Cabazitaxel Demonstrates Potent Antitumour Activity Against Canine Large‐Cell Alimentary Lymphoma In Vitro and In Vivo

ABSTRACT

Canine large‐cell alimentary lymphoma (LCAL) is an aggressive malignancy with limited effective treatment options and a poor prognosis. In a preliminary drug screening study using three canine LCAL cell lines (CLC, Ema, and Nody‐1), the taxane cabazitaxel (CBZ) was identified as a promising candidate drug. This study aimed to evaluate the antitumour effects of CBZ in vitro and in vivo and determine its potential as a novel therapeutic candidate for canine LCAL. Cell viability, tubulin polymerisation, cell cycle distribution, and apoptosis were assessed in CLC, Ema, and Nody‐1 cells following treatment with CBZ or vehicle control. In addition, the effect of CBZ was evaluated in a xenotransplantation mouse model established using CLC cells. CBZ reduced cell viability across all cell lines in a dose‐dependent manner, with sub‐nanomolar half‐maximal inhibitory concentrations. A tubulin polymerisation assay demonstrated that CBZ increased polymerised tubulin levels, indicating microtubule stabilisation. CBZ treatment induced G2/M cell cycle arrest and significantly increased apoptotic cell populations, accompanied by decreased procaspase‐3 levels and increased cleaved caspase‐3 expression. In vivo, CBZ treatment reduced ascites accumulation and significantly prolonged survival relative to the vehicle control. These findings suggest that CBZ induces G2/M cell cycle arrest and caspase‐dependent apoptosis through tubulin stabilisation and suppresses tumour progression in vivo. CBZ represents a promising therapeutic candidate for canine LCAL.

1. Introduction

Lymphoma is among the most frequently diagnosed neoplasms in dogs, accounting for approximately 7%–24% of all canine neoplasms and 83% of all canine haematopoietic malignancies [1]. Canine lymphoma occurs in various anatomical forms, including multicentric, mediastinal, abdominal (alimentary, hepatic, splenic, and renal), cutaneous, ocular, central nervous system, and pulmonary lymphomas. Alimentary lymphoma (AL) primarily affects the gastrointestinal tract and frequently extends to extraintestinal organs such as the lymph nodes, liver, and spleen. It accounts for approximately 5%–7% of all canine lymphomas [2, 3]. Based on cell morphology, canine AL is broadly classified into large‐cell and small‐cell subtypes. Medical therapy remains the mainstay of treatment for both, but large‐cell AL (LCAL) is particularly aggressive and highly resistant to treatment, resulting in a poor prognosis with a median survival time of only a few months [4, 5, 6, 7]. Therefore, the development of novel therapeutic strategies for canine LCAL represents an important unmet clinical need.

In our preliminary study, a stepwise drug screening of 1134 US Food and Drug Administration (FDA)‐approved agents was performed using three canine LCAL cell lines (CLC, Ema, and Nody‐1). First, treatment with all 1134 compounds at 2 μM for 48 h identified 37 agents that reduced cell viability to less than 10% across all cell lines (Table S1). Next, these 37 compounds were evaluated at 0.2 μM for 48 h, and 12 agents reduced cell viability to less than 10% across all cell lines (Table S2). Finally, these 12 compounds were further evaluated at 0.02 μM for 48 h, and cabazitaxel (CBZ) showed the lowest mean cell viability among the three cell lines (Figure 1). Based on these results, CBZ was selected for further investigation.

FIGURE 1.

Heatmap of cell viability in CLC, Ema, and Nody‐1 cells after treatment with 12 selected candidate compounds at 0.02 μM for 48 h. HCl, hydrochloride.

Taxanes are a class of antineoplastic agents that stabilise microtubules by inhibiting microtubule depolymerisation, thereby disrupting mitotic spindle function and inducing cell cycle arrest and apoptosis [8]. CBZ is a second‐generation taxane developed to overcome resistance to first‐generation taxanes such as paclitaxel and docetaxel. Previous studies have shown that CBZ exhibits reduced affinity for the drug efflux transporter P‐glycoprotein, which is a major mechanism of taxane resistance in cancer cells [9, 10]. Clinically, CBZ has demonstrated significant antitumour activity and improved overall survival in human patients with metastatic castration‐resistant prostate cancer whose disease has progressed during or after docetaxel‐based therapy [11], leading to its approval by the FDA in 2010. In addition, the antitumour activity of CBZ has been demonstrated in a variety of human cancer cell lines and experimental tumour models [12, 13, 14, 15, 16, 17, 18, 19]. However, no studies have investigated the effects of CBZ in canine malignancies including LCAL. This study aimed to evaluate the effects of CBZ on canine LCAL cells and assess its potential as a therapeutic agent for this disease.

2. Materials and Methods

2.1. Reagents and Antibodies

CBZ and doxorubicin were purchased from Selleck Chemicals (Houston, TX, USA) and FUJIFILM Wako Pure Chemical (Osaka, Japan), respectively. The reagents were dissolved in dimethyl sulfoxide.

For western blot analysis, rabbit polyclonal antibodies against caspase‐3 and p62/SQSTM1 were obtained from Cell Signalling Technology (Danvers, MA, USA) and Medical and Biological Laboratories (Tokyo, Japan), respectively. A rabbit monoclonal antibody against LC3A/B (clone: D3U4C) was obtained from Cell Signalling Technology. Mouse monoclonal antibodies against α‐tubulin (clone: DM1A) and β‐actin (clone: AC‐74) were purchased from Novus Biologicals (Centennial, CO, USA) and Sigma‐Aldrich (Saint Louis, MO, USA), respectively. The antibodies against p62/SQSTM1, LC3A/B, α‐tubulin, and β‐actin were validated for use in dogs based on the manufacturer's specifications or previous studies [20, 21]. Horseradish peroxidase‐conjugated goat anti‐rabbit and anti‐mouse immunoglobulin G secondary antibodies were purchased from Bio‐Rad Laboratories (Hercules, CA, USA). All antibodies were diluted according to the manufacturer's recommendations.

2.2. Cell Line Validation and Culture Conditions

Three canine LCAL cell lines (CLC, Ema, and Nody‐1) were obtained directly from the original investigators who established them. Their growth characteristics and morphology were consistent with those described in a previous study [22]. All cell lines were confirmed to be free of Mycoplasma contamination using the Takara PCR Mycoplasma Detection Set (Takara Bio, Kusatsu, Japan). The cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical) supplemented with 10% foetal bovine serum (Gibco, Carlsbad, CA, USA) at 37°C with 5% CO₂. A human lung cancer cell line (A549) was purchased from RIKEN BRC (Tsukuba, Japan). The cells were maintained in DMEM (Gibco) supplemented with 10% foetal bovine serum under the same culture conditions.

2.3. Water‐Soluble Tetrazolium Salt Assay

Cell viability was assessed using a Cell Counting Kit‐8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. CLC, Ema, and Nody‐1 cells were seeded in 96‐well plates at densities of 5.0 × 10³, 4.0 × 10⁴, and 1.5 × 10⁴ cells per well, respectively. The cells were treated with various concentrations (0–10 nM) of CBZ for 48 h, followed by the addition of cell counting kit‐8 reagent to each well. Cell viability was determined by measuring absorbance at 450 nm using a microplate reader (Infinite 200 Pro; Tecan, Männedorf, Switzerland). All samples were analysed in quintuplicate, and three independent experiments were performed.

2.4. Tubulin Polymerisation Assay

A cell‐based tubulin polymerisation assay was performed according to a previously described method [16]. CLC, Ema, and Nody‐1 cells were seeded in Petri dishes at densities of 3.0 × 10⁶, 1.0 × 10⁷, and 5.0 × 10⁶ cells per dish, respectively. After treatment with 1 nM CBZ or vehicle control for 1 h, the cells were washed with Hanks' balanced salt solution (HBSS; FUJIFILM Wako Pure Chemical) and incubated with a low‐salt buffer containing 20 mM Tris–HCl (pH 6.8), 1 mM MgCl₂, 2 mM EGTA, 0.5% NP‐40, and a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) at room temperature for 5 min. The lysates were centrifuged at 20000 × g for 30 min, and the supernatant was used as the soluble tubulin fraction. The pellet was lysed in RIPA buffer (Cell Signalling Technology) containing protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics) at 4°C for 5 min and used as the polymerised tubulin fraction. The soluble and polymerised fractions from the same aliquot of cell lysates were subjected to western blot analysis using anti‐α‐tubulin antibody. A549 cell lysate served as a positive control. Two independent experiments were performed.

2.5. Cell Cycle Analysis

CLC, Ema, and Nody‐1 cells were seeded in 6‐well plates at a density of 4.0 × 10⁵ cells per well. The cells were treated with 1 nM CBZ or vehicle control for 12 h. Subsequently, the cells were washed with HBSS, centrifuged at 125 × g for 5 min, fixed in 75% ethanol, and stored at −30°C until the analysis. After washing with HBSS, the cells were incubated at 37°C for 40 min in the dark with a staining solution containing 50 μg/mL propidium iodide (PI; Sigma‐Aldrich), 0.1 mg/mL RNase A (NIPPON GENE, Tokyo, Japan), and 0.05% Triton X‐100 (Sigma‐Aldrich). The DNA content was analysed using a Cell Sorter SH800S (SONY, Tokyo, Japan). All samples were analysed in triplicate, and three independent experiments were performed.

2.6. Apoptosis Assay

A MEBCYTO Apoptosis Kit (Medical and Biological Laboratories) was used according to the manufacturer's instructions. CLC, Ema, and Nody‐1 cells were seeded in 6‐well plates at a density of 4.0 × 10⁵ cells per well. The cells were treated with 1 nM CBZ or vehicle control for 12 h. After washing with HBSS, the cells were centrifuged at 125 × g for 5 min and incubated at room temperature for 15 min in the dark with a staining solution containing FITC‐conjugated annexin V and PI. Viable (double negative), early apoptotic (FITC‐positive/PI‐negative), and late apoptotic (double positive) cells were quantified using a Cell Sorter SH800S. All samples were analysed in triplicate, and three independent experiments were performed.

2.7. Western Blot Analysis

CLC, Ema, and Nody‐1 cells were seeded in Petri dishes at densities of 3.0 × 10⁶, 1.0 × 10⁷, and 5.0 × 10⁶ cells per dish, respectively. The cells were treated with 1 nM CBZ or vehicle control for 0–12 h. After washing with HBSS, the cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics) on ice for 5 min. The lysates were centrifuged at 13000 × g at 4°C for 15 min, and the supernatants were collected. Two independent experiments were performed.

Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated on 10% or 8%–16% Mini‐PROTEAN TGX Precast Gels (Bio‐Rad Laboratories) and transferred to Trans‐Blot Turbo Mini PVDF membranes (Bio‐Rad Laboratories). The membranes were blocked with 3% bovine serum albumin (Nacalai Tesque, Kyoto, Japan) at room temperature for 1 h and incubated with primary antibodies at 4°C for 12 h. After washing with Tris‐buffered saline containing 0.2% Tween 20 (Sigma‐Aldrich), the membranes were incubated with appropriate secondary antibodies at room temperature for 1 h. Protein bands were visualised using Clarity Western ECL Substrate (Bio‐Rad Laboratories) and detected with Mi‐II 600CB (BioTools, Gunma, Japan). Cell lysates from Ema cells treated with 0.2 μM doxorubicin or vehicle control for 24 h were used to validate the antibody against caspase‐3 for canine application.

2.8. Xenotransplantation Mouse Model

Twenty female NOD/SCID mice (5 weeks old) were purchased from the Jackson Laboratory (Kanagawa, Japan). All mice were housed in autoclaved isolator cages within a specific pathogen‐free facility and acclimated for 1 week prior to the experiment. To establish the xenotransplantation model, 1.0 × 10⁷ CLC cells were injected intraperitoneally into each mouse, as previously described [22], in which CLC demonstrated the highest tumour engraftment efficiency among the three cell lines. After inoculation, the mice were monitored daily for clinical signs including weakness, ascites accumulation, and dyspnoea, and body weight was recorded daily. Six days after tumour cell inoculation, the mice were administered CBZ (10 mg/kg) or vehicle control intraperitoneally once a week [18]. The vehicle consisted of 56.5% sterile ultrapure water, 40% polyethylene glycol 300 (Selleck Chemicals), 1% Tween 80 (Selleck Chemicals), and 2.5% dimethyl sulfoxide. Euthanasia was performed when humane endpoints were reached, including severe lethargy, body weight loss exceeding 20% of the pre‐inoculation weight, or severe respiratory distress associated with ascites accumulation. Survival time was defined as the interval from treatment initiation to the humane endpoint. All experimental procedures involving mice were approved by the Animal Care Committee of Kitasato University (authorisation number: 25‐032).

2.9. Statistical Analysis

Half‐maximal inhibitory concentration (IC₅₀) values were calculated using nonlinear regression analysis with a four‐parameter logistic model. Comparisons between two groups were performed using Welch's t‐test. Survival curves were generated using the Kaplan–Meier method and compared using the log‐rank test. All statistical analyses were conducted using Prism software version 8.4.3 (GraphPad Software, Boston, MA, USA). Statistical significance was set at p < 0.05.

3. Results

3.1. CBZ Inhibits Proliferation of Canine LCAL Cells

CBZ reduced cell viability in all canine LCAL cell lines in a dose‐dependent manner relative to the vehicle control (Figure 2). The estimated IC₅₀ values of CBZ were 1.8 × 10² pM for CLC cells, 9.6 pM for Ema cells, and 3.3 × 10² pM for Nody‐1 cells. However, because the viability data for Nody‐1 cells lacked sufficient intermediate response points within the dynamic range of the dose–response curve, the reliability of the calculated IC₅₀ value may be limited.

FIGURE 2.

Cell viability of CLC, Ema, and Nody‐1 cells after treatment with cabazitaxel (CBZ; 0–10 nM) for 48 h. Experiments were performed in quadruplicate. Data are presented as mean ± standard error.

3.2. CBZ Promotes Tubulin Polymerisation in Canine LCAL Cells

As shown in Figure 3, the soluble tubulin levels did not differ between the CBZ‐ and vehicle‐treated canine LCAL cell lines. In contrast, CBZ treatment increased the polymerised tubulin levels relative to the vehicle control.

FIGURE 3.

Western blot analysis of soluble and polymerised tubulin in CLC, Ema, and Nody‐1 cells after treatment with cabazitaxel (CBZ; 1 nM) or vehicle control for 1 h. A human lung cancer cell line (A549) was used as a positive control.

3.3. CBZ Alters Cell Cycle Distribution in Canine LCAL Cells

Cell cycle analysis by flow cytometry revealed a significant increase in the G2/M phase fraction in Ema and Nody‐1 cells after treatment with CBZ for 12 h relative to the vehicle control (Figure 4). In contrast, CLC cells showed a significant decrease in the G2/M phase fraction. A significant increase in the sub‐G1 phase fraction was also observed in all cell lines.

FIGURE 4.

(A) Representative data on cell cycle analysis of CLC, Ema, and Nody‐1 cells after treatment with cabazitaxel (CBZ; 1 nM) or vehicle control for 12 h. (B) Quantitative comparison of cell cycle phase distribution in CLC, Ema, and Nody‐1 cells after treatment with CBZ or vehicle control. Experiments were performed in triplicate. Data are presented as mean ± standard error.

3.4. CBZ Induces Caspase‐Dependent Apoptosis in Canine LCAL Cells

Flow cytometric analysis revealed significant increases in both early and late apoptotic cell populations in all cell lines after treatment with CBZ for 12 h relative to the vehicle control, whereas the proportion of viable cells was significantly decreased (Figure 5).

FIGURE 5.

(A) Representative data on flow cytometric analysis of CLC, Ema, and Nody‐1 cells stained with FITC‐conjugated annexin V and propidium iodide (PI) following treatment with cabazitaxel (CBZ; 1 nM) or vehicle control for 12 h. The cells were classified as double negative (viable cells), FITC‐positive/PI‐negative (early apoptotic cells), or double positive (late apoptotic cells). (B) Quantitative comparison of viable, early apoptotic, and late apoptotic cell populations after treatment with CBZ or vehicle control. Experiments were performed in triplicate. Data are presented as mean ± standard error.

As shown in Figure 6, CBZ treatment for 12 h slightly decreased procaspase‐3 levels and markedly increased cleaved caspase‐3 levels in all canine LCAL cell lines relative to the vehicle control. In contrast, the LC3A/B and p62/SQSTM1 levels in the CBZ‐ and vehicle‐treated cells did not differ at any of the time points examined (0–12 h) (Figure S1).

FIGURE 6.

Western blot analysis of procaspase‐3 and cleaved caspase‐3 in CLC, Ema, and Nody‐1 cells after treatment with cabazitaxel (CBZ; 1 nM) or vehicle control for 12 h. Ema cells treated with doxorubicin (DOX; 0.2 μM) were used as a positive control. β‐Actin served as a loading control.

3.5. CBZ Suppresses Tumour Progression and Prolongs Survival in a Xenotransplantation Mouse Model

The body weight of the mice gradually increased with progressive abdominal distension after tumour cell inoculation. No significant differences in body weight were observed between the CBZ‐treated and vehicle‐treated groups prior to treatment initiation. From 1 day after treatment initiation until the first mouse reached the humane endpoint, body weight was significantly lower in the CBZ‐treated group than in the vehicle‐treated group (Figure 7A). During the same period, the vehicle‐treated group exhibited greater abdominal distension than the CBZ‐treated group (Figure 7B). Survival time from treatment initiation was significantly prolonged in the CBZ‐treated group relative to the vehicle‐treated group (Figure 7C).

FIGURE 7.

(A) Body weight changes in canine large‐cell alimentary lymphoma (LCAL)‐engrafted mice treated with cabazitaxel (CBZ, 10 mg/kg) or vehicle control. Data are presented as mean ± standard error (n = 10). N. S., not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. (B) External appearance of the mouse with body weight closest to the median in each group at 5 days after treatment initiation. (C) Kaplan–Meier curves of survival from the treatment initiation to the humane endpoint in the canine LCAL‐engrafted mice. A dot indicates a censored case.

4. Discussion

In the present study, CBZ exhibited antitumour activity against canine LCAL cells both in vitro and in vivo. CBZ reduced cell viability in three LCAL cell lines at sub‐nanomolar concentrations and induced tubulin stabilisation, G2/M arrest, and caspase‐dependent apoptosis. In addition, CBZ significantly prolonged survival in a xenotransplantation mouse model. These findings indicate that CBZ effectively suppresses tumour progression in canine LCAL.

CBZ exhibited antitumour activity against canine LCAL cells in vitro, with estimated IC₅₀ values in the subnanomolar range. In a previous study using an MTT colorimetric assay, the IC₅₀ values of CBZ after 48 h of treatment were 5.4 × 10 nM and 6.6 × 10 nM for two human prostate cancer cell lines (DU145 and PC3), respectively, and ranged from 2.9 × 10 to 5.3 × 10² nM for four human colon cancer cell lines (HCT116, LoVo, HCT8, and DLD1) [19]. These findings suggest that canine LCAL cells are highly sensitive to CBZ. Because CBZ is already an FDA‐approved drug for the treatment of human prostate cancer, it may also have clinical potential for the treatment of canine LCAL.

CBZ treatment significantly increased polymerised tubulin levels relative to the vehicle control in all canine LCAL cell lines. This is consistent with the results of previous studies in human prostate cancer and renal cell carcinoma cells [16, 17]. Microtubules are composed of α‐ and β‐tubulin heterodimers and exhibit dynamic instability characterised by continuous cycles of polymerisation and depolymerisation. Through this property, microtubules contribute to various cellular processes, including intracellular transport, maintenance of cell morphology, cell motility, and formation of the mitotic spindle responsible for chromosome segregation. CBZ binds to tubulin and promotes tubulin polymerisation, thereby stabilising microtubules and suppressing microtubule dynamics driven by dynamic instability [23]. Furthermore, suppression of microtubule dynamics activates the spindle assembly checkpoint, thereby inhibiting the transition from metaphase to anaphase during mitosis. Consistent with this mechanism, CBZ has been reported to induce G2/M phase arrest and exert antitumour effects in various human cancer cell lines [13, 14, 15, 19]. In the present study, CBZ induced G2/M phase arrest in Ema and Nody‐1 cells, indicating that stabilisation of microtubules by CBZ disrupts normal cell cycle progression in canine LCAL cells. However, G2/M phase arrest was not observed in CLC cells. This discrepancy may reflect the transient nature of G2/M arrest that could not be captured under the experimental conditions used in this study.

In addition to inducing cell cycle arrest, CBZ promoted apoptosis in canine LCAL cells. Cell cycle analysis revealed an increased proportion of cells in the sub‐G1 phase fraction following CBZ treatment in all canine LCAL cell lines, suggesting the induction of apoptotic cell death. Consistent with this finding, annexin V/PI staining followed by flow cytometric analysis demonstrated significant increases in both early and late apoptotic cell populations after CBZ treatment. Furthermore, western blot analysis revealed decreased procaspase‐3 expression and increased cleaved caspase‐3 expression, indicating activation of the caspase‐dependent apoptotic pathway. These findings are consistent with previous reports showing that CBZ can induce apoptosis following prolonged mitotic arrest in human prostate and colorectal cancer cell lines [19]. Although a previous study has reported that CBZ induces autophagy in a human lung cancer cell line [14], no apparent changes in the expression of the autophagy‐related proteins LC3A/B or p62/SQSTM1 were observed in any of the cell lines examined in the present study, suggesting that autophagy was not induced under our experimental conditions.

Body weight in the canine LCAL‐xenografted mice gradually increased with progressive abdominal distension after tumour cell inoculation, likely reflecting ascites accumulation as previously reported [22]. From 1 day after treatment initiation until the first mouse reached the humane endpoint, body weight was significantly lower in the CBZ‐treated group than in the vehicle‐treated group. Abdominal distension was also less pronounced in the CBZ‐treated mice, suggesting that CBZ reduced ascites accumulation and consequently lowered body weight. Although treatment‐related toxicity could potentially contribute to body weight loss, previous studies using tumour‐bearing mouse models showed significant weight loss at 25 mg/kg but not at 15 mg/kg [12, 24]. Because the dose used in the present study was 10 mg/kg, CBZ‐induced toxicity is unlikely to explain the observed weight reduction. In addition to reducing ascites accumulation, CBZ significantly prolonged survival in the xenotransplantation model of canine LCAL, suggesting that CBZ has therapeutic potential not only at the cellular level but also in vivo.

In a previous study, toxicity evaluation of CBZ was conducted in healthy dogs [25]. When CBZ was administered intravenously once every 3 weeks for 13 cycles, the highest non‐severely toxic dose was reported to be 0.5 mg/kg. In addition, the maximum plasma concentration after a single intravenous administration of CBZ at 0.5 mg/kg ranged from 7.8 × 10 nM to 1.2 × 10² nM. These concentrations are much higher than the estimated IC₅₀ values observed in the canine LCAL cell lines in the present study (range, 9.6 pM–3.3 × 10² pM), suggesting that CBZ may exert therapeutic effects in clinical cases. Further investigations, including clinical trials in dogs with naturally occurring LCAL, are warranted to evaluate the safety and therapeutic efficacy of CBZ in clinical settings.

Canine LCAL shares features including cell morphology, immunophenotype, lesion localisation, biological behaviour, and prognosis with human enteropathy‐associated T‐cell lymphoma (EATL) [26], which is an aggressive peripheral T‐cell lymphoma. No validated and standardised treatment protocols for human EATL exist because of the rarity of this disease [27]. Therefore, identifying novel therapeutic strategies is an urgent need. Because the effect of CBZ on human EATL remains unclear, the present findings in canine large‐cell AL may also be relevant to the development of novel therapeutic strategies for human EATL.

In conclusion, CBZ represents a promising therapeutic candidate for canine LCAL and warrants further investigation in clinical trials.

Funding

This work was supported by Japan Society for the Promotion of Science, JP23K14087, JP25K09422.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Western blot analysis of LC3A/B and p62/SQSTM1 in CLC, Ema, and Nody‐1 cells after treatment with cabazitaxel (CBZ; 1 nM) or vehicle control for 0–12 h. A human lung cancer cell line (A549) was used as a positive control. β‐actin served as a loading control.

Table S1: Cell viability of three canine large‐cell alimentary lymphoma cell lines after treatment with 1134 compounds at 2 μM for 48 h.

Table S2: Cell viability of three canine large‐cell alimentary lymphoma cell lines after treatment with 37 compounds at 0.2 μM for 48 h.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.