Cannabinoids have been extensively studied for their various biological effects in human cell models, but their potential applications in veterinary oncology have not been thoroughly examined. In particular, research on the impact of CBD on tumor cells of animal origin is limited, emphasizing the need for additional studies using in vitro models in species of veterinary interest. In our study, we first investigated whether CBD has specific effects on equine sarcoid cells, measuring its impact on cell viability, apoptosis, and cytotoxicity. Our analyses indicated a significant impact of CBD dose and exposure time on cell viability, apoptosis, and cytotoxicity. Although not all comparisons between groups were significant, our results demonstrate a dose- and time-dependent decrease in cell viability, with significant decreases occurring at higher concentrations and prolonged exposure to CBD. These findings are consistent with previous studies,^23–25 that showed that CBD decreases the viability of various cancer cell lines, including human colorectal cancer cells and gingival squamous cell carcinoma cells (OECM-1). Similar to our data, more pronounced effects were reported at higher concentrations. The observed cytotoxicity was minimal at 6 hours, but a notable increase was observed at the lowest concentration (0.75 μM), suggesting that cell death mechanisms at this early stage may involve mild cellular damage that does not yet substantially decrease viability. Mechanisms such as necrosis or autophagy may have been at play during this period, warranting further investigation. These findings are consistent with results from other studies,^10,23,26,27 supporting the hypothesis that CBD induces cell death through multiple pathways. Interestingly, no significant cytotoxicity was observed at 6-24 hours, potentially indicating cellular adaptation, activation of repair mechanisms, or transition to a more orderly form of cell death, such as apoptosis. The decrease in cytotoxicity at this time also could be related to activation of repair mechanisms or a transient compensatory effect in the cell population. Additionally, the lack of significant activation of caspase-3/7 at the early stage (6 hours) suggests that the cells may undergo delayed apoptosis. The time-dependent peak in apoptosis, with caspase-3/7 activity reaching 75% of cells at 0.75 μM after 72 hours, emphasizes apoptosis as the dominant mechanism of cell death after prolonged exposure. In agreement with our study, previous studies reported that treatment with CBD induces cytotoxicity in a time-dependent manner by selectively decreasing cell viability and inducing apoptosis in ovarian carcinoma cells²⁸ or in human and canine glioma cells.²⁹ The concentration of CBD also plays a crucial role in its cytotoxicity. The obtained results show a cytotoxic effect at the lowest dose (0.75 μM) after 6 hours, which is less common in other studies where higher concentrations (eg, 5-20 μM) are typically required to observe significant early effects.^28,30,31 This finding suggests that equine sarcoid cells may be susceptible to CBD, perhaps because of unique metabolic or signaling pathways in these cells. Although high concentrations of CBD (eg, 20 μM) significantly activated apoptosis in equine sarcoid cells after 24 hours, cytotoxicity itself was not significant at this time point. This delayed cytotoxicity at higher doses contrasts with many studies in cancer cells, where high CBD concentrations (10-25 μM) frequently induce rapid and robust cytotoxic effects within the first 24 hours. For example, studies on MCF-7 breast cancer cells³⁰ and glioma cells¹⁵ reported significant cytotoxicity at concentrations exceeding 10 μM within 24 hours, attributed to mechanisms such as mitochondrial dysfunction and oxidative stress. In contrast, our data suggest that equine sarcoid cells might exhibit an adaptive response to higher doses during the early phase, delaying overt cytotoxic effects until 48-72 hours, when significant cell death occurs across all tested concentrations.

Our results indicate that CBD significantly modulates the secretion of MMP-1, MMP-2, and MMP-9 in equine sarcoid cells. These matrix metalloproteinases are central to extracellular matrix (ECM) degradation and tumor cell invasion, and their dysregulation is closely linked to enhanced tissue infiltration and local aggressiveness. In equine sarcoids, MMPs are known to be upregulated and associated with ECM remodeling and tumor progression, particularly in the context of bovine papillomavirus involvement.^32–38 Matrix metalloproteinase-1, expressed in neoplastic sarcoid cells, is implicated in local tumor aggressiveness and has been shown to be upregulated by BPV-1 activity.^33,36 In our study, CBD significantly decreased MMP-1 concentrations across all tested CBD concentrations and time points, with the most pronounced effect (approximately 48.9%) observed at 24 hours. Although limited evidence is available on CBD’s effect on MMP-1 in cancer cells, a biphasic response has been described in non-tumor models such as human gingival fibroblasts, where low CBD concentrations increased, and higher doses decreased MMP-1 concentrations.³⁹ This finding suggests that CBD’s effects on MMP-1 are dose-dependent and highly context-specific. Our findings indicate a consistent inhibitory action of CBD on MMP-1 in sarcoid cells, supporting its relevance as a regulatory target in tumor-associated ECM degradation. Cannabidiol also decreased the concentrations of MMP-2 and MMP-9, two proteolytic enzymes strongly implicated in tumor cell invasion and motility. Our data indicated robust early inhibition of both proteins at higher CBD concentrations, with some delayed effects at lower concentrations. These results are in agreement with previous studies that found MMP-2/9 suppression by CBD in glioma and triple-negative breast cancer models.^40,41 Taken together, our findings suggest a dose- and time-dependent molecular impact of CBD on ECM remodeling in equine sarcoid cells, suggesting its potential to disrupt invasion-associated mechanisms at the biochemical level.

To further explore CBD’s influence on invasive behavior, we conducted a cell invasion assay using a reconstituted ECM membrane. The changes (34%-59% at 24 hours), however, did not reach significance. At the 24-hour time point, cell viability remained high (77.5%), and no significant apoptosis or cytotoxicity was observed, indicating that the decreased invasion was not an artifact of cell loss. These findings are consistent with results observed in other tumor models, including highly invasive bladder cancer cells. In a study of T24 bladder cancer cells, CBD at a concentration as low as 2.5 μM significantly decreased invasion, emphasizing its potential to inhibit invasive behavior even in aggressive cancer models.⁴² Similarly, several studies have shown that CBD can decrease invasion as well as migration in various cancers, including breast cancer, lung cancer, glioblastoma, and head and neck squamous cell carcinoma.^26,31 Although equine sarcoids are not metastatic by nature, their classification as locally invasive skin tumors emphasizes the importance of targeting mechanisms driving their tissue-specific invasiveness.

Our study had several limitations that should be taken into account when interpreting our findings. One important constraint is limited sample diversity, because the study included only three primary equine sarcoid cell lines. This narrow representation may not fully capture the biological heterogeneity of sarcoid tumors and therefore could limit the generalizability of the results. Another limitation concerns the range of experimental conditions. Although the selected CBD concentrations (0.75, 6.75, and 20 μM) and exposure durations (6, 24, and 48 hours) provided valuable insights, they may not reflect the complete spectrum of potential cellular responses, particularly at lower or higher doses or with prolonged exposure.

In addition to these considerations, we sought to determine whether the effects observed in sarcoid cells were specific to neoplastic transformation. To this end, we conducted parallel analyses using primary normal equine dermal fibroblasts. Notably, CBD treatment did not decrease cell viability at any concentration or time point in these non-tumor cells. Similarly, fibroblast migration in the scratch assay proceeded comparably to that of vehicle-treated controls, with no detectable impairment in wound closure. These observations are consistent with previous findings in fibroblasts from humans, which likewise exhibited no cytotoxic response to similar CBD concentrations.^42,43 Collectively, these data support the interpretation that the effects seen in sarcoid cells likely reflect tumor-specific responsiveness rather than non-selective cytotoxicity against equine dermal cells.

Finally, it is important to acknowledge that the use of an in vitro system inherently limits the extrapolation of results to in vivo contexts, where factors such as the tumor microenvironment and immune interactions may significantly influence cellular responses.^43,44 In conclusion, our study provides preliminary evidence that CBD may exert anti-proliferative effects on equine sarcoid cells, as indicated by decreased cell viability and increased caspase 3/7 activity under specific experimental conditions, suggesting the involvement of apoptotic mechanisms. Additional studies are needed to confirm and extend these findings across a broader range of models and conditions. Notably, CBD also significantly suppressed the concentrations of matrix metalloproteinases MMP-1, MMP-2, and MMP-9, which are essential for ECM degradation and are closely associated with the invasive behavior of tumor cells. However, the decrease in cell invasion observed in the functional assay did not reach significance. These findings suggest that CBD may interfere with matrix-associated mechanisms relevant to tissue infiltration, even at sub-cytotoxic concentrations. Importantly, complementary analyses using normal equine dermal fibroblasts showed no decrease in viability or migration, indicating that the effects observed in sarcoid cells likely reflect tumor-associated responsiveness rather than nonspecific toxicity. Overall, equine sarcoid cells exhibit clear biological responsiveness to CBD, supporting its relevance as a modulator of matrix remodeling and invasive potential in this tumor model.