Gilvetmab is a caninised monoclonal antibody against canine PD-1. Concurrent or sequential administration of anti-PD-1 antibodies and radiation therapy (RT) improves survival time in various species. Eighteen dogs with mast cell tumours (MCTs) were prospectively treated with 24 Gy of RT over Days 0, 7 and 21 and gilvetmab every 2 weeks beginning on the second fraction of RT (Day 7) for up to 10 doses. Best tumour response was complete in one (6%), partial in eight (44%) and stable disease in nine dogs (50%) for an ORR of 50%. Median time to best response was 7 days after the first fraction of RT. Thirteen dogs (72%) developed progressive disease with a median progression free interval (PFI) of 116 days after the first fraction of RT. Nine adverse events (AEs) attributed to gilvetmab were recorded in seven dogs (39%) consisting of infusion reactions in four, metabolic changes in three and gastrointestinal signs and pancreatitis in one each. Overall, five dogs (28%) experienced Grade 3 AEs. Radiation AEs were limited to Grade 1 for both acute and late effects in seven dogs (39%). There was a significant difference in PFI and MST for dogs with and without lymph node metastasis with a median PFI of 64 versus 292 days and MST of 103 versus 587 days (p = 0.02 and 0.005, respectively). This sequence, or combination, of therapies did not provide durable tumour control for canine MCT and the AE profile warrants further evaluation.
Cytarabine intravenous constant rate infusion (IV CRI) toxicity in cats with lymphoma has not been well documented. We aimed to assess toxicities following cytarabine IV CRI as a single-agent or within a multiagent protocol in cats with lymphoma. Medical records of cats with lymphoma treated with cytarabine IV CRI, with documented dose, route, duration and at least one follow-up visit between 1990 and 2024, were retrospectively reviewed. Adverse events (AEs) were retrospectively graded according to VCOG-CTCAE v2. Thirty-eight cats were included, and 70 doses were evaluated for AEs. Median cytarabine dose was 389.8 mg/m2 (range 100.1-697.3 mg/m2) over a median infusion duration of 6 h (range 2-17 h). A total of 103 AEs were documented across 70 administrations. Gastrointestinal AEs were the most common category: altered appetite (21/38 cats, 55.2%), vomiting (9/38, 23.7%; one grade 3, the rest grades 1-2), diarrhoea (2/38, 5.3%) and dysphagia (2/38, 5.3%). Anaemia was the most common hematologic AE (14/38 cats, 36.8%; five grade 1, two grade 2, five grade 3, two grade 4); six cats (15.8%) developed thrombocytopenia (four grade 1, two grade 2), and one cat (2.6%) developed grade 2 neutropenia. Lethargy was the most common constitutional AE (12/38 cats, 31.6%). IV CRI of cytarabine was generally tolerated, with predominantly low-grade AEs, although grade 3-4 anaemia was not uncommon. Response rate and clinical benefit remain unknown; investigation of alternative dosing regimens may help clarify its therapeutic potential.
DNA mismatch repair (MMR) deficiency is a clinically important biomarker in human oncology, yet its relevance in feline neoplasia remains poorly understood due to limited characterisation and the absence of validated reagents. In this study, we established a practical immunohistochemistry (IHC) approach for evaluating feline MMR proteins by confirming the cross-reactivity of anti-human MLH1, MSH2 and MSH6 antibodies using CRISPR/Cas9-engineered feline tumour cell lines lacking each target gene. Using these validated antibodies, we conducted a pilot screening to assess MMR protein expression in 83 feline tumours and observed variable reductions or losses of MLH1, MSH2 and MSH6 across several tumour types. Notably, MLH1 expression was lost in all melanoma cases (6/6, 100%) and in most osteosarcomas (14/15, 93%), indicating that MMR perturbations may be particularly relevant in these malignancies. These findings provide the first analytically validated framework for assessing MMR proteins in feline oncology and suggest that MMR dysregulation may be more widespread than previously recognised. Although the clinical sample size is limited, this initial pilot screening maps the preliminary landscape of feline MMR protein expression. Further large-scale validation studies incorporating microsatellite instability testing, methylation analysis and genomic profiling are warranted to clarify the biological significance of these alterations.
Human mucosal melanoma (hMM) is a rare but highly aggressive malignancy with poor clinical outcomes and limited responsiveness to current immunotherapeutic strategies. Unlike cutaneous melanoma, hMM is characterized by an ultraviolet-independent pathogenesis, low tumour mutational burden, and pronounced molecular and immunological heterogeneity, which collectively inhibit effective translational modelling and therapeutic development. Naturally occurring canine oral melanoma (cOM) shares key biological features with hMM, including spontaneous tumour development in immunocompetent hosts, low mutational burden, extensive structural genomic alterations, and a highly immunosuppressive tumour microenvironment. These shared characteristics support the positioning of cOM as a relevant comparative oncology model for investigating the mechanisms underlying tumour aggressiveness, immune evasion, and therapeutic resistance in mucosal melanoma. In this review, we synthesize evidence suggesting that hMM and cOM are driven less by single dominant oncogenic mutations than by a complex convergence of pathway-level dysregulation arising from pervasive copy-number alterations, structural variants, and epigenetic plasticity. We further highlight the influence of these molecular features on immune visibility and immune suppression, as well as their contributions to the limited efficacy of single-agent targeted therapies and immune checkpoint blockade. Insights from canine immunotherapy studies provide preliminary translational support for combination-based therapeutic strategies and biomarker development. By integrating molecular, epigenetic, and immunological perspectives across species, this review supports the use of cOM as a comparative and translational platform for advancing therapeutic innovation in mucosal melanoma.
With the development and increased accessibility of diagnostic methods in veterinary medicine, the possibilities for early detection of proliferative lesions in the canine large intestine have expanded over the past decade. Since in human medicine, topoisomerase II alpha (Topo IIα) is considered a prognostic and predictive marker in various tumours, including colorectal carcinoma, we aimed to verify the role of this enzyme in proliferative epithelial lesions of the canine large intestine. In this study, we examined and compared the expression of Topo IIα in normal rectal mucosa, hyperplastic polyps, adenomas, and adenocarcinomas of the large intestine in dogs. Each type of lesion included 20 cases, in which the expression of Topo IIα was evaluated using immunohistochemistry. The median percentage of Topo IIα positive cells was 29.4% (IQR 24.9-37.1) in polyps, 30.3% (IQR 24.6-34.1) in adenomas, 36% (IQR 26.2-42.0) in adenocarcinomas, and 7.8% (IQR 5.9-10.6) in normal intestinal mucosa. Statistical analysis using the Kruskal-Wallis test followed by Dunn's post hoc tests with Bonferroni correction revealed significantly higher Topo IIα expression in hyperplastic polyps (p = 0.013, r = 0.62), adenomas (p = 0.033, r = 0.57) and adenocarcinomas (p = 0.002, r = 0.73) compared with normal rectal mucosa, with large effect sizes. Our results may serve as a preliminary basis for the potential use of anthracyclines in the treatment of canine colorectal adenocarcinomas, as well as in multiple adenomas or polyps when surgical options are limited. This study presents new insights of Topo IIα expression in canine large intestine carcinogenesis and could provide a valuable foundation for further clinical research.
Anatomic location is a known prognostic factor for canine mast cell tumours (MCTs). Previous publications have reported conflicting results about the clinical significance of MCT location in the inguinal and perineal regions and male genitalia. Hence, the goal of this study is to evaluate the grade, breed distribution and clinical outcome of canine perivulvar MCT. Surgical biopsy reports and associated medical records submitted between 2013 and 2024 to Michigan State University Veterinary Diagnostic Laboratory were retrospectively evaluated. Information regarding age, reproductive status, breed, tumour location, microscopic description, histopathologic grade, and survival was collected. Kaplan-Meier analysis was used to estimate median survival time (MST) with dogs lost to follow-up (LTF) or alive censored from analysis. Biopsy reports from 64 dogs with 68 perivulvar MCT were evaluated. The most common phylogenetic classification was Mastiff-Terrier (47%). The majority (97%) of evaluated MCT were cutaneous, with 85% categorised as Kiupel low grade, while the remaining 15% were high grade. Three of 19 tumours (16%) that had c-KIT PCR performed had mutations in either Exon 8 or Exon 11. KIT expression pattern was classified as Pattern 2 or 3 in 71% of evaluated tumours. Median Ki-67 proliferation index and Ki-67 × AgNOR scores were 7 and 13, respectively. MST for the entire population was 868 days. Dogs LTF had a median follow-up time of 442 days. Dogs with perivulvar MCT may achieve prolonged survival time with surgical excision, and prospective studies are warranted to determine the effect of adjuvant therapy on outcome.
Vasculogenic mimicry (VM) is a tumour cell-lined vessel-like structure associated with poor prognosis, metastasis, and treatment resistance in human breast cancer. Furthermore, it has been described in canine mammary tumours (CMTs). Hypoxia is an established driving factor for VM formation in tumours with this capability. This study aimed to compare VM-like structure formation between two CMT carcinoma cell lines, CF41.Mg and CMT-U27. VM-like tubular structure formation was evaluated using a Matrigel-based 3D cell culture system under normal conditions and after CoCl2 preconditioning. VM-like tubular structures were observed only in CF41.Mg cells, and CoCl2 preconditioning increased the number of meshes and segments without a time-dependent difference between 24 and 48 h. In contrast, CMT-U27 cells did not form VM-like structures under the tested conditions. Subsequently, RNA sequencing, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot analyses were conducted. Exploratory RNA sequencing of untreated cells showed differences in the expression patterns of selected genes involved in angiogenesis, epithelial-to-mesenchymal transition, cell migration, hypoxia-associated responses, and extracellular matrix organization between the two cell lines. CoCl2 treatment was associated with VM-related transcriptional and protein-level changes in CF41.Mg cells. In contrast, CoCl2 treatment was associated with E-cadherin upregulation and no significant changes in MMP2 or CDH2 expression in CMT-U27 cells. Taken together, under the tested conditions, CoCl2 preconditioning enhanced VM-like structure formation in CF41.Mg cells but did not induce VM-like tubular structures in CMT-U27 cells.
Tumour cells commonly exhibit aerobic glycolysis and produce lactate despite oxygen availability. Lactate dehydrogenase (LDH) catalyses pyruvate-lactate interconversion and regulates intracellular lactate levels. Endothelial cells also depend on glycolysis for ATP production, which prompted us to investigate LDH in canine hemangiosarcoma (HSA), a malignant endothelial tumour. We inhibited LDH with (R)-GNE-140 or sodium oxamate in two canine HSA cell lines (HU-HSA-2 and HU-HSA-3) and generated HU-HSA-3 clones with knockout of LDHA or LDHB to evaluate the effects of LDH perturbation. (R)-GNE-140 and sodium oxamate suppressed proliferation and reduced global histone lactylation levels in both cell lines. mRNA-sequencing (mRNA-seq) of (R)-GNE-140-treated HU-HSA-2 cells identified cholesterol/lipid metabolism-related gene sets among the top negatively enriched pathways. Representative cholesterol/lipid metabolism genes such as SREBF2, SQLE and LDLR responded differently depending on cell lines and inhibitors. (R)-GNE-140 decreased these genes in HU-HSA-2 but not HU-HSA-3, whereas sodium oxamate decreased them in HU-HSA-3 with limited effects in HU-HSA-2. In HU-HSA-3, LDHA and LDHB knockout clones decreased SREBP2 expression and reduced the number of lipid droplets. Fluvastatin, a cholesterol metabolism inhibitor, inhibited HSA cell growth in vitro but did not significantly suppress tumour growth in two HSA patient-derived xenograft (PDX) models. In contrast, combined fluvastatin and dipyridamole treatment inhibited proliferation in vitro and tumour growth in PDX models. Collectively, these results suggest a context-dependent association between LDH and cholesterol/lipid metabolism in canine HSA cell lines and provide a rationale for further evaluation of combined cholesterol pathway inhibition.
Accurate preoperative identification of cervical lymph node (LN) metastasis is essential for staging and treatment planning in dogs with oral malignancy, yet conventional imaging offers limited diagnostic sensitivity. This retrospective study evaluated whether CT-derived radiomic features, combined with machine learning classifiers, could predict the metastatic status of mandibular and retropharyngeal LNs. Forty-nine dogs with histopathologically confirmed oral malignancy underwent contrast-enhanced CT, and four bilateral cervical LN sites were manually segmented on images resampled to 1.0 mm isotropic spacing, yielding 195 LN observations (18 metastatic, 9.2%). One hundred and seven radiomic features were extracted per LN using PyRadiomics, and variance filtering, Spearman redundancy removal (|ρ| > 0.95), and Mann-Whitney U testing with Benjamini-Hochberg correction were performed within each fold of leave-one-patient-out cross-validation (LOOCV) to eliminate selection-leakage bias. Logistic regression, random forest (RF), support vector machine, and XGBoost were trained with SMOTE applied within training folds only, and two endpoints were evaluated on the same out-of-fold predictions: per-LN (primary) and per-patient (secondary, via maximum-probability aggregation across the four sites). Confidence intervals were derived from 5000-iteration patient-level bootstrap, and sensitivity analyses included repeated StratifiedGroupKFold cross-validation and cluster-robust generalised estimating equation (GEE) inference. At the per-LN level, RF achieved an AUC of 0.649 (95% CI 0.474-0.831; sensitivity 0.444, specificity 0.898) and XGBoost an AUC of 0.631 (0.501-0.772; sensitivity 0.944, specificity 0.379). Patient-level aggregation yielded an RF AUC of 0.613 (0.431-0.786; 9/13 metastatic-positive dogs identified) and an XGBoost AUC of 0.485. Four GLSZM/GLCM texture features were selected in 92%-100% of LOOCV folds and remained significant under GEE (adjusted p ≤ 0.012). CT-derived texture features therefore carry a reproducible, biologically interpretable signal for cervical LN metastasis, but leakage-controlled performance is modest in this proof-of-concept study, and external validation in larger, multi-institutional cohorts is required before clinical translation.
Companion canines need advances in therapeutic options for solid tumour malignancies. Natural killer (NK) cell infusions offer a promising treatment modality in canines with solid tumours. Autologous natural killer products are limited by dysfunctional immunity and a manufacturing process that delays care whereas allogeneic NK cells offer the possibility of 'off-the-shelf' therapy to be administered from healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 during expansion. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. Flow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumours were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. Canine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro and safety in vivo. Further studies will optimise the NK cell product and escalate dosing to reach the maximal tolerable dose.
Canine mammary tumours (CMTs) and human breast cancer (HBC) share highly similar pathological characteristics. Cancer stem cells (CSCs) are critical to breast cancer invasion, metastasis, drug resistance, and recurrence. The experiment utilised CMT cell line CMT-U27 and HBC cell line MDA-MB-231. Cancer stem cell spheres were isolated from the two cell lines by serum-free culture respectively, named as CMT-U27 microspheres (CMT-U27S) and MDA-MB-231 microspheres (MDA-MB-231S), and the proportion of CSCs with the CD44+/CD24- phenotype was identified by flow cytometry. Monensin (MON), one of Polyether ionophore antibiotics, has been demonstrated to effectively suppress various types of CSCs, while erlotinib (ERL), as a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR), effectively inhibits cancer cell growth. This study investigates the synergistic inhibitory effects of MON-ERL combination on CSCs derived from CMTs. Cell viability was analysed by CCK-8 assay, while cell invasion and mammosphere formation assays were conducted to evaluate changes. An orthotopic tumour model in nude mice using CMT-U27S was established for in vivo validation. The results suggested that MON and ERL combination synergistically inhibited CSCs viability and significantly suppress their invasion and mammosphere formation abilities. The expression of key proteins of the EGFR pathway-p-EGFR, PI3K, and p-AKT was significantly reduced. In animal experiments, tumour volumes in the combination treatment group were markedly reduced compared to those in all other groups (p < 0.05), and no lung metastases were observed only in the combination group. Immunofluorescence and immunohistochemistry results indicated a marked reduction of CD44+/CD24- cells in the combination group, along with suppressed expression of stem cell markers and proliferation/apoptosis proteins. In conclusion, MON combined with ERL effectively inhibits the proliferation, self-renewal, and metastatic capabilities of CSCs from CMTs by synergistically downregulating the EGFR/PI3K pathway, demonstrating promising antitumor potential and offering a new strategy for CMTs treatment.
Electrochemotherapy is increasingly used as a locoregional treatment for feline squamous cell carcinoma, but its role in advanced nasal planum disease remains poorly defined. This retrospective study evaluated the long-term outcome of ECT as a sole treatment modality in 18 cats with T3-T4N0M0 nasal planum squamous cell carcinoma. Adverse events were assessed according to VCOG-CTCAE v2. Response was assessed using RECIST v1.1 criteria, and progression-free survival (PFS), overall survival (OS) and time to progression (TTP) were analysed using Kaplan-Meier methods. At the end of treatment, 72.2% of cats achieved complete response, 16.7% partial response and 11.1% stable disease, resulting in an overall response rate of 88.8%. The 1-year complete response rate was 61.1%. Treatment response was significantly associated with PFS (p = 0.006), OS (p < 0.001) and TTP (p = 0.006), while recurrence was associated with shorter PFS (p = 0.003) and OS (p = 0.002). The 1-year risk of recurrence was 50%. Adverse events were mostly low-grade (VCOG Grade I-II) and self-limiting, the most common being transient local pain, erythema/crusting and pruritus; no grade ≥ 3 complications such as tissue necrosis or fistula formation were observed. These results suggest that ECT is an effective and well-tolerated treatment also in advanced stages nasal planum squamous cell carcinoma, with high response rates and long-term control disease. The risk of recurrence underscores the need for prolonged follow-up, as additional treatment sessions may be performed upon relapse, while ECT can also be used with palliative intent to prolong survival while preserving quality of life.
Canine mammary gland tumours (MGT) represent a common malignancy in intact female dogs, with a high metastasis rate approaching 50% and a poor prognosis. However, metastasis-related genes remain largely unelucidated. Accordingly, we aimed to identify differentially expressed mRNAs and miRNAs in primary canine MGT tissues from dogs with or without confirmed metastasis. We compared mRNA and microRNA (miRNA) expression profiles between malignant MGT tissue samples with and without metastasis. Differentially expressed genes and miRNAs were identified using next-generation sequencing and analysed using the Empirical Analysis of DEGs tool within CLC Genomics Workbench. We identified 119 genes and eight miRNAs as differentially expressed in primary MGTs from dogs with confirmed metastasis. The RIPPLY1 gene was significantly downregulated in primary MGT samples with confirmed metastasis and this result was validated by RT-qPCR. The Cancer Genome Atlas (TCGA) database showed decreased RIPPLY1 expression in human breast cancer, supporting its possible relevance to tumour biology. In silico analysis (miRWalk) revealed that miR-187 is a predicted candidate miRNA that may interact with RIPPLY1 and both NGS data and RT-qPCR showed increased cfa-miR-187 expression in primary tumour samples from dogs with confirmed metastasis. The inverse expression pattern of RIPPLY1 and cfa-miR-187, together with RNAhybrid binding prediction, suggests a possible candidate miRNA-mRNA relationship. STRING analysis suggested a possible pathway context involving beta-catenin/TCF-related proteins, but this computational result requires further validation. Based on these findings, we identified RIPPLY1 and cfa-miR-187 as potential candidate transcripts associated with primary canine MGTs from dogs with confirmed metastasis but functional validation is required.
Canine cutaneous mast cell tumours (MCTs) demonstrate variable clinical behaviour. Histological grading remains the primary prognostic approach, but some low-grade tumours behave aggressively, and interobserver variability persists. This underscores the need for complementary and objective biomarkers. Raman spectroscopy (RS) is a non-destructive optical technique based on the inelastic scattering of monochromatic light capable of detecting subtle biochemical changes in tissues. This exploratory study evaluates whether RS could differentiate low-grade from high-grade cutaneous MCTs based on characteristic vibrational signatures. Eighteen canine cutaneous mast cell tumours were graded according to the Kiupel two-tier system, comprising 10 low-grade and 8 high-grade cases. Formalin-fixed tumour samples were analysed, yielding a total of 68 Raman spectra. Spectra were acquired with a 785 nm laser, pre-processed using baseline correction, and normalized to the 1007 cm-1 phenylalanine band. Data normality was tested with the Shapiro-Wilk test, and group comparisons used the Mann-Whitney U test (α = 0.05). The 1007 cm-1 band showed no significant difference between Low- and High-Grade tumours (p = 0.068), validating its use for normalization. Conversely, the 860 cm-1 tyrosine-associated band was significantly higher in high-grade tumours (p = 0.033). Ratios normalized to the 1007 cm-1 peak consistently discriminated between grades, with significantly higher values in low-grade tumours across multiple spectral regions-particularly 1007/1299 and 1007/1301-1320 (all p < 0.05). Overall, RS identified distinct biochemical fingerprints between tumour grades in canine cutaneous MCTs, especially in spectral regions associated with amino acids, lipids, and nucleic acids. These findings support the potential of RS as a complementary tool for MCT characterization and encourage further validation in larger cohorts with clinical outcome data.
Moderately hypofractionated definitive-intent radiotherapy protocols can be used for canine intracranial meningiomas to shorten overall treatment time while maintaining tumour control and limiting normal tissue toxicity. This retrospective study evaluated oncologic outcomes, toxicity, and radiation dose-volume-related prognostic factors in dogs with imaging-based intracranial meningiomas treated with a 10-fraction definitive-intent protocol. Medical records from 2015 to 2025 were reviewed. All dogs received 10 daily fractions of 4 Gy delivered with a homogeneous dose prescription, risk-adapted heterogeneous dose distributions, or an 11% simultaneously integrated boost (SIB) to the gross tumour volume. One hundred and ten dogs were included, with a median follow-up of 498 days. Median time to progression was 873 days (95% CI: 552-1195), median overall survival after first radiotherapy was 756 days (95% CI: 474-1039), and median DSS was 1078 days (95% CI: 915-1242). Time to progression differed among treatment subgroups (p = 0.024). Larger volumes of normal brain receiving ≥ 32 Gy (> 11.5 cm3) were associated with shorter overall (p < 0.001) and disease-specific survival (p = 0.002). Use of an SIB was associated with shorter disease-specific survival (HR 2.47; p = 0.012), and post-radiotherapy seizures were associated with reduced time to progression (HR 2.01; p = 0.026). Tumour location was not identified as a prognostic factor. Acute or early delayed neurologic toxicity occurred in 10% of dogs and was generally transient and generally corticosteroid responsive. In conclusion, the definitive-intent, moderately hypofractionated 10-fraction radiotherapy protocol achieves long lasting tumour control and survival outcomes in dogs with intracranial meningiomas, provided that evidence-based brain dose-volume constraints are respected.
Osteosarcoma is the most common primary bone tumour in dogs, with approximately 75% of cases occurring in the appendicular skeleton. Standard-of-care therapy consists of limb amputation followed by carboplatin chemotherapy. A minority of dogs have limb-sparing treatments including stereotactic body radiation therapy (SBRT) or surgery. SBRT has a high incidence of pathologic fracture (41%-80% of cases) following treatment, which often results in amputation or euthanasia. Prophylactic bone stabilisation following SBRT has not been recommended due to high complication rates. The IlluminOss system (IS) is an intramedullary implant consisting of a light-curable polymer within an inflatable balloon catheter, which is approved in humans for traumatic fractures and prevention of fractures secondary to bone metastases. We performed a study to assess the safety and feasibility of using the IS following SBRT in six dogs with suspected appendicular osteosarcoma. The IS was placed the day after completion of SBRT. Five of six dogs received adjuvant carboplatin chemotherapy. The IS was well tolerated with no implant-associated infections or immediate post-operative complications occurring. Pathologic fractures occurred in four dogs with a median fracture-free interval of 107 days. Three dogs ultimately underwent amputation due to pathologic fractures. No other post-operative complications occurred. The median survival time was 156 days. These findings suggest that the IS may be a palliative option for dogs following SBRT, but further research to optimise use in the dog and evaluation of the use of the IS with ancillary fixation is necessary.
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.
Gene expression profiling (GEP) is being used with increasing frequency to characterise canine tumours, but canine samples are often not collected in a medium to preserve RNA. Cytology samples from human tumours have been shown to provide adequate RNA for GEP using NanoString nCounter technology, suggesting a similar approach might be viable for canine samples. Determine the quantity and quality of RNA extracted from canine cytology slides, evaluate the time frame for RNA integrity and assess the utility of performing GEP with extracted RNA. Cytological specimens were derived from reactive lymph nodes (n = 10) and lymphoma cases (n = 34) and characterised via flow cytometry or PCR for antigen receptor rearrangements. RNA was extracted from stained and unstained lymph node cytology slides using a RNeasy Mini Kit and subsequently evaluated for quality and quantity. GEP was carried out using nCounter technology with the NanoString Canine IO Panel. Extracted RNA from lymphoma and reactive cytology samples generated concentrations ranging from 27.5 to 543.9 ng/μL (median: 168.5 ng/μL; interquartile range (IQR): 108.15-275.95 ng/μL). RNA integrity numbers (RINs) ranged from 1.0 to 7.2 (median: 2.9; IQR: 2.10-4.45) and DV200 values ranged from 50.5% to 93.2% (median: 81.65%; IQR: 71.95%-87.85%). RIN and DV200 values were highest for extractions within 7 days of sample collection. GEP revealed overexpression of 17 genes in lymphoma samples such as MYC, TERT and BRCA2. Canine lymph node cytologic specimens provide sufficient RNA quality for GEP. The nCounter Canine IO Panel demonstrates the utility of cytology specimens for evaluating gene expression.
Rational, mechanism-based drug selection for canine diffuse large B-cell lymphoma (DLBCL) remains limited, despite extensive molecular work in human DLBCL. We asked whether human and canine DLBCL share a conserved transcriptional network that could anchor therapeutic hypotheses for dogs. Using public human and canine DLBCL transcriptomes, we derived a 409-gene cross-species module defined by concordant tumour-normal dysregulation and projected it onto pathway and protein-protein interaction (PPI) networks. The module was dominated by DNA replication, cell-cycle checkpoint and DNA damage-response programmes and contained a compact PPI core centred on TOP2A and PARP1. Querying the Connectivity Map/LINCS library with the human component of the module highlighted anthracycline-like topoisomerase II poisons (valrubicin, etoposide, amsacrine) and the PARP inhibitor rucaparib among the ~0.2% most negative connectivity scores, while directly targeting TOP2A and/or PARP1. A simple up-down module score strongly separated tumour from normal lymph nodes in both species and was associated with outcome: higher scores predicted longer progression-free survival in CHOP-treated dogs and better overall survival in an independent human cohort, with a similar but underpowered trend in TCGA-DLBC. Finally, extracellular-vesicle microRNA (EV-miRNA) profiling in human DLBCL showed that differentially expressed EV-miRNAs, including let-7 family members, miR-21-5p, miR-124-3p and miR-205-5p, converge on the same TOP2A/PARP1-centred core. These cross-species, multi-layer data support topoisomerase II poisons and PARP inhibition as coherent, network-anchored candidate therapies for canine DLBCL, with module scores and EV-miRNAs as candidate biomarkers.
Effective chemotherapy for canine histiocytic sarcoma (CHS) has yet to be established. In our previous study, CHS cell lines were subclassified into two groups based on their gene expression profiles: Group A and Group B. This study aimed to identify novel therapeutic agents that are effective against each CHS subgroup, and we performed high throughput screening, which examined the effects of 1824 chemical compounds on the 10 CHS cell lines. Among the compounds examined, 73 compounds were identified as candidates to show antiproliferative effects in all CHS cell lines examined, and the median IC50 values of 5-fluorouracil and sunitinib were 7.49 μM and 608 nM, respectively, across all CHS cell lines. While there was no compound that was specifically effective for Group B CHS cell lines, duvelisib was identified as a selective antitumor agent for CHS cell lines in Group A (median IC50 = 287 nM), with minimal effects on the other group (median IC50 > 5 μM) and normal peripheral blood mononuclear cells (IC50 = 7.46 μM). The amount of phosphorylated Akt protein decreased following duvelisib treatment in all eight CHS cell lines in Group A, but not in cell lines in Group B. Furthermore, duvelisib induced cell cycle arrest and increased cell death in a subset of Group A cell lines. These findings demonstrated that duvelisib exerted antiproliferative effects through inhibition of the Akt pathway in a subset of CHS cell lines while causing minimal cytotoxicity to normal cells.
