In the recent scientific literature within the field of veterinary oncology, there has been an increasing emphasis on the research and utilization of biomarkers. Despite MCTs being among the two most common tumors in dogs, studies on biomarker identification have been limited, and to date, no biomarkers have been characterized that can be obtained from blood samples [23,24,25]. The only predictive marker for which research has been published to date in canine MCT is c-KIT mutational status [26]. c-KIT mutations are a tissue marker that can indicate more aggressive forms of MCT, can predict outcomes, including the likelihood of recurrence or metastasis, and can guide treatment choices [27].

Recently, Rinaldi and co-authors reported that CB1 and CB2 are expressed in cMCTs and are inversely correlated with tumor grade [22]. The current study expanded this research from cannabinoid receptors to eCBs, endogenous ligands able to bind to and functionally activate cannabinoid receptors. Although in recent years the circulating levels of eCBs have been determined in canine chronic enteropathy [18] and lymphoma [19], there are no data regarding the circulation of eCBs in the context of MCTs in dogs. In this study population, past authors have evaluated WHO clinical stage [3], histological grading [4,5], and clinical characteristics like ulcers and size [1]. In agreement with what has been reported previously in the literature [1,28], the size (i.e., >3 cm) and/or the presence of ulceration were characterized by more aggressive biological behavior except for in one case, where an ulcer was present in a low-grade cMCT in clinical stage I.

In this study, the authors observed a significant increase in 2-AG in dogs affected by cMCT compared to the control group. The increase in 2-AG levels observed in this study is in agreement with the alteration of eCB levels highlighted in other types of tumors. Indeed, 2-AG was increased in human tissues in endometrial carcinoma [29], colorectal cancer [30], pituitary adenomas [31], glioma [32], and hepatocellular carcinoma [33]. Additionally, the authors unveiled a reduction in AEA content in the plasma of MCT patients, clearly providing evidence in favor of the distinct involvement of the two main eCBs in the disease. Furthermore, it should also be mentioned that an inverse regulation, characterized by increased 2-AG and decreased AEA levels, was observed in the human plasma of 304 cancer patients [20], as well as in human tissue glioma [32].

In veterinary medicine, the only other study on eCBs in canine neoplasia showed a reduction in 2-AG and an increase in AEA serum levels in dogs affected by DLBCL [19], with these results being in opposition to the alteration of eCB levels highlighted in human patients suffering from the same tumor [34].

Although the molecular mechanism underlying the alteration of eCBs was not investigated in this study, it is possible to hypothesize that dysregulations of eCBs’ biosynthetic and/or degradative processes could account for the changes in eCB levels reported here. For instance, the upregulation of diacylglycerol lipase (DAGL) or the downregulation of monoacylglycerol lipase (MAGL) could be consistent with the increase in 2-AG reported in the tissue in hepatocellular carcinoma [33] and in endometrial carcinoma [29], respectively.

In order to critically evaluate their diagnostic potential in cMCT, the remarkable accuracy of eCBs in distinguishing between control and cMCT dogs was thoroughly investigated. Notably, 2-AG demonstrated an exceptional ability to help discriminate between healthy and cMCT dogs, as evidenced by an impressive area under the ROC curve (AUC) of 0.98, highlighting its strong potential as a biomarker for cMCT detection. Similarly, the significant decrease in AEA levels in cMCT dogs, with an AUC of 0.85, further supports its promise as a diagnostic marker for cMCT. Further investigations are warranted to determine whether similar accuracy can be observed between plasma eCB levels and different groups of other spontaneous tumors in dogs. Moreover, in the present study, we also observed that PEA levels in the plasma of dogs affected by MCT were lower compared to healthy dogs, with an impressive area under the ROC curve (AUC) of 0.81. The trend towards a reduction in AEA and PEA plasma levels in cMCT dogs appears to be in agreement with the well-documented observation that AEA shares with PEA a common biosynthetic pathway, and suggests that a comprehension of the alteration of specific NAEs-mediated signaling could contribute to the unveiling of pathophysiological mechanisms of cMCT. Despite its structural similarity to AEA, PEA does not bind to CBs, and its therapeutic effects are mainly based on the activation of other molecular targets [35]. Of note, we showed that none of the compounds quantified in this study, PEA included, seem to be affected by treatment with antihistamines. Indeed, four dogs had received antihistamine therapy before the visit, but neither cetirizine nor famotidine oral administration significantly impacted eCB levels. PEA is extensively studied for its anti-inflammatory effects, particularly for its role in attenuating the degranulation of mast cells (MCs) triggered by substance P [36]. Moreover, PEA has been shown to prevent IgE-induced degranulation in isolated canine skin MCs (histamine, PGD2, and TNF-α release). The decrease in PEA may suggest a dysfunction within the intrinsic response mechanism intended to moderate the inflammatory reactions of MCs [37]. The mechanisms underlying its anti-inflammatory effects are mainly due to its ability to bind the peroxisome proliferator-activated receptor α (PPAR-α) [38] that also mediates its effect in several carcinogenesis mechanisms [39,40]. Moreover, PEA modulates AEA effects by increasing AEA endogenous levels and potentiating its binding at CB1 and CB2 receptors or at transient receptor potential vanilloid type-1 (TRPV1) channels, by downregulation of fatty acid amide hydrolase (FAAH), the enzyme responsible of AEA inactivation, or by a positive allosteric modulation of TRPV1 channels [35].

Finally, even though it has been previously reported that low concentrations of OEA promote the migration of neoplastic cells through the activation of Erk signaling pathways [41] and that high concentrations inhibit tumor cell migration [42,43], in this study, no differences were reported in OEA levels in cMCT dogs compared to healthy dogs. This supports a different involvement of N-acylethanolamines and their receptor targets in cMCT, as OEA is not a ligand at classical CB receptors.

Despite the insights gained from this study, several limitations should be acknowledged. Firstly, the sample size was relatively small, patients in the IV clinical stage were not enrolled, and a limited number of dogs affected by high-grade cMCT was included. Moreover, the study was conducted using a cross-sectional design, and samples were not matched for age and BCS, although these factors do not appear to affect the results regarding circulating eCBs [18]. Based on the above-mentioned considerations, it seems evident that future studies are needed to understand the molecular mechanism underlying the alterations of eCBs observed in this study. In particular, it will be necessary to assess if the dysregulation of eCBs’ metabolism in terms of expression, and/or the activity of biosynthetic (DAGL and NAPE) and/or degradative (FAAH and MAGL) enzymes could account for the changes in eCB levels reported here. It will be important to evaluate whether systemic changes in the different elements of the ECS are the mirror image of those in bioptic tissues. Finally, longitudinal studies are also required to ascertain whether eCB tone and signaling undergo specific alterations during the different clinical stages of MCTs, and whether they are associated with disease progression or prognosis. On the other hand, prospective studies are needed to evaluate the modulation of eCBs’ plasmatic tone as a potential examination of the efficacy of pharmacological therapy. Therefore, it is possible to foresee that a complete knowledge of the action and function of the ECS in cutaneous neoplastic diseases might lead to the development of alternative therapeutic strategies based on the pharmacological modulation of the ECS. Indeed, synthetic pharmacological tolls such as receptor agonists and/or inhibitors of eCB hydrolytic enzymes, as well as antagonists and/or inhibitors of eCB biosynthetic enzymes, might enhance or reduce, respectively, the activity of the ECS. On this basis, it will be possible to hypothesize their use in the fine-tuning of the ECS’s therapeutic potential in MCTs. Lastly, data reported here, together with previous evidence that cannabinoid receptors are expressed in cMCT [22] and the above-suggested future studies, can provide the biochemical and molecular basis to justify the increasing interest and use of cannabinoids, particularly CBD products, in veterinary medicine [44,45].