HMPOS cell lines, both with relatively high baseline COX2 expression (Fig. 3a),were treated with meloxicam. ­PGE2 concentrations in the culture supernatant were significantly reduced after
3 d of meloxicam treatment compared to untreated control cells (Fig. 4a). In the tumor microenvironment,
infiltrating immune cells can be an additional source of P
­ GE238. Indeed, canine PBMCs stimulated in vitro with
SEB and anti-CD28 antibody produced detectable amount of ­PGE2 that was significantly reduced by meloxicam(Fig. 4b).
To evaluate the immunostimulatory potential of meloxicam, PBMCs were treated with meloxicam alone orin combination with the anti-PD-L1 antibody c4G12. Both meloxicam and anti-PD-L1 antibody enhanced IL-2
production from stimulated canine PBMCs when applied individually, whereas the increase in IFN-γ production was not statistically significant. However, combination treatment further enhanced IL-2 production andsignificantly increased IFN-γ production (Fig. 4c,d), suggesting that dual blockade of PD-1/PD-L1 and COX-2/
PGE2 pathways is a promising strategy to enhance antitumor immune responses in dogs.

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Figure 3.  PGE2 is a potential immunosuppressive factor in canine cancer. (a) COX2 mRNA expression incanine cancer cell lines. Total RNA was extracted from each cancer cell line and COX2 mRNA expression
was measured by RT-qPCR. HPRT1 expression was used as an internal control. The mean values of triplicate
measurement are shown. Error bars indicate the standard deviation (SD). (b) Plasma ­PGE2 concentrationin dogs with tumors. Plasma samples were collected from healthy (n = 8) and tumor-bearing dogs (n = 21).
Red bars indicate the median values. Statistical analysis was performed using Mann–Whitney U test. (c,d)
Suppression of cytokine production from canine peripheral blood mononuclear cells (PMBCs) by P
­ GE2. Canine
PBMCs were cultured for 3 d with or without 2.5 μM ­PGE2, and concentrations of (c) IL-2 (n = 7) and (d) IFN-γ(n = 15) in the supernatant were measured by ELISA. Red bars indicate the median values. Statistical analysis
was performed using Wilcoxon signed rank test.

Discussion

Immunotherapy is now recognized as the “fourth pillar of human cancer treatment”, with clinical importanceequivalent to surgery, radiotherapy, and traditional chemotherapy. The applications of cancer immunotherapy
are spreading, particularly as combination treatment, including with molecular-targeted drugs. In veterinary
medicine, a few clinical studies, including from our group, have examined the efficacy of immune checkpoint
blockade for dogs with cancer; however, as in human ­cancer2,3,39, only a subset of dogs responded well to ICI­therapy11,16,40. The identification of biomarkers predictive of ICI response will not only optimize treatment efficacy
but may also provide clues to the mechanisms underlying tumor immune evasion in dogs as well as humans.
Indeed, dogs are an excellent animal model for human cancer research because they develop naturally occurring
tumors that share many features with the corresponding human cancers, such as invasiveness and metastatic
propensity, molecular aberrations, heterogeneity, and response/resistance to ­treatment41. As dogs are immunocompetent and genetically outbred, they could be especially useful for testing immunotherapy and precision
medicine for cancers. Therefore, the identification of biomarkers and combination therapies for canine cancer
may not only improve veterinary practice but also benefit human clinical oncology.
Serum ­PGE2, IL-12p40, IL-8, MCP-1, and SCF concentrations were higher in dogs with OMM than healthy
controls, providing clues to the immunological changes associated with tumorigenesis and progression of OMM,
including resistance to endogenous antitumor mechanisms. These findings are consistent with previous reports
showing elevated serum IL-12p40 in ­osteosarcoma42, IL-8 in mammary tumor and ­osteosarcoma42–44, and MCP-1
in lymphoma, histiocytic sarcoma, urothelial carcinoma, and malignant ­melanoma45–49 compared to healthy
dogs. Upregulation of these factors may reflect immunological responses in the tumor microenvironment, but
the cellular and locoregional sources of these factors remain to be investigated.
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Figure 4.  The cyclooxygenase-2 (COX-2) inhibitor meloxicam reduces P­ GE2 production and enhances
cytokine production by canine PBMCs with or without anti-programmed death ligand 1 (PD-L1) antibody
treatment. (a,b) Suppression of P
­ GE2 production by the COX-2 inhibitor meloxicam. Cells were cultured
for 3 d with 5 μM meloxicam, and ­PGE2 concentration in the supernatant was measured by ELISA. (a) ­PGE2
production from canine cancer cell lines CMM-1 and HMPOS. Mean values of triplicate measurement are
shown. Error bars indicate the SD. Statistical analysis was performed using Mann–Whitney U test. (b) ­PGE2
production from canine PBMCs (n = 7). Red bars indicate the median values. Statistical analysis was performed
using Wilcoxon signed rank test. (c,d) Enhancement of cytokine production from canine PBMCs by meloxicam
treatment. Canine PBMCs were cultured for 3 d with 5 μM meloxicam and/or 20 μg/mL anti-PD-L1 antibody
c4G12, and (c) IL-2 (n = 11) and (d) IFN-γ (n = 15) concentrations in the supernatant were measured by ELISA.
Each point represents the relative cytokine concentration compared to no treatment control prepared from thesame individual. Red bars indicate the median values. Statistical analysis was performed using Steel–Dwass test(*P < 0.05).
Higher serum MCP-1 and VEGF-A concentrations were associated with poorer OS in OMM dogs receiving
c4G12 therapy, in accordance with previous reports on human melanoma patients under ICI ­therapy21,50. MCP-1
is an inducible chemokine regulating macrophage infiltration into t­ umors51. In humans, both primary and metastatic melanomas, but not normal skin, express MCP-152, and antagonism of its receptor CCR2 enhanced the therapeutic efficacy of anti-PD-1 antibody in a murine melanoma m­ odel53. Considering the protumor propensity oftumor-associated ­macrophages54, it is plausible that canine OMM tissues overexpress MCP-1 and thereby attract
monocytes/macrophages, which in turn create an immunosuppressive microenvironment and confer resistanceto anti-PD-L1 therapy. Similarly, VEGF-A is also immunosuppressive in the tumor ­microenvironment55 inaddition to acting as the primary driver of tumor angiogenesis. Numerous human clinical studies have revealed
that anti-VEGF agents in combination with ICIs show promising efficacy against neoplasms such as renal cell
carcinoma and hepatocellular ­carcinoma56. Although a similar immunosuppressive activity has not been demonstrated in dogs, VEGF overexpression has been demonstrated by immunohistochemistry in canine ­OMM57. Thus,
combined VEGF targeting and ICI therapy warrants further investigation. The prolonged OS observed in OMM
subgroups exhibiting high serum IL-2, IL-12p40, and SCF concentrations is also suggestive of a strong association
between antitumor immune response and clinical outcome. Both IL-2 and IL-12 contribute to T cell activation,
so the baseline serum concentrations of these cytokines may indicate antitumor immune status in OMM dogs.
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In contrast, the mechanistic implications of elevated SCF, a growth factor/cytokine involved in hematopoiesis
through binding to its receptor c-Kit58, are still unclear as relevance of SCF to antitumor immunity has not been
established. As a limitation of the survival analyses, only a small number of dogs (n = 27) were included, allowing
only exploratory univariate analyses. Because the univariate analysis may contain multiple biases (e.g., breeds,
age, and sex), the results should carefully be interpreted. Multivariate analyses of a larger cohort and validation in
an independent cohort are needed to determine which of these aforementioned factors can be used as predictive
biomarkers in veterinary practice. In addition, further investigation is needed to clarify whether these potential
biomarkers are specific to anti-PD-L1 therapy or also applicable to other treatment modalities of canine OMM.
Prostaglandin ­E2 is a well-known mediator of acute local ­inflammation38. However, P­ GE2 also serves as an
immunosuppressive factor in late or chronic phases by reducing T cell, NK cell, and dendritic cell functions
and by promoting regulatory T cell ­development38. The ­PGE2 biosynthetic enzyme COX-2 is often upregulated
in premalignant and malignant t­ issues59, and promotes cancer cell survival, metastasis, and ­angiogenesis59,60 as
well as immune e­ vasion61. Therefore, pharmacological targeting of COX-2 is considered a promising approachfor cancer prevention and ­therapy59. Recently, crosstalk between the PD-1/PD-L1 and COX-2/PGE2 pathways
was ­reported28,62,63. In mouse tumor models, COX inhibition by aspirin or celecoxib enhanced the efficacy ofanti-PD-1 ­antibody28, implying that inhibition of the COX-2/PGE2 pathway could be a useful adjuvant to ICI
treatment. Indeed, we previously reported that dual blockade of these pathways enhanced ICI therapeutic efficacy in cattle with bovine leukemia virus infection and in a mouse lymphoma m­ odel64,65, suggesting that thiscombination strategy is applicable across animal species and diseases.
To our knowledge, this is the first report to demonstrate that, in any animal species, baseline serum ­PGE2 concentration is associated with clinical outcome of ICI therapy and that P­ GE2 is a suppressor of canine immune cell
activation. We also demonstrate that COX-2 inhibition plus anti-PD-L1 antibody treatment enhances immune
cell activation, further supporting the potential of this combination therapy for treatment of canine cancers
including OMM. Given that COX-2 overexpression is a common feature of various cancer types, that COX-2
inhibitors are already widely used in clinical practice with well-known and manageable side effects profiles, andthat COX-2 activity is involved in malignant phenotype independent of immune suppression, we suggest that
COX-2 inhibitors are broadly applicable, safe, and effective drugs for ICI combination therapy. Nonetheless,careful attention should be paid to possible side effects of the combination treatment. Treatment-related adverse
events of c4G12 included pneumonitis, elevated liver enzymes and lipase, vomiting, and d­ iarrhea16, while meloxicam treatment is reported to be associated with gastrointestinal (vomiting, diarrhea, and ulceration), urinary(azotemia and renal failure), hepatic (elevated liver enzymes), and dermatologic (pruritus) ­abnormalities66.
Because gastrointestinal and hepatic toxicities are suggested for both treatments, frequency and severity of these
events must be monitored in particular in future studies on the combination therapy.
In conclusion, we have identified several serum factors including P­ GE2 as potential predictive biomarkers of
prolonged survival in dogs with OMM receiving anti-PD-L1 therapy. Further, we identified the COX-2/PGE2
axis as a potential target to enhance the efficacy of ICI therapy in dogs. The overall consistency with humanbiomarker analyses and preclinical studies suggests that dogs could be a clinically relevant, large animal modelfor ICI therapy. The clinical study of this combination therapy for canine cancer is now in progress in our veterinary hospital.
Received: 12 November 2021; Accepted: 13 May 2022

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Author contributions

N.M., S.K., T.O., S.M., and K.O. designed and supervised the project. N.M., Y.A., Y.S., T.D., K.W. and H.T. performed the experiments. N.M., S.K., Y.A., and Y.S. analyzed the data. T.D., S.T., K.H., S.K., H.O., Y.K., and Y.S.
provided materials, reagents and/or analysis tools. N.M., S.K., and Y.A. prepared the manuscript. All authors
reviewed and approved the manuscript.

Funding

This work was supported by the Grant-in-Aid for Scientific Research (Grant Numbers 16K15042, 19H03114,
19K15969, and 21K1498301) and Grant-in-aid for JSPS Fellows (Grant Number 15J01989) from the Japan Society
for the Promotion of Science (JSPS) and by Japan Agency for Medical Research and Development (AMED) underGrant Number: JP21am0101078. This work was also supported by Clinical Research Promotion Fund 2020 by
Hokkaido University Veterinary Teaching Hospital. Funders had no role in the study design, data collection andanalysis, decision to publish, and manuscript preparation.

Competing interests

S Konnai, KO, SM, TO, NM, and Y Suzuki are authors of a patent application that covers materials and techniques
described in this paper (PCT/JP2018/27041). All other authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​022-​13484-8.
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