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Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function

Background Monocytes/macrophages are likely key cells in immune modulation in dogs with osteosarcoma (OSA). Increased peripheral monocyte counts are negatively correlated with shorter disease‐free intervals in dogs with OSA. Understanding the monocyte/macrophage's modulatory role in dogs with OSA can direct further studies in immunotherapy development for OSA. Hypothesis/Objectives That OSA evades the immune response by down‐regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses. Animals Eighteen dogs with OSA that have not received definitive treatment and 14 healthy age‐matched controls Methods Clinical study—expression of peripheral blood monocyte cell surface receptors, monocyte mRNA expression and cytokine secretion, monocyte chemotaxis, and survival were compared between clinical dogs with OSA and healthy control dogs. Results Cell surface expression of multiple chemokine receptors is significantly down‐regulated in peripheral blood monocytes of dogs with OSA. The percentage expression of CCR2 (median 58%, range 2–94%) and CXCR2 expression (median 54%, range 2–92%) was higher in control dogs compared to dogs with OSA (CCR2 median 29%, range 3–45%, P = 0.0006; CXCR2 median 23%, range 0.2–52%, P = 0.0007). Prostaglandin E2 (PGE 2) (OSA, median 347.36 pg/mL, range 103.4–1268.5; control, 136.23 pg/mL, range 69.93–542.6, P = .04) and tumor necrosis factor‐alpha (TNF‐α) (P = .02) levels are increased in OSA monocyte culture supernatants compared to controls. Peripheral blood monocytes of dogs with OSA exhibit decreased chemotactic function when compared to control dogs (OSA, median 1.2 directed to random migration, range 0.8–1.25; control, 1.6, range of 0.9–1.8, P = .018). Conclusions and Clinical Importance Dogs with OSA have decreased monocyte chemokine receptor expression and monocyte chemotaxis, potential mechanisms by which OSA might evade the immune response. Reversal of monocyte dysfunction using immunotherapy could improve survival in dogs with OSA.

A ppendicular osteosarcoma (OSA), the most common primary bone malignancy in dogs, has a high rate of metastasis and a poor prognosis. 1,2 The overall median survival time from time of diagnosis for dogs that receive standard-of-care therapy (primary tumor removal and adjuvant chemotherapy) is approximately 10-12 months, with a 20% 2-year survival rate. 1 Metastasis is the primary cause of death in dogs with OSA, with approximately 90% of dogs already harboring occult metastatic disease at diagnosis. 1 Controlling metastatic disease is crucial to improving outcomes in OSA.
Tumor-induced immune suppression is one of the mechanisms by which malignancies ensure survival. For example, tumors recruit monocytes and macrophages and induce pro-tumorigenic changes in their phenotypes and function leading to a microenvironment that favors tumor growth. 3 OSA cells from dogs secrete Transforming Growth Factor-beta (TGF-b), which likely prostaglandin E 2 qPCR quantitative RT-PCR SSC side scatter TGF-b transforming growth factor-beta TNF-a tumor necrosis factor-alpha contributes to immune suppression as TGF-b is considered an inhibitory immunoregulatory cytokine. [4][5][6] Interestingly, dogs with OSA who develop infections after limb-spare surgery have been observed to almost double their median survival time, potentially via up-regulation of antitumor immunity. 7 Dogs with OSA and infections were half as likely to die, half as likely to develop metastasis, and these survival effects were because of a delay in metastasis rather than control of local tumor recurrence. 7 Studies in mice have confirmed these observations and suggest that the antitumor effects of infection could be modulated via monocytes and macrophages. Mice with chronic osteomyelitis that developed OSA experienced improved survival, had increased numbers of inflammatory monocytes, and depletion of monocytes/macrophages in these mice reversed the survival benefit afforded by osteomyelitis. 8 Understanding the role of immunomodulatory cells such as monocytes in creating a protumorigenic environment in dogs with OSA will inform future investigations to identify therapeutic targets to reverse tumor-induced immune dysfunction and inhibit metastatic disease. To this end, we sought to identify phenotypic and functional differences in monocytes between dogs with OSA and healthy dogs. The objectives of our study were (1) to compare cell surface receptor expression between monocytes from OSA and healthy dogs, (2) to compare mRNA expression and cytokine secretion between monocytes from OSA and healthy dogs, (3) to compare chemotactic differences between monocytes from OSA and healthy dogs, and (4) to analyze associations between OSA survival and peripheral blood monocyte counts/receptor expression. Our hypothesis was that OSA evades the immune response by down-regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses.

Patient Enrollment and Sample Collection
Client-owned dogs diagnosed with OSA that had not received any tumor-directed therapy or immunomodulatory drugs were enrolled into the study. Because of the enrollment of dogs at a tertiary referral center, some dogs with OSA had been prescribed anti-inflammatory medications by their primary referring veterinarian. A similar population of age-matched medium and largebreed control dogs without known disease that were also not receiving any anti-inflammatory or immunomodulatory medications were recruited for the study by the North Carolina State University (NCSU) Clinical Studies Core. Verbal and written owner consent were obtained for all dogs participating in the study, and peripheral blood samples were collected under an approved Institutional Animal Care and Use Committee (IACUC) protocol.

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and of a Purified Monocyte Population
Approximately 25 mL of peripheral blood was collected into EDTA blood collection tubes from 14 control dogs and 18 dogs with OSA via venipuncture. Five mL blood was submitted for complete blood counts (CBC) in 10 healthy controls and 18 dogs with OSA. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using HistopaqueÒ-1077. a A portion of the PBMCs from 13 healthy controls and 18 dogs with OSA was preserved for cell surface staining, and a portion underwent cell sorting to isolate a purified monocyte population using a MoFloÒ high-speed cell sorter. b Monocyte cell isolation was performed based on forward (FSC) versus side scatter (SSC), and CD14+ expression, and the phenotype of the sorted monocytes was confirmed via light microscopy. 9 Approximately 1 9 10 6 sorted monocytes from 7 healthy controls and 7 dogs with OSA were centrifuged and resuspended in RNAprotect c cell reagent for PCR analysis. Sorted monocytes from 8 healthy controls and 7 dogs with OSA were used for stimulation cultures, and sorted monocytes from 14 healthy controls and 5 dogs with OSA were used for chemotaxis assays, as outlined below.

Flow Cytometry (FACS) Analysis
A portion of the PBMCs from 13 healthy controls and 18 dogs with OSA were centrifuged, resuspended in 2 mL Phosphate-Buffered Saline (PBS), then stained with the following antibodies against cell surface receptors: anti-CD14, d anti-CD16, e anti-CD62L, f anti-CD32 e , anti-CD11c, g anti-CCR2, h anti-CCR7 h , anti-CD43 h , anti-CX 3 CR1 h , and anti-CXCR2 h ( Table 1). A tube containing unstained cells served as a negative control, and the appropriate isotype controls (IgG2a PE f , IgG1 FITC f , rabbit polyclonal e conjugated antibodies) were applied. Single color compensation controls were established at the start of these experiments, and the data shown here represent multicolor analysis. All antibodies were directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC). The stained cells were incubated for 20 minutes on a shaker at lowspeed, in the dark, at room temperature, after which the cells were washed twice with PBS. A FacsCaliber i flow cytometer was used to perform FACS analysis, and the data were analyzed using the accompanying CellQuest TM software. Monocytes were gated based on FSC and SSC (Fig 1a), and gates for determining the percentage of monocytes that stained positive for antibody were set using isotype controls as guidelines.
NanoDrop 2000c spectrophotometer g , and also used gel electrophoresis to confirm RNA quality ( Figure S1a). cDNA was subsequently synthesized in a Techne TC4000 j thermal cycler using the Promega© RT kit k according to manufacturer's directions. Primers for qPCR to detect mRNA expression levels of Interleukin-10 (IL-10), Interleukin-12 (IL-12), Tumor Necrosis Factor-alpha (TNF-a), and Cyclooxygenase-2 (COX-2) were selected based on previous reports (Table 2), 10,11 and purchased from Integrated DNA Technologies. l The PerfeCTa Ò SYBR Ò Green FastMix Ò for iQ TM PCR kit m was used for mRNA expression detection, and the reactions performed with the iCycler iQ TM real-time PCR detection system. n All samples were run in triplicate. The thermal cycling conditions were as follows: cycle 1 (95.0°C for 8 minutes) 1X, cycle 2 (95.0°C for 30 seconds, 56.0°C for 20 seconds, 72.0°C for 40 seconds) 40X, cycle 3 (melting curve starting at 60.0°C for 10 seconds, with stepwise increases of 0.5°C for 70X to achieve 95°C), and cycle 4 (hold at 4.0°C). The housekeeping gene, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), was used for normalization of results. The CT value of GAPDH was consistent across experiments and between animals for all PCR results reported here (CT =~21). The PCR product was separated using agarose gel electrophoresis, the DNA was sequenced by GENE-WIZ Ò DNA sequencing services, and the sequences were subjected to alignment searches in the NCBI database to confirm matches with the gene of interest ( Figure S1b). The 2 ÀDDCT method was used to derive the normalized relative mRNA expression level of the target genes.

Prostaglandin ELISA
Sorted monocytes from 8 healthy controls and 7 dogs with OSA were used for stimulation experiments-half of these monocytes served as unstimulated controls and incubated for 6 hours at 37°C and 5% CO 2 . The other half were stimulated with 100 ng/ 10 6 monocytes of lipopolysaccharide (LPS a ) and incubated similarly for 6 hours at 37°C and 5% CO 2 . The resultant supernatants were collected, stored at À20°C until they were assessed for PGE 2 levels using a commercially available ELISA kit o according to manufacturer's directions.

IL-10, IFN-c, TNF-a Magnetic Bead Panel Assay
Sorted monocytes from 5 healthy controls and 5 dogs with OSA were used for stimulation experiments as described for the prostaglandin ELISA. The resultant supernatants were collected, stored at À20°C until they were assessed for IL-10, IFN-c, and TNF-a levels using a commercially available canine cytokine magnetic bead panel kit (MILLIPLEX Ò MAP) p according to manufacturer's directions. IL-12 was not available as an analyte in the kit, thus IFN-c levels were analyzed as a surrogate, as IL-12 is known to induce IFN-c secretion.

Chemotaxis Assay
Sorted monocytes from 14 healthy controls and 5 dogs with OSA were used for migration assays. Monocyte migration was assessed using a NeuroProbe ChemoTx q 96-well disposable cell migration system with 8 lm polycarbonate framed filters. Sorted monocytes were resuspended in 10 6 /mL of cell culture media, labeled with 1 lg calcein-AM r /10 6 monocytes, and the cells incubated for 30 minutes in the dark at room temperature. Following incubation, the cells were resuspended at 10 6 /mL in Dulbecco's Modified Eagle Medium (DMEM s ). Twenty-five lL of the cell suspension were deposited on each filter. The lower chamber, ie, the 96-well plate, contained the following: 100% heat-inactivated fetal bovine serum (FBS g ), 100 ng/mL monocyte chemoattractant protein-1 (MCP-1 t ), 100 nM N-Formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP a ), 100 ng/mL CCL19 t as positive chemoattractants, and PBS as a negative control. The positive chemoattractants were either placed individually in separate wells, or combined together in a single well. For total cell counts, 25 lL of cells was placed directly into the lower well to quantify maximal fluorescence. The plate was incubated for 4 hours at 37°C, after which the nonmigrated cells on top of the filters were gently wiped off using a cell scraper. Cellular fluorescence in the bottom wells was quantified using a fMax fluorescence microplate reader u at 485 and 538 nm wavelength, and the corresponding SoftMax Pro software. Filter fluorescence was also quantified to confirm that cells were not adherent to the underside of the filter. Degree of migration was calculated as a percentage of migrated cells out of the known cell numbers placed in bottom wells. Cell migration was expressed as a migration index, calculated by dividing directed cell chemotaxis (cells migrating in response to a chemokine, quantified by degree of fluorescence) by random cell chemotaxis (cells migrating in response to media alone, quantified by degree of fluorescence). Each sample was assayed in triplicate.

Statistical Analyses
Statistical analyses were performed to compare monocyte cell surface receptor expression levels between groups using the nonparametric Mann-Whitney U-test because of the non-normal distribution of the data. Comparison of relative mRNA expression of each gene and of PGE 2 levels between groups were analyzed with the Mann-Whitney U-test. All statistical tests were carried out as 2-sided tests, and a P-value of <.05 was considered significant. Comparison of peripheral blood monocyte counts between dogs with OSA and healthy controls was performed using a 2-tailed T-Test. Pearson's correlation coefficient was used to determine the strength and direction of a possible linear relationship between monocyte surface receptors and peripheral blood monocyte counts. Two-way ANOVAs and least-squares means were used to test for differences in monocyte surface receptors because of status and monocyte counts (high versus low). To analyze the chemotaxis data, one-way ANOVAs were run and means were compared using Welch's adjustment for heterogeneous variation. Kaplan-Meier survival analysis was used to compare survival between groups, with proportional hazards regression analysis to test the effect of chemokine receptor expression on survival. Survival was calculated as the number of days from diagnosis of disease to death attributed to disease, or last follow-up if the patient was still

Study Population
The mean (AESD) age at time of OSA diagnosis was 8.7 (AE2.1) years. All dogs with OSA were purebred, with the most common pure breeds being Labrador Retriever

Monocyte Surface Receptor Expression
The primary flow cytometry canine monocyte gates were based on typical forward and side scatter characteristics. Once that forward versus side light scatter gate was established, we ensured that greater than 98% of the CD14+ cells were found within this gate. As CD14 expression on peripheral blood monocytes varies greatly, this ensured that we included the CD14 l,o monocytes for surface receptor analysis (Fig 1a). Monocytes exhibited positive surface expression of CD14, CD16, CD32, CD62L, CCR2, CCR7, CD43, CX 3 CR1, CXCR2, with low levels of CD11c. Irrelevant isotype controls were used for each experiment-the isotype control binding of monocytes was <5%. The median percentages and ranges of positively staining cells for each antibody are listed in the legend accompanying Fig 1b. Comparison of monocyte surface receptors between untreated dogs with OSA and healthy controls revealed a significant decrease in surface receptor expression of CD62L (P < .00001), CCR2 (P = .0006), CCR7 (P = .0004), CD43 (P = .008), CX 3 CR1 (P = .002), CXCR2 (P = .0007) in dogs with OSA, and a significant increase in CD16 expression in dogs with OSA (P = .005) (Fig 1b). Fig 1c depicts representative dot plots with the percentage of cell surface receptor expression in monocytes of a healthy control versus an OSA dog.
A peripheral blood monocyte count of >400 cells/lL has been significantly associated with decreased diseasefree interval in dogs with OSA. 12 Others have reported associations between peripheral blood monocyte counts and outcomes in humans with OSA. 13 Hence, we evaluated the flow cytometry data in conjunction with peripheral blood monocyte counts, using 400 cells/lL as a differentiation point between high and low monocyte counts according to previously reported findings. 12 There was no significant difference in the peripheral blood monocyte counts of dogs with OSA (mean = 514.7 cells/lL, SD = 377.9 cells/lL) compared to healthy controls (mean = 331.9 cells/lL, SD = 192.6 cells/lL; P = .16). Comparison of receptor expression between dogs with high (>400 cells/lL) versus low (<400 cells/lL) monocyte counts revealed a dichotomy of CD14, CD16 expression in dogs with OSA-CD14 expression is high (P = .01) and CD16 expression is low (P = .02) when monocyte counts are high (Table 3). Least-squares means from the 2-way ANOVA comparing relationships between monocyte counts and receptor expression are shown in Table S1.
In healthy controls, there were strong significant positive correlations between chemokine receptors, whereas in dogs with OSA, there were fewer and less robust significant positive correlations between chemokine receptors (Fig 2). The Pearson's correlation values with corresponding P-values are represented in Table S2.
Relative mRNA Expression of Monocyte IL-10, IL-12, TNF-a, and COX-2 TNFa, IL-10, and IL-12 are inflammatory mediators secreted by monocytes and macrophages, and differential levels of TNFa, IL-10, and IL-12 secretion have been described for the various human monocyte subsets, 14,15 reflecting subset-specific functional differences. COX-2 expression in monocytes reflects a proinflammatory state. [16][17][18] We compared relative mRNA expression of IL-10, IL-12, TNFa, and COX-2 in dogs with OSA versus healthy controls to discern potential variation in cytokine secretion that might reflect functional differences. mRNA expression analysis of monocytes from untreated dogs with OSA and healthy controls revealed no significant differences in relative mRNA expression levels of IL-10 (P = .44), IL-12 (P = .41), and TNF-a (P = .14) between the 2 groups. The relative mRNA expression of COX-2 was decreased in dogs with OSA compared to healthy controls, but the difference was not significant (P = .08).
Evaluation of IL-10, IFN-c, TNF-a, and prostaglandin E 2 secretion from monocytes As mRNA levels might not consistently correspond with protein secretion, we analyzed monocyte secretion of the cytokines IL-10, IFN-c, TNF-a, and PGE 2 . Similar to our mRNA evaluation, we compared cytokine secretion from monocytes of dogs with OSA versus healthy controls to assess for functional differences. Secretion of cytokines such as TNF-a, IL-10, IFN-c, and PGE 2 has been noted to differ between human monocyte subsets, and can be affected by disease. 15,17 PGE 2 ELISA and the MILLIPLEX Ò assay revealed increased PGE 2 levels (P = .04) and increased TNF-a levels (P = .02), respectively in supernatants of cultured monocytes stimulated with LPS of untreated dogs with OSA compared to normal controls (Fig 3a, b). The median concentrations and ranges of PGE 2 concentrations were 347.36 pg/mL, 103.4-1268.5 pg/mL for dogs with OSA, and 136.23 pg/ mL, 69.93-542.6 pg/mL for healthy controls. The median concentrations and ranges of TNF-a levels were 934.7 pg/mL, 644.3-2709 pg/mL for dogs with OSA, and 267.2 pg/mL, 70.4-670.4 pg/mL for healthy controls. Levels of IL-10 and IFN-c were low to undetectable in both groups.

Monocyte Chemotaxis
The results of our phenotypic analysis of monocytes between dogs with OSA and healthy controls revealed marked decreases in chemokine receptor expression in dogs with OSA (Fig 1b,c). Therefore, we asked whether monocyte chemotaxis might be altered by OSA. As described in our methods, monocytes from OSA and healthy dogs were assessed by the NeuroProbe Che-moTx Ò cell migration system. Comparison of monocyte chemotaxis between dogs with OSA and healthy controls revealed significantly decreased chemotaxis in dogs with OSA when CCL19 (P = .018), and when a mixture of all 4 chemoattractants (P = .018) was used (Fig 4). With CCL2 or fMLP as chemoattractants, there was also decreased monocyte chemotaxis in dogs with OSA compared to healthy controls, but the difference did not reach significance (CCL2 P = .067; fMLP P = .067).

Survival Analysis
As a peripheral monocyte count of >400 cells/lL has been significantly associated with decreased disease-free interval in dogs with OSA, 12 we performed a Kaplan-Meier survival analysis comparing dogs with OSA with high or low monocyte counts, using 400 cells/lL as a cut-off point. One dog died of unknown cause, and was assumed to have died from OSA. We did not find a statistically significant difference in median survival between the 2 groups (P = .15, Fig 5). Our regression analysis revealed that increasing percentages of CCR2 expression on peripheral monocytes was significantly associated with increasing survival in dogs with OSA (P < .0001).

Discussion
The goal of this study was to determine phenotypic and functional differences between peripheral blood monocytes from untreated dogs with OSA versus healthy age-matched controls. Our hypothesis was that evasion of the immune response by OSA is due in part to down-regulation of monocyte chemokine receptor expression and migratory function, and suppression of host immune responses. We demonstrated a significant decrease in monocyte chemokine receptors, increased PGE 2 and TNF-a secretion by monocytes, and decreased monocyte chemotaxis in dogs with OSA.
Monocytes display a variety of cell surface receptors, including leukocyte chemokine receptors. CD62L, also known as L-selectin, is a cell adhesion molecule present on immune cells such as monocytes, lymphocytes, and NK cells. CD62L mediates the trafficking of these cells to sites of inflammation and peripheral lymphoid tissue by binding to glycoproteins. 19,20 CCR2, a chemokine receptor for MCP-1, is especially prominent in mediating monocyte migration. 21 Similarly, CCR7 regulates migration of leukocytes to secondary lymphoid organs via binding of its ligands CCL19 and CCL21. 20 CD43 can be found on a variety of immune cells including lymphocytes, granulocytes, monocytes, macrophages, and NK cells, and it plays a role in cell adhesion and migration. 22,23 CX 3 CR1, and CXCR2, both G proteincoupled chemokine receptors, mediate lymphocyte, monocyte and granulocyte migration. 20 There are few reports describing the effect of OSA on monocyte surface receptor expression in dogs. For example, down-regulation of MHC class II and CD80 in canine myeloid cells when exposed to tumor cell lines including OSA in vitro has been observed. 24 In humans, the prevalence of an immunosuppressive phenotype in peripheral blood monocytes of OSA patients has been observed. 13 However, to the authors' knowledge, there are no reports evaluating phenotypic changes in monocytes of clinical dogs with OSA.
We assessed the expression of monocyte surface receptors using flow cytometry, and demonstrated that peripheral blood monocytes from dogs with OSA exhibited significantly decreased chemokine receptor expression, namely of CD62L, CCR2, CCR7, CD43, CX 3 CR1, and CXCR2. This pronounced down-regulation of monocyte chemokine receptors of dogs with OSA suggests a peripheral sequestration of monocytes, inhibited from migrating to sites of need such as the primary tumor or metastatic lesions. Two of the chemokine receptors, CCR2 and CXCR2, had significantly higher expression on monocytes of healthy controls with low monocyte counts compared to monocytes of dogs with OSA that had high or low monocyte counts. This supports our theory that the decreased expression of chemokine receptors in dogs with OSA reflects a peripheral sequestration. There was no difference in the mean peripheral monocyte counts between dogs with Table 3. Comparison of receptor expression between dogs with high (>400 cells/lL, n = 11) versus low (<400 cells/lL, n = 17) monocyte counts. In dogs with OSA, when monocyte counts are high, there is a dichotomy of CD14, CD16 expression-CD14 is high (P = .01) and CD16 is low (P = .02).  OSA and healthy controls, which could indicate that this parameter is not as sensitive for detecting a peripheral sequestration compared to analyzing the relationship between chemokine receptors and monocyte counts in both groups of dogs. Alternatively, the lack of difference in mean monocyte counts could also refute our theory of peripheral monocyte sequestration in OSA. We found a strong positive correlation between expression of chemokine receptors in healthy controls, whereas the correlation was less robust in dogs with OSA, suggesting a disturbance in the regulation of these chemokine receptors. A retrospective study of dogs with OSA found that dogs with peripheral monocyte counts above 400 cells/lL had a significantly shorter disease-free interval. 12 Similarly, in human OSA patients, a low peripheral lymphocyte-to-monocyte ratio (≤3.43) was associated with poorer prognosis for survival and disease-free interval. 25 One possible explanation is that higher monocyte counts reflect a peripheral sequestration of monocytes in dogs that had more rapid tumor progression, and our findings of suppressed expression of multiple monocyte chemokine receptors in dogs with OSA suggest such an association. We did not detect a significant overall difference in peripheral monocyte counts of dogs with OSA versus healthy controls in our study population. Our Kaplan-Meier survival analysis comparing dogs with OSA with high (>400 cells/lL) versus low (<400 cells/lL) monocyte counts did not reveal significant differences in survival between groups. However, we did find that increasing percentages of CCR2 expression on monocytes of dogs with OSA was significantly associated with increasing survival, which, in conjunction with our chemotaxis data, suggests that suppression of chemokine receptors and chemotaxis is a form of tumor-mediated monocyte dysfunction.
Additionally, we found that dogs with OSA with a peripheral monocyte count of >400 cells/lL exhibited a dichotomy of CD14, CD16 expression on monocytes-CD14 expression was high and CD16 expression was low, when compared to dogs with monocyte counts <400 cells/lL. Human monocytes have been classified into subsets, with different phenotypes and functions. Earlier classification schemes split human monocytes into CD14 high CD16-"inflammatory, classical" and CD14 low CD16+ "resident, nonclassical" monocytes. 26,27 Recent studies have shown that the CD16+ monocytes can be further characterized into intermediate and nonclassical subsets, with the intermediate subset expressing higher CD14 and lower CD16 levels, and the nonclassical subset conversely expressing lower CD14 and higher CD16 levels. 15 The function of these monocyte subsets has not been fully defined, but the intermediate subset appears to produce proinflammatory cytokines such as TNF-a, IL-1b, IL-6, express high levels of MHC II antigen processing and presentation genes, and are potent stimulators of T cells. 15 There are no corresponding studies differentiating canine monocytes into subsets. Based upon human classification, our data suggest that there is an increase in intermediate monocytes in dogs with OSA with monocyte counts >400 cells/lL. Potentially, these intermediate monocytes, with their proinflammatory and immunostimulatory capacity, are effective antitumor monocytes that are sequestered in the periphery in OSA.
Monocyte migration can reportedly be affected by neoplasia. Peripheral monocyte migration is inhibited by lactate concentrations common in tumor microenvironments, and this loss of migratory ability is not because of a reduction in cell viability. 28 Chemokine receptors such as CCR5 are down-regulated in monocytes and macrophages in human patients with head and neck squamous cell carcinoma, leading to dysregulation of chemotactic receptor-ligand signaling and decreased monocyte/macrophage migratory ability. 29 Based on findings by other investigators and our demonstration of profound down-regulation of monocyte chemokine receptors in dogs with OSA, we hypothesized that OSA inhibits monocyte chemotaxis as an immunosuppressive strategy. Using an in vitro migration assay, we demonstrated decreased monocyte chemotaxis in dogs with OSA compared to healthy controls. This difference was significant when CCL19 was used as a chemoattractant, and when supraphysiologic combinations of chemoattractants were used-a combination of FBS, CCL2, CCL19, and fMLP. CCL19 is a ligand for the chemokine receptor CCR7, and is a known monocyte chemoattractant. 30 There was also decreased monocyte migration in dogs with OSA when other known chemokines such as CCL2 or fMLP were used, and even though the differences were not significant, the proximity of the P-value (.067) to significance (.05) suggests that use of these chemokines deserves further evaluation. CCL2 is also known as monocyte chemotactic protein, binds CCR2, and together with fMLP, which binds to G protein-coupled receptors, is used as chemoattractants for monocytes. The significant down-regulation of CCR2 and CCR7 on monocytes of dogs with OSA demonstrated by our flow cytometry data supports the observation that monocyte migration is decreased in dogs with OSA. The primary disadvantage of our method for assessing migration was the high level of random migration observed, potentially because of excessive stimulation of monocytes by the cell sorting process. As a result, we included supraphysiologic combinations of chemoattractants to counter this potential issue. Monocyte chemotaxis is decreased in OSA: Box-and-whisker plot comparing the mean levels of random: directed monocyte migration for the various chemokines used, either alone or in combination (CCL2, CCL19, FBS, fMLP) between healthy controls (n = 14) and dogs with OSA (n = 5). Monocyte migration is significantly reduced in dogs with OSA when CCL19 (P = .018) or a combination of chemokines (P = .018) was used as chemoattractants. Decreased migration in dogs with OSA was also observed with CCL2 or fMLP as chemoattractants, but the difference did not reach significance (CCL2 P = .067; fMLP P = .067). There could be a multi-faceted explanation for the down-regulation of monocyte chemokine receptors in dogs with OSA. Similar to our findings, the loss of CX 3 CR1 expression in human monocytes increased glioma growth. 31 However, in contrast to our observations, this tumor-promoting effect is not because of loss of migratory capability. In fact, the absence of CX 3 CR1 was associated with an increase in monocyte infiltration into the tumor microenvironment, where the monocytes were theorized to differentiate into tumor-associated macrophages for promotion of tumor growth. Evaluation of the relationship between monocyte chemokine receptor expression and the infiltration of monocytes/macrophages in the tumor microenvironment in OSA is needed to explore whether this phenomenon occurs in dogs.
The higher PGE 2 levels that we observed in monocyte culture supernatants of dogs with OSA are consistent with our hypothesis that OSA suppresses monocyte chemotaxis and the host immune response. PGE 2 is a main metabolite resulting from the conversion of arachidonic acid by cyclooxygenases (COX-1 and COX-2), and increased levels of COX-2 and PGE 2 have been observed in various tumors. 32-35 PGE 2 secretion from human carcinoma cell lines inhibits monocyte chemotaxis by down-regulating cell surface expression of chemokine and adhesion receptors CCR5 and Mac-1, respectively. 36 Our finding of increased PGE 2 secretion from monocytes of dogs with OSA correlates with our observation that monocyte chemotaxis is inhibited in OSA. Increased PGE 2 levels have been shown to effect global immunosuppression, contributing to immunopathology in many cancers. 37 For example, PGE 2 disables the innate immune response by inhibiting neutrophils, monocytes and macrophages, and disrupts the cross-talk between dendritic cells and T cells that is essential for activation of the adaptive immune response. PGE 2 also skews T helper cells toward a type 2 pro-tumorigenic instead of a type 1 antitumor response, and promotes the accumulation of T regulatory and myeloid-derived suppressor cells, which can be utilized by the tumor to suppress an effective antitumor response. 37 A recent study in human OSA showed inhibition of human OSA cells by decreasing PGE 2 levels via microRNA modulation, demonstrating the role of PGE 2 in tumor promotion. 38 Specifically in OSA of dogs, PGE 2 is increased in both canine OSA cell lines and in naturally occurring tumors, 39 COX-2, microsomal PGE 2 synthase-1, and PGE 2 receptor are demonstrated to be increased in OSA lesions using immunohistochemistry staining, 40 and OSA cells from dogs secrete PGE 2 . 41 Our findings of increased PGE 2 in dogs with OSA are in accordance with these previous reports, and emphasize the ability of PGE 2 to induce immunopathology in OSA.
We did not find significant differences in relative mRNA expression of IL-10, IL-12, TNF-a, and COX-2 in monocytes from OSA compared to healthy dogs. There was a decreasing trend in COX-2 expression in dogs with OSA, but the difference from healthy controls was not significant. This is contrary to what we expected, given the higher PGE 2 secretion from monocytes of dogs with OSA, and the overexpression of COX-2 that has been reported in OSA of dogs. 40,42 Potentially, there might be other enzymes such as microsomal PGE 2 synthase-1 that could be concurrently influencing the level of PGE 2 secretion. However, as the difference in COX-2 expression between OSA and healthy dogs was not statistically significant, whether COX-2 expression in monocytes of dogs with OSA is truly decreased needs to be further evaluated, as other influences such as miRNA activity can affect mRNA transcription. Because of potential disparities between mRNA and protein expression, we evaluated monocyte secretion of TNF-a, IFN-c, and IL-10, using IFN-c as representative of IL-12 activity. There were low to undetectable levels of IFN-c and IL-10 from both groups, and higher levels of TNF-a secreted from monocytes of dogs with OSA. This was an unexpected finding, as TNF-a is a proinflammatory cytokine. However, TNF-a induces a number of other changes, particularly in the context of the tumor microenvironment. TNF-a induces the up-regulation of IL-34 expression in OSA cell lines, and IL-34 has been associated with progression of disease and promotion of angiogenesis in OSA. 43 TNF-a has also been shown to increase the production of PGE 2 as a mechanism for effecting bone resorption. 44 Potentially, the increase in TNF-a in monocytes of dogs with OSA reflects tumor-driven mechanisms for OSA promotion rather than induction of a proinflammatory monocyte response. Other immunomodulatory cytokines such as TGF-b are utilized by OSA for tumor promotion. TGF-b, generally known to dampen immune responses and promote tumor growth, is secreted by OSA cells from dogs, 6 and has also been implicated in the pathogenesis of human OSA. 45 Collectively, these findings indicate a complex microenvironment that promotes immune dysfunction and tumor growth.
One potential limitation of this study is the use of clinical dogs and random source healthy controls, instead of purpose-bred dogs which provide a genetically uniform study population. However, one strong advantage of using nonpurpose-bred dogs is the genetic diversity that accurately reflects that of the general pet population. This diversity, together with the exposure to shared environmental variables with humans, contributes to the unique canine model of spontaneously occurring OSA that bears striking similarities to the human disease. Such a model is extremely valuable for studying the disease to improve outcomes for both humans and dogs.
In conclusion, this study demonstrated that monocyte chemokine receptors are markedly down-regulated in dogs with OSA compared to healthy controls, and monocytes from dogs with OSA exhibit decreased chemotactic capacity and secrete higher levels of PGE 2 and TNF-a . Our findings strongly suggest that OSA in dogs evades the immune system by suppressing chemokine receptor expression and increasing PGE 2 secretion in monocytes, thus inhibiting their migration, leading to sequestration in the periphery. Increased PGE 2 levels could also promote immunosuppression, as PGE 2 can exert a multitude of immunopathologic effects such as interfering with lymphocyte proliferation and the antitumor cytotoxicity of T cells, inhibit NK cell, dendritic cell, neutrophil, monocyte and macrophage effector functions. Increased TNF-a secretion could be a tumorpromoting mechanism through manipulation of the tumor microenvironment and elevating PGE 2 production. Understanding the mechanisms by which OSA causes dysregulation of the immune response will provide insight into developing novel immunotherapeutics to reverse tumor-induced immunopathology and reduce metastatic disease in OSA.

Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1. Agarose gel electrophoresis for RNA and DNA assessment. Table S1. Least-squares means from the 2-way ANOVA comparing relationships between monocyte counts and receptor expression. Table S2. Pearson's correlations between chemokine receptors in OSA dogs (n = 18) compared to healthy controls (n = 13).