Establishment of canine acanthomatous ameloblastoma (CAA) cell lines has profound importance in pre-clinical in vitro testing. The goal of this study was to develop, authenticate, and characterize three CAA cell lines. Cell lines were developed with standard cell culture techniques from dogs with naturally occurring CAA. Each cell line was authenticated as canine with a validated PCR panel. Epithelial cell origin was confirmed with pan-CK flow cytometry. Next generation DNA and RNA sequencing characterized features of the cell lines and compared them to the parent tumor. We confirmed that all cell lines maintained mutations seen in the parent tumors including a missense HRAS mutation and RTK-RAS pathway upregulation across all samples. Other common mutations (2/3 cell lines and their parent tumors) included ADGRA3, DNAH7, F10, KIAA1671, OGFR, SLC6A17, SNX7, SPTBN5, TENM4, and YEATS2. We compared the transcriptional profiles from our parent tumors and established cell lines to historical transcriptomic analysis of CAA and healthy gingiva. We confirmed the same canonical CAA molecular features of primary tumors across studies with distinct clustering from healthy gingiva. Further, we documented that the cell lines maintained upregulated genes that are seen within in vivo CAA across both studies including upregulated GPC4 and ETV5 as well as gene sets associated with the presence of epithelial-mesenchymal transformation, KRAS, Pi3K-AKT signaling, and hedgehog signaling pathways. Thorough characterization of the mutational and transcriptional profiles of the established cell lines, especially in context to how they differ from the parent tumors, sets the platform for translational in vitro testing.

IntroductionGlioma stem cells (GSCs) have been implicated in radio- and chemotherapeutic resistance of glioblastoma (GBM). Therapeutic targeting of GSCs has shown promise in immunocompromised rodent models but have not been translated into effective therapies for human patients. These failures underscore the translational limitations of rodent models and highlight the need for complementary models that accurately and reliably predict therapeutic translation for human HGG. Spontaneous canine high-grade gliomas (HGGs) may provide a complementary translational model for human therapeutic development. While described in canine HGGs, little is known about canine glioma stem cell biology.MethodsHere, we evaluated cellular metabolism, cytosine modifications, gene expression, and functional tests of malignancy to interrogate differences between canine high-grade astrocytoma-derived glioma stem-cell like cells (GSLC) and a traditional non-stem cell glioma cell line following exposure to hypoxia. ResultsHypoxia increased oxygen consumption rates in GSLCs and augmented features of malignancy in GSLCs. We observed variable cytosine modifications and mRNA expression across cell lines, and our data did not correlate cytosine modification patterns with oxygen consumption capacity following hypoxia. However, we did demonstrate a positive correlation between up-regulated genes in human GBM GSCs and hypomethylation of orthologous canine genes following hypoxia.DiscussionTogether, these data support that hypoxia enhances distinct stem-like traits in canine astrocytoma GSLCs, similar to human GSCs.

Equine Juvenile Spinocerebellar Ataxia (EJSCA) is a novel autosomal recessive neurologic disease in Quarter Horses. Affected foals display a progressive proprioceptive ataxia by 1-5 weeks of age, leading to recumbency and necessitating euthanasia. Whole genome sequencing was performed on 7 EJSCA cases and unaffected horses that included 4 obligate carriers, 4 unaffected half or full-siblings, and 28 unrelated, unaffected control Quarter Horses. An 82 kb region of association was identified (EquCab3.0, chr11: 6963986-7045999), containing 9 candidate SNPs across four genes (FADS6, FDXR, GRIN2C and TMEM104). Decreased FDXR mRNA expression and a cryptic exon was identified in spinal cord tissue from EJSCA cases via RNA-sequencing. One of the 9 associated SNPs (FDXR-203 c.177 + 1778G > C) was the eighth base pair of this cryptic exon. Affected foals were all homozygous for the variant. Protein concentrations of FDXR were lower in EJSCA cases in spinal cord and liver compared to unaffected controls. The FDXR-203 c.177 + 1778G > C mutation represents the first non-coding neurological genetic variant in horses. Additionally, this is the first genetic cause of a degenerative axonopathy in the horse and a spontaneous disease model to study FDXR pathology in humans.

STAT3 is an oncogenic transcription factor that activates cancer cell signaling and induces an immunosuppressive immune environment, making it an attractive therapeutic target. Transcription factors are exceptionally challenging targets and there are no Food and Drug Administration-approved STAT3 inhibitors. We previously reported positive pharmacodynamics of a linear STAT3 decoy oligonucleotide administered intratumorally in a phase 0 trial in patients with head and neck cancer squamous cell carcinoma (HNSCC). Here, we describe the anti-tumor and immune effects of a systemically administered cyclic STAT3 decoy (CS3D) in immunocompetent HNSCC murine models and the safety and efficacy of CS3D in a clinical trial in pet cats with HNSCC. Responders in the clinical trial (35% disease control rate) showed significant differences in selected peripheral blood immune parameters as well as elevated PD-1 expression in the tumors compared with non-responders. These findings support a clinical trial of CS3D in HNSCC patients.

Canine osteosarcoma (OSA) and melanoma are highly aggressive cancers with a high propensity for lung metastasis. A recent phase 1 trial demonstrated that inhaled recombinant human interleukin-15 (rhIL-15) immunotherapy is safe and may be effective against lung metastases in dogs with metastatic OSA or melanoma. Notably, baseline lymphopenia correlated with clinical benefit in that trial. Building on these findings, this study evaluates whether preconditioning with doxorubicin to induce transient lymphodepletion before inhaled rhIL-15 immunotherapy improves the overall response rate in dogs with metastatic melanoma or tumors of bone. Dogs with established pulmonary metastases from melanoma or bone tumors were treated with doxorubicin (30 mg/m2 or 1 mg/kg) 7 days before starting inhaled rhIL-15 (50 µg twice daily x 14 days). Response rate was the primary objective of this study. Secondary objectives included toxicity assessment, immune correlative analyses via hematological monitoring, and NanoString transcriptomics analysis. Ten dogs were enrolled, with eight reaching the Day 35 evaluable response assessment. One dog achieved a complete response lasting over 1 year, and two had stable disease, yielding an overall clinical benefit rate (CBR) of 30

Background:We have previously shown inhaled IL-15 is associated with anti-tumor responses in dogs with metastatic osteosarcoma (OSA) and melanoma. We evaluated inhaled IL-15 combined with amputation and chemotherapy for localized canine OSA eligible for treatment with curative intent. Methods:In a multicenter COTC phase-II-trial for dogs with limb OSA, we hypothesized 2 weeks of inhaled rhIL-15 after amputation and prior to chemotherapy would reduce the risk of metastatic failure at the completion of chemotherapy from a historical rate of 40% to 20%. Using a 2-sided alpha of 0.05, we planned an accrual of 40 dogs to test this hypothesis with 80% power. We performed immune correlative assays and sequencing of peripheral blood mononuclear cells (PBMCs) and primary amputation specimens. Results:Unexpectedly, disease-free survival and overall survival were statistically inferior for dogs in the intent-to-treat population compared to a well-validated historical control cohort, so the trial was halted for futility. Cytotoxicity assays of PBMCs showed significant decreases after both surgery and chemotherapy with an overall decrease from the start to end of therapy (-18.2 ± 16.1%, P<0.001). Some dogs demonstrated positive fold change in PBMC cytotoxicity, which correlated significantly with improved dog survival (P = 0.004, r=0.62). Although plasma concentrations of key cytokines varied markedly with no significant differences between disease-free and metastatic-failure patients, inflammatory cytokines such as IL-6 showed absolute increases post-amputation and post-chemotherapy, correlating with decreases in cytotoxicity. Tumor sequencing data reproduced immune signatures as observed in both human and canine cohorts, and PBMC single cell sequencing data showed that gene expression profiles of NK and T cells were significantly different between short and long disease-free interval subjects. Conclusions:Inhaled rhIL-15 combined with amputation and chemotherapy is associated with worse outcomes in dogs with OSA. Correlative assays suggest significant immunological effects of amputation and chemotherapy on immune responses. These data have important implications on novel immunotherapy strategies involving multimodality approaches including surgery and chemotherapy.

Glioma stem cells (GSCs) have been implicated in radio- and chemotherapeutic resistance of glioblastoma (GBM). Therapeutic targeting of GSCs has shown promise in immunocompromised rodent models but have not been translated into effective therapies for human patients. The limited success of translating therapies from rodent models to GBM patients may be attributed, in part, to the lack of co-evolution between tumors and the tumor microenvironment in immunocompromised rodent models. Thus, spontaneous canine high-grade gliomas (HGGs) may provide a complementary translational model for human therapeutic development. While described in canine HGGs, little is known about canine glioma stem cell biology. In this study, we evaluatedcellular metabolism, DNA methylation, gene expression, and functional tests of malignancy to interrogate differences between canine glioma stem-cell like (GSLC) lines and a traditional serum grown glioma cell line following exposure to hypoxia. Hypoxia increased oxygen consumption rates in GSLCs, which correlated with hypoxia-induced hypomethylation and increased mRNA levels of genes important in cellular metabolism and stemness (e.g., KCNN3, KDM4B, TCF7L2), as well as augmented features of malignancy in GSLCs. Importantly, we were able to demonstrate a positive correlation between up-regulated genes in human GBM GSCs and hypomethylation of orthologous canine genes following hypoxia. Together, these data highlight similarities between canine and human GBM GSC cellular metabolism and their epigenetic regulation.

Genome-wide association studies (GWAS) in long-lived human populations have led to identification of variants associated with Alzheimer’s disease and cardiovascular disease, the latter being the most common cause of mortality in people worldwide. In contrast, naturally occurring cancer represents the leading cause of death in pet dogs, and specific breeds like the Golden Retriever (GR) carry up to a 65% cancer-related death rate. We hypothesized that GWAS of long-lived GRs might lead to the identification of genetic variants capable of modifying longevity within this cancer-predisposed breed. A GWAS was performed comparing GR dogs ≥ 14 years to dogs dying prior to age 12 which revealed a significant association to ERBB4 , the only member of the epidermal growth factor receptor family capable of serving as both a tumor suppressor gene and an oncogene. No coding variants were identified, however, distinct haplotypes in the 5′UTR were associated with reduced lifespan in two separate populations of GR dogs. When all GR dogs were analyzed together ( n = 304), the presence of haplotype 3 was associated with shorter survival (11.8 years vs. 12.8 years, p = 0.024). GRs homozygous for haplotype 3 had the shortest survival, and GRs homozygous for haplotype 1 had the longest survival (11.6 years vs. 13.5 years, p = 0.0008). Sub-analyses revealed that the difference in lifespan for GRs carrying at least 1 copy of haplotype 3 was specific to female dogs ( p = 0.009), whereas survival remained significantly different in both male and female GRs homozygous for haplotype 1 or haplotype 3 ( p = 0.026 and p = 0.009, respectively). Taken together, these findings implicate a potential role for ERBB4 in GR longevity and provide evidence that within-breed canine lifespan studies could serve as a mechanism to identify favorable or disease-modifying variants important to the axis of aging and cancer.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

Purpose Although recombinant human interleukin-15 (rhIL-15) has generated much excitement as an immunotherapeutic agent for cancer, activity in human clinical trials has been modest to date, in part due to the risks of toxicity with significant dose escalation. Since pulmonary metastases are a major site of distant failure in human and dog cancers, we sought to investigate inhaled rhIL-15 in dogs with naturally occurring lung metastases from osteosarcoma (OSA) or melanoma. We hypothesized a favorable benefit/risk profile given the concentrated delivery to the lungs with decreased systemic exposure. Experimental design We performed a phase I trial of inhaled rhIL-15 in dogs with gross pulmonary metastases using a traditional 3+3 cohort design. A starting dose of 10 µg twice daily × 14 days was used based on human, non-human primate, and murine studies. Safety, dose-limiting toxicities (DLT), and maximum tolerated dose (MTD) were the primary objectives, while response rates, progression-free and overall survival (OS), and pharmacokinetic and immune correlative analyses were secondary. Results From October 2018 to December 2020, we enrolled 21 dogs with 18 dogs reaching the 28-day response assessment to be evaluable. At dose level 5 (70 μg), we observed two DLTs, thereby establishing 50 µg twice daily × 14 days as the MTD and recommended phase 2 dose. Among 18 evaluable dogs, we observed one complete response >1 year, one partial response with resolution of multiple target lesions, and five stable disease for an overall clinical benefit rate of 39%. Plasma rhIL-15 quantitation revealed detectable and sustained rhIL-15 concentrations between 1-hour and 6 hour postnebulization. Decreased pretreatment lymphocyte counts were significantly associated with clinical benefit. Cytotoxicity assays of banked peripheral blood mononuclear cells revealed significant increases in peak cytotoxicity against canine melanoma and OSA targets that correlated with OS. Conclusions In this first-in-dog clinical trial of inhaled rhIL-15 in dogs with advanced metastatic disease, we observed promising clinical activity when administered as a monotherapy for only 14 days. These data have significant clinical and biological implications for both dogs and humans with refractory lung metastases and support exploration of combinatorial therapies using inhaled rhIL-15.

The BMI1 proto-oncogene, polycomb ring finger protein (BMI1) is a key component of the epigenetic polycomb repressor complex 1, and has been associated with aggressive behaviour and chemotherapeutic resistance in various malignances including human gliomas. Similar to humans, spontaneous canine gliomas carry a poor prognosis with limited therapeutic options. BMI1 expression and the effects of BMI1 inhibition have not been evaluated in canine gliomas. Here, we demonstrate that BMI1 is highly expressed in canine gliomas. Although increased BMI1 protein expression correlated with higher glioma grade in western blot assays, this correlation was not observed in a larger sample set using immunohistochemical analysis. The BMI1 inhibitor, PTC-209, suppressed BMI1 expression in established canine glioma cell lines and resulted in antiproliferative activity when used alone and in combination with chemotherapeutic agents. PTC-209 targeting of BMI1 activated the retinoblastoma (RB) pathway through downregulation of total and phosphorylated RB, independent of INK4A/ARF signalling, likely through BMI1-inhibition mediated upregulation of p21. These data support the rationale for targeting of BMI1 signalling and the use of canine glioma as a translational therapeutic model for human disease.

Gene retrocopies arise from the reverse transcription and insertion into the genome of processed mRNA transcripts. Although many retrocopies have acquired mutations that render them functionally inactive, most mammals retain active LINE-1 sequences capable of producing new retrocopies. New retrocopies, referred to as retro copy number variants (retroCNVs), may not be identified by standard variant calling techniques in high-throughput sequencing data. Although multiple functional FGF4 retroCNVs have been associated with skeletal dysplasias in dogs, the full landscape of canid retroCNVs has not been characterized. Here, retroCNV discovery was performed on a whole-genome sequencing data set of 293 canids from 76 breeds. We identified retroCNV parent genes via the presence of mRNA-specific 30-mers, and then identified retroCNV insertion sites through discordant read analysis. In total, we resolved insertion sites for 1911 retroCNVs from 1179 parent genes, 1236 of which appeared identical to their parent genes. Dogs had on average 54.1 total retroCNVs and 1.4 private retroCNVs. We found evidence of expression in testes for 12% (14/113) of the retroCNVs identified in six Golden Retrievers, including four chimeric transcripts, and 97 retroCNVs also had significantly elevated FST across dog breeds, possibly indicating selection. We applied our approach to a subset of human genomes and detected an average of 4.2 retroCNVs per sample, highlighting a 13-fold relative increase of retroCNV frequency in dogs. Particularly in canids, retroCNVs are a largely unexplored source of genetic variation that can contribute to genome plasticity and that should be considered when investigating traits and diseases.

Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state, in vitro activation with cytokines and co-culture, and in vivo activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and in vitro versus in vivo treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.

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Dogs exhibit a wide variety of coat color types, and many genes have been identified that control pigment production, appearance, and distribution. Some breeds, such as the Nova Scotia Duck Tolling Retriever (NSDTR), exhibit variation in pheomelanin pigment intensity that is not explained by known genetic variants. A genome-wide association study comparing light red to dark red in the NSDTR identified a significantly associated region on canine chromosome 15 (CFA 15:23 Mb–38 Mb). Coverage analysis of whole genome sequence data from eight dogs identified a 6 kb copy number variant (CNV) 152 kb upstream of KITLG. Genotyping with digital droplet PCR (ddPCR) confirmed a significant association between an increased copy number with the dark-red coat color in NSDTR (p = 6.1 × 10−7). The copy number of the CNV was also significantly associated with coat color variation in both eumelanin and pheomelanin-based Poodles (p = 1.5 × 10−8, 4.0 × 10−9) and across other breeds. Moreover, the copy number correlated with pigment intensity along the hair shaft in both pheomelanin and eumelanin coats. KITLG plays an important role in melanogenesis, and variants upstream of KITLG have been associated with coat color variation in mice as well as hair color in humans consistent with its role in the domestic dog.

Background Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells impede translation. Outbred dogs offer an important link for NK-based cancer immunotherapy studies. We compared gene expression profiles of dog NK signatures in vitro and from a phase I clinical trial of inhaled IL-15, and analyzed dog, mouse and human NK cells using a novel orthologous transcriptome. Methods We performed differential gene expression (DGE) using resting healthy donor CD5dim NK populations and following ex vivo activation using recombinant human (rh)IL-15 or co-culture with irradiated feeder cells. Eight dogs with naturally-occurring pulmonary metastases were enrolled on a Phase I clinical trial of inhaled rhIL-15 using a 3+3 cohort design with escalating doses of inhaled rhIL-15. Blood was collected from study dogs before, during, and after therapy. We compared DGE among healthy and cancer-bearing dogs and then across mouse, dog and human NK cells in resting and activated states using ~7000 1:1 orthologous genes. Results DGE revealed distinct transcriptional profiles between the ex vivo resting, IL-15 and co-cultured canine NK cells. Among treated patients, hierarchical clustering revealed that in vivo NK cell transcriptional signatures grouped by individual dog, and not amount of time exposed to treatment. PCA showed in vivo profiles of the clinical responders were distinctly separate from the non-responding patients (PC1 38%, PC2 12%). Patient in vivo NK cell transcription profiles most closely resembled those of ex vivo resting NK cells and not IL-15 treated or co-culture activated (PC1 43%, PC2 19%), likely reflecting key differences in activation. In cross-species analysis, PCA showed within-species spatial clustering of resting NK cells. After activation, variance between dog and human NK cells decreased, while variance between human and mouse NK cells increased (PC1 40%, PC2 28%). Conclusions In this first transcriptomic sequencing of dog NK cells, we demonstrate distinct gene profiles of ex vivo activated NK cells from healthy donors compared to circulating NK cells from dogs receiving inhaled rhIL-15 on a clinical trial. Baseline in vivo NK cell profiles appear to predict response to therapy more than changes over time. We also show distinct gene profiles of NK cells across the most commonly used mouse, dog, and human NK populations, with convergence of dog and human NK cells after activation. By defining the canine NK cell DGE signatures, these data fill a gap in translational NK studies. Ethics Approval The canine clinical trial study was approved by IACUC and Clinical Trials Review Board (Inhaled IL-15 Immunotherapy for Treatment of Lung Metastases, Protocol #20179).

Abstract Introduction: Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have been a barrier to translation. Outbred dogs are an important link for NK-based cancer immunotherapy studies, so better characterization of canine NK cells is needed. We used RNAseq to compare gene expression profiles of ex vivo dog NK cells to in vivo NK signatures from dogs with pulmonary metastases receiving inhaled recombinant human (rh) IL-15 in a phase I clinical trial. Methods: Eight dogs with spontaneous pulmonary metastases were enrolled on an IACUC and clinical trials review board-approved Phase I clinical trial of inhaled rhIL-15 using a 3+3 cohort design with escalating doses of inhaled rhIL-15. Blood was collected from study subjects immediately pre-treatment and on days 7, 14 and 21 after treatment initiation for isolation of NK cells (CD5dim) and RNAseq. We performed differential gene expression (DGE) comparing trial patients to healthy beagle CD5dim NK populations (resting) and ex vivo activated beagle NK cells using IL-15 and feeder line co-culture. We assessed global transcriptional profile, specific activating and inhibitory receptors of NK function, and principal component analysis (PCA) for variation between treatment groups (FDR <0.05). Results: Of 8 dogs, 2 demonstrated > 100-day survival with 1 stable disease and 1 partial response based on RECIST criteria. DGE revealed distinct transcriptional profiles between the ex vivo resting, IL-15 and co-cultured canine NK cells. Among treated patients, hierarchical clustering revealed that in vivo NK cell transcriptional signatures grouped by individual dog, and not amount of time exposed to treatment. PCA showed in vivo profiles of the long term survivors were distinctly separate from the non-responding patients (PC1 38%, PC2 12%). This suggests response to therapy could be determined by baseline NK cell characteristics rather than changes over time following treatment. Patient in vivo NK cell transcription profiles most closely resembled those of ex vivo resting NK cells and not IL-15 or co-culture activated (PC1 43%, PC2 19%), likely reflecting key differences in activation ex vivo vs in vivo. Key genes induced in vivo (>20X) post inhalation of rhIL-15 include DLA-DRA, B2M, and thymosin beta 4, while key genes induced ex vivo post rhIL-15 exposure include CD96, KLRB1, and SPP1/OPN. Conclusion: In this first transcriptomic sequencing of dog NK cells, we demonstrate distinct gene profiles of ex vivo activated NK cells from healthy donors compared to circulating NK cells from dogs receiving inhaled rhIL-15 on clinical trial. Baseline NK cell profiles appear to predict response more than changes over time. These data highlight the strength of the outbred dog model in speeding novel immunotherapy and biomarker studies. Citation Format: Alicia A. Gingrich, Taylor Reiter, Sean Judge, Daniel York, Mio Yanagisawa, Ian Sturgill, Rachel Brady, Kevin Stoffel, Arta Monjazeb, C. Titus Brown, Robert Rebhun, Robert J. Canter. Transcriptomic profiles improve characterization natural killer cells and predict response to cytokine therapy in clinical trial for dogs with pulmonary metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1339.

Our lack of understanding of the immune microenvironment in canine osteosarcoma (cOSA) has limited the identification of potential immunotherapeutic targets. In particular, our ability to utilize readily available tissue from a dog's primary tumour to predict the type and extent of immune response in their pulmonary metastatic lesions is unknown. We, therefore, collected 21 matched pairs of primary tumours and pulmonary metastatic lesions from dogs with OSA and performed immunohistochemistry to quantify T-lymphocyte (CD3), FOXP3+ cell, B-lymphocyte (Pax-5), and CD204+ macrophage infiltration. We found that T-lymphocytes and FOXP3+ infiltrates in primary tumours positively correlated with that of metastatic lesions (ρ = 0.512, P = 0.038 and ρ = 0.698, P = 0.007, respectively), while a strong trend existed for CD204+ infiltrates (ρ = 0.404, P = 0.087). We also observed T- and B-lymphocytes, and CD204+ macrophages to be significantly higher in a dog's pulmonary metastasis compared to their primary tumour (P = 0.018, P = 0.018, P = 0.016, respectively), while FOXP3+ cells were only significantly higher in metastases when all primary tumour and metastasis lesions were compared without pairing (P = 0.036). Together, these findings suggest that the metastatic immune microenvironment may be influenced by that of the primary cOSA, and that primary tumour immune biomarkers could potentially be applied to predict immunotherapeutic responses in gross metastatic disease. We, therefore, provide a rationale for the treatment of cOSA pulmonary metastases with immunotherapeutics that enhance the anti-tumour activity of these immune cells, particularly in dogs with moderate to high immune cell infiltration in their primary tumours.

Curcumin has well-established anti-cancer properties in vitro, however, its therapeutic potential has been hindered by its poor bioavailability. Lipocurc is a proprietary liposome-encapsulated curcumin formulation that enables intravenous delivery and has been shown to reach its highest concentration within lung tissue. The goal of this study was to characterize the anti-cancer and anti-angiogenic activity of Lipocurc in vitro, in addition to evaluating Lipocurc infusions in dogs with naturally occurring cancer. We therefore evaluated the effect of Lipocurc, relative to free curcumin, on the viability of canine osteosarcoma, melanoma and mammary carcinoma cell lines, as well as the ability of Lipocurc to inhibit endothelial cell viability, migration and tube formation. We also undertook a pilot clinical trial consisting of four weekly 8-hour Lipocurc infusions in 10 cancer-bearing dogs. Tumour cell proliferation was inhibited by curcumin at concentrations exceeding those achievable in the lung tissue of dogs. Similarly, equivalent high concentrations of Lipocurc and curcumin also inhibited endothelial cell viability, migration and tube formation. Four out of six dogs completing planned infusions of Lipocurc experienced stable disease; however, no radiographic responses were detected.

BACKGROUND:Activating transcription factor 5 (ATF5) is a transcription factor that is highly expressed in undifferentiated neural progenitor/stem cells as well as a variety of human cancers including gliomas.AIMS:In this study, we examined the expression and localization of ATF5 protein in canine gliomas, and targeting of ATF5 function in canine glioma cell lines.MATERIALS AND METHODS:Paraffin-embedded canine brain glioma tissue sections and western blots of tumours and glioma cells were immunoassayed with anti-ATF5 antibody. Viability of glioma cells was tested with a synthetic cell-penetrating ATF5 peptide (CP-d/n ATF5) ATF5 antagonist.RESULTS:ATF5 protein expression was in the nucleus and cytoplasm and was present in normal adult brain and tumour samples, with significantly higher expression in tumours as shown by western immunoblotting. CP-d/n ATF5 was found to decrease cell viability in canine glioma cell lines in vitro in a dose-dependent manner.CONCLUSION:Similarities in expression of ATF5 in rodent, dog and human tumours, and cross species efficacy of the CP-d/n ATF5 peptide support the development of this ATF5-targeting approach as a novel and translational therapy in dog gliomas.