Canine Osteosarcoma Research Articles

Methadone does not potentiate the effect of doxorubicin in canine tumour cell lines.

Opioid receptor activation was shown to enhance the efficacy of anti‐neoplastic drugs in several human cancer cell lines. In these cell lines, doxorubicin increased the number of opioid receptors and methadone concurrently enhanced cellular doxorubicin uptake. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti‐cancer therapy. Especially in veterinary medicine, where side effects of chemotherapy are tolerated to a lesser extent and hence smaller doses are given, agents potentiating chemotherapeutic agents would be an optimal approach to treatment. Canine transitional cell carcinoma cells (TCC, K9TCC), canine osteosarcoma cells (OSA, Abrams) and canine hemangiosarcoma cells (HSA, DAL‐4) were incubated with different combinations of methadone, buprenorphine and doxorubicin, in order to test inhibition of cell proliferation. Opioid receptor density was assessed with fluorescence‐activated cell sorting in drug native and doxorubicin pretreated cells. In TCC and OSA cell lines opioid receptor density increased after doxorubicin pretreatment. In combination treatment, however, we did not find significant potentiation of doxorubicin's inhibitory effect on proliferation in these cell lines. Neither was there a significant increase of the effect of doxorubicin when the opioids were added 24 hr before doxorubicin. Hence, we could not confirm the hypothesis that opioids increase the anti‐proliferative effect of the anti‐neoplastic drug doxorubicin in any of these canine tumour cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer.

S100A4 mRNA-protein relationship uncovered by measurement noise reduction

Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and “splicing weakness” involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman’s coefficient ρ = 0.72 (p = 0.006)).Key messages• RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour.• Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation.• HNRNPL is stably expressed across various cancer tissues and osteosarcoma.• Logit transformed qIHC score better associates with mRNA amount.• Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.

Molecular Landscape of Canine Osteosarcoma

Osteosarcoma affects about 2.8% of dogs with spontaneous cancer and its occurrence is significantly higher than observed in humans. The prognosis of this disease is relatively poor, where the survival rate of ~45% of dogs with bone cancer is one year. With the recent advancement in characterizing mutational landscapes of various cancer types, personalized medicine has become increasingly important for cancer treatment. The purpose of this study is to characterize the mutation and expression profile of canine osteosarcoma. Using whole exome capture (Agilent Sure Select Canine V2) we sequenced 26 primary tumors and matched normal samples, with coupled treatment and outcome data. These samples were previously analyzed for gene expression using Affymetrix Canine 2.0 microarrays.The BWA tool was used to map Illumina reads to the CanFam3.1 genome and short variants were called and annotated using Mutect2 and SnpEff tools, respectively. Additionally, copy number variations were called and annotated using Sequenza and GISTIC tools. The total number of somatic variants identified across 26 samples ranged from 148 to 3,785 The protein coding mutations per Megabase ranged from 0.25 to 11.92. Among the protein coding somatic variants identified as SNVs and INDELS, an average of 80% were missense mutations (range 64% to 92%). The top recurrent mutations were observed in TP53 (82% of the samples), SETD2 (22%). We have also identified 56 known cancer genes with copy number variations (amplification or deletion) in at least 58% of the samples. When compared to pediatric osteosarcoma, there was an overlap of 38 genes (eg: TFEB, MYC) that were recurrently amplified and 69 genes (eg: RB1, MSH3) were recurrently deleted in both canine and human osteosarcoma.Using the microarray data, we have identified 585 differentially expressed genes between normal bone and osteosarcoma samples. Using GeneSet Expression Analysis (GSEA) we observed down‐regulation of myogenesis genes and up‐regulation of extracellular matrix genes in tumor samples. Additionally, we have quantified T‐cell and macrophage infiltration in the tumors via immunohistochemistry. Across 20 samples, the median percentage of tumor area with CD3+ and MAC387 positive cells was 0.14% and 0.21%, respectively. There was no significant correlation between disease‐free interval (DFI) and median dichotomized T‐cell or macrophage infiltration. However, Kaplan‐Meier analysis revealed that dogs with TP53 missense mutations had significantly longer DFIs than those with TP53 wildtype/null mutations. Further, correlation of gene expression and DFI can provide us with prognostic biomarkers for osteosarcoma in dogs.Support or Funding InformationAnschutz Foundation and Morris Animal Foundation

Fibroblastic Subtype has a Favourable Prognosis in Appendicular Osteosarcoma of Dogs

Osteosarcoma (OS) is an aggressive malignant bone neoplasm that occurs mostly in the appendicular skeleton of dogs and people. OS is classified based on the presence of malignant stroma and the formation of extracellular matrix into osteoblastic, chondroblastic and fibroblastic forms. This study investigated the correlation between the three histological subtypes of canine OS and clinical outcome. Additionally, we examined whether there was any difference in the immunolabelling of desmin, S100 and neuron-specific enolase (NSE) between the three histological subtypes. Formalin-fixed and paraffin wax-embedded tissues from 87 dogs with primary OS were available for this study. The survival times were correlated with appendicular OS subtypes in dogs that were treated surgically, received adjuvant chemotherapy and had no pulmonary metastasis at the time of diagnosis. Dogs with an appendicular fibroblastic OS had significantly prolonged mean average survival times (546±105 days) in comparison with dogs having appendicular osteoblastic (257±48 days) or appendicular chondroblastic (170±28 days) OS (P=0.003, Log Rank). The results also revealed that the appendicular chondroblastic subtype is a significant indicator for poor prognosis in dogs compared with the fibroblastic or osteoblastic subtypes (P=0.006, Cox regression). Moreover, the findings indicated that there was no significant correlation between the localization of desmin, NSE or S100 and histological subtypes. Importantly, dogs with appendicular fibroblastic OS were found to have a better prognosis when compared with dogs with other subtypes. This may suggest that histological subtypes of appendicular OS have diverse behaviour and could be used to categorize patients for risk-based assessment.

Evaluations of phylogenetic proximity in a group of 67 dogs with osteosarcoma: a pilot study

Aim of the Research The aim of this study is to analyze a group of dogs of different breeds affected by osteosarcoma (OSA), to document any prevalence of this primary bone neoplasia in breeds that are phylogenetically close and to help with further research human medicine, as a model of study for prevalence and epidemiology of human OSA in multiple populations. Study Design Pilot study from two canine surgery databases between 2002 and 2013. Materials and Methods Breeds were classified in groups based on their phylogenetical proximity. Differences in prevalence of OSA between breeds and groups were evaluated with a permutation test. For each breed and each group, a ranking was made by calculating 95 % confidence intervals and counting the no-overlapping between breeds and groups. The relation between the dogs’ heights and the prevalence of OSA was analyzed using a logistic regression between the disease status and dog size. Results A total of 67 dogs with OSA, in 26 different breeds were included. Ten breeds were overrepresented and, the majority of these, were classified in 2 predisposed groups phylogenetically close to each other. The prevalence of OSA was associated with the dogs’ height within predisposed breeds, but, in general, taller breeds were not the most affected. Conclusions and Clinical Relevance In this study, despite the small number of dogs, we observed that the most commonly affected breeds with OSA are phylogenetically closely related. This highlights the importance of genetics in the aetiology of canine OSA . In this preliminary study, indications are given on breeds, samples and genome locations to be further investigated. This could allow identification of pathogenic alleles in dogs, and potentially in humans. Furthermore this pilot research can represent a model of epidemiologic study of human OSA.

Impact of radiation dose and pre-treatment pain levels on survival in dogs undergoing radiotherapy with or without chemotherapy for presumed extremity osteosarcoma.

The purpose of this bi-institutional retrospective study was to determine whether survival for dogs with extremity osteosarcoma (OS) is improved through the use of stereotactic radiotherapy (SRT; a single fraction of 25 Gy, or 36 Gy total given in three consecutive daily fractions) plus chemotherapy, vs lower dose conventionally planned and delivered hypofractionated radiotherapy (CHRT; 14-20 Gy total in 1-2 consecutive daily fractions) plus chemotherapy. We also sought to determine whether baseline pain severity influences oncologic outcomes following radiotherapy for canine extremity OS. The medical records of 82 dogs undergoing radiotherapy for confirmed or presumed OS were reviewed. In dogs receiving combinations of both chemotherapy and radiotherapy, survival was significantly longer with SRT vs CHRT (median overall survival time: 350 vs 147 days; P = .031). In a univariate analysis, dogs with pulmonary metastases and high pain at the time of irradiation had short overall survival times; use of high radiation doses and chemotherapy were associated with improved survival. Separate multivariable models were built to assess the predictive nature of various factors that might influence event-free or overall survival in dogs treated with radiotherapy, with or without chemotherapy; for dogs treated with both chemotherapy and radiotherapy, overall survival times were significantly longer when baseline pain scores were 'low' (vs 'high'; hazard ratio: 0.258; P = .030), radiation doses were high (hazard ratio: 0.943; P = .034). Neither pain nor radiation dose were associated with survival in dogs treated with radiotherapy, without chemotherapy.

Assessment of PDGFRβ promoter methylation in canine osteosarcoma using methylation-sensitive high-resolution melting analysis.

Platelet-derived growth factor signalling pathways play a fundamental role in inducing and sustaining the proliferative and prosurvival stimuli in canine osteosarcomas (cOSAs). The increased expression of platelet-derived growth factor receptors (PDGFRs) α and β, and their cognate ligands, were almost invariably observed in cOSAs and OSA-derived cell lines. In particular, overexpression of PDGFRβ-mediated signalling pathways was found in both the tumour microenvironment, where it drives stromal cell recruitment, and in neoangiogenesis, such as in tumour cells where it triggers aberrant proliferation, migration and local invasion. The majority of the pathological consequences of PDGFRβ signalling are because of aberrant expression. In fact, epigenetic dysregulation of oncogenes throughout demethylation of their promoter has emerged as a pivotal mechanism driving oncogenesis. The aim of this study was to assess the methylation status of the PDGFRβ promoter and to clarify its role in modulating the expression of the tyrosine kinase receptor in canine osteosarcoma. The CpG island of the PDGFRβ promoter was identified using a mixed in silico and experimental approach, and a method based upon the methylation-sensitive high-resolution melting assay for quantitatively and precisely assessing the methylation status of the promoter was then set up. The method herein described was then exploited to assess the methylation status of the promoter in a case series of cOSAa. COSAs consistently but variably expressed PDGFRβ. However, the promoter was almost completely demethylated, and its methylation status did not correlate with the expression levels. This finding supported the hypothesis that post-transcriptional regulatory mechanisms may act in cOSAs.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

Using machine learning to identify chaperones for sialidosis

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups (“healthy”, “osteosarcoma”, “other bone tumor”, or “non-neoplastic disease”). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as “osteosarcoma detected” or “osteosarcoma-NOT detected”. Dogs in a validation set whose post-treatment samples were classified as “osteosarcoma-NOT detected” had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

Cytological and Histopathological Correlation of Feline Giant Cell-Rich Osteosarcoma

Background: Primary bone tumours are uncommon and poorly reported in cats but osteosarcoma (OSA) is the most frequent, mostly in elderly animals. Giant cell-rich OSA is considered rare in the literature representing 3% of all OSA in humans. The mitotic index seems to have a significant effect on the survival time of cats affected by this neoplasm as well as the tumour histopathological grade. The objective of this study was to report the cytological and histopathological findings of a giant cell rich OSA in a 4-year old cat with persistent feline leukemia virus (FeLV) antigenaemia.Case: A 4-year-old male neutered cat was referred with a history of persistent FeLV viraemia and pelvic limb lameness with a firm swelling. Previous radiographs of the affected limb revealed bone lysis in the third and fourth metatarsals and increased soft tissue radiopacity in the tarsal region. The referral veterinary assumed it to be osteomyelitis and initiated clinical treatment with antibiotic and anti-inflammatory. The cat was referred after there was no response to medical treatment. The cat was presented with a 5cm diameter ulcerated mass, with putrid odor in the pelvic limb. Complementary exams were performed, and abnormalities were found, including increased urea, creatinine, calcium and potassium, and decreased sodium and phosphorus. A new radiograph showed exuberant bone proliferation, with increased radiopacity involving tarsal, metatarsal, distal third of tarsal I and II, and distal diaphysis of metatarsal V, without compromising the metaphyseal region of distal diaphysis of metatarsal IV. Chest radiographs and abdominal ultrasound were unremarkable. Fine-needle aspiration was performed for cytological analysis, which reavealed a moderate amount of pleomorphic mesenchymal cells with moderate adhesion, cytoplasm with a format ranging from fusiform to stellate, pronounced anisocytosis and cellular pleomorphism, and elevated nucleus:cytoplasm ratio. Nucleus was oval and presented loose chromatin, single to double large and evident nucleolus, frequent karyomegaly, along with marked anisocariosis and nuclear pleomorphism. Multinucleated giant cells were and there was a single mitotic figure in 12 high power fields (0.196 mm2 FN20/400x). Therefore, it was suggestive of malignant mesenchymal neoplasia with possibility of OSA, fibrosarcoma oe undifferentiated sarcoma. Limb amputation with femoral disarticulation was performed uneventfully. The material was conserved in 10% formalin, submitted to macroscopic and microscopic evaluation, which showed a large number of fusiform and stellate cells, with indistinct edges, scarce eosinophilic cytoplasm, high nucleus:cytoplasm ratio and oval nucleus, presenting moderate to marked anisocariosis, loose chromatin, with unique and evident nucleoli, besides of bone trabeculae. Nuclear pleomorphism was moderate and there was four mitotic figures in three random high-power fields (400x). It was observed areas of vascular ectasia, and neoplastic embolization in lymphatic and blood vessels. Among the cells, collagenous stroma was predominant but in some areas there was an eosinophilic amorphous material with the possibility of osteoid matrix or collagen. There was also a large numbers of multinucleated giant cells. The histopathological result was compatible with a grade III giant cell rich OSA.Discussion: Although bone tumours are uncommon in cats, OSA is the most frequent, affecting maingly middle-aged to elderly cats, with a mean age of 10 years, which is different from the present report in a 4-year old cat, with FeLV persistent viraemia. Retroviral status may have influenced the development of the disease at na early onset. FeLV induces uncontrolled cell proliferation through insertional mutagenesis (usually near myc) inducing malignant neoplasias, mainly lymphoma, but also multiple cartilaginous exostosis, which, along with osteomyelitis and bone cyst were included in this patient´s list of differential diagnoses. Despite the macroscopic and radiographical andagressiveness there was no metastasis identified through chest radiographs or abdominal ultrasound, and feline OSA is associated with a lower metastatic rate, if compared to canine OSA. Cytological analysis was compatible with malignant mesenchymal neoplasia, being suggestive of giant cell rich OSA. There is no cytological classification for OSA, however cytological findings of malignancy may be correlated with the patient's clinical course. Cytological features were in agreement with the histopathological findings, compatible with a grade III giant cell rich OSA. Therefore, it is concluded that the characteristics of malignancy presented by cytology were sufficient for the recommendation of amputation of the affected limb, once there was no imaging sign of metastases in the chest or abdomen.

Outcome and Metastatic Behavior of Canine Sinonasal Osteosarcoma (2005-2015).

Sinonasal neoplasms in dogs behave locally aggressively, and metastatic disease has not been a common cause of death. The metastatic rate of sinonasal osteosarcoma (OSA) is not well characterized, and reported outcomes of these patients are variable. The purpose of this study is to evaluate the outcome and metastatic behavior of canine sinonasal OSA. Medical records of canine patients diagnosed with sinonasal OSA via histopathology between January 2005 and December 2015 were reviewed. Patients with any form of treatment or no treatment were included. Time to local progression, time to metastasis, and overall survival data were evaluated. Variables that may impact outcome, such as tumor stage and treatment type, were evaluated. Twenty-seven dogs were identified that fit the inclusion criteria. Overall, 30.0% of dogs developed metastasis over the disease course, with a median time to metastasis of 458 days (95% confidence interval [CI] 318-758 days). The median time to local progression was 335 days (95% CI 264-544 days). The overall median survival time was 410 days (95% CI 341-627 days). Regarding metastasis, sinonasal OSA behaves similarly to sinonasal neoplasms of other histologies and dissimilarly to appendicular OSA. The outcome of treated patients appears similar to that of sinonasal tumor patients with other histologies.

Delineation of hypoxia-induced proteome shifts in osteosarcoma cells with different metastatic propensities

Osteosarcoma (OS) is the most common bone cancer in children and young adults. Solid tumors are characterized by intratumoral hypoxia, and hypoxic cells are associated with the transformation to aggressive phenotype and metastasis. The proteome needed to support an aggressive osteosarcoma cell phenotype remains largely undefined. To link metastatic propensity to a hypoxia-induced proteotype, we compared the protein profiles of two isogenic canine OS cell lines, POS (low metastatic) and HMPOS (highly metastatic), under normoxia and hypoxia. Label-free shotgun proteomics was applied to comprehensively characterize the hypoxia-responsive proteome profiles in the OS cell phenotypes. Hypothesis-driven parallel reaction monitoring was used to validate the differential proteins observed in the shotgun data and to monitor proteins of which we expected to exhibit hypoxia responsiveness, but which were absent in the label-free shotgun data. We established a “distance” score (|zHMPOS − zPOS|), and “sensitivity” score (|zHypoxia − zNormoxia) to quantitatively evaluate the proteome shifts exhibited by OS cells in response to hypoxia. Evaluation of the sensitivity scores for the proteome shifts observed and principal component analysis of the hypoxia-responsive proteins indicated that both cell types acquire a proteome that supports a Warburg phenotype with enhanced cell migration and proliferation characteristics. Cell migration and glucose uptake assays combined with protein function inhibitor studies provided further support that hypoxia-driven adaption of pathways associated with glycolytic metabolism, collagen biosynthesis and remodeling, redox regulation and immunomodulatory proteins typify a proteotype associated with an aggressive cancer cell phenotype. Our findings further suggest that proteins involved in collagen remodeling and immune editing may warrant further evaluation as potential targets for anti-metastatic treatment strategies in osteosarcoma.

MiR-106B-25 Cluster expression: a comparative human and canine osteosarcoma study

BackgroundOsteosarcoma (OS) is the most common primary malignant bone tumour in dogs and human beings, characterised by similar genetic and clinical features. With the aim to define similarities and differences...

Abstract B026: The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability

Abstract Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. While the genomic landscape of human OS has been interrogated, limited data exist regarding that of dogs. Using whole genome sequencing (WGS) we identified the presence of mutations in DMD, the gene encoding dystrophin, in canine OS. The purpose of this work was to expand upon these findings and begin to interrogate the functional consequences of these mutations. Methods: Whole-genome sequencing (WGS) of 24 canine primary OS tumor samples was completed on an Illumina platform. Somatic single nucleotide and structural variants, and germline variants were identified using Cerberus, a canine cancer genomics pipeline. Expression of the Dp71 DMD isoform was modulated using CRISPR-Cas9 and lentiviral overexpression approaches; the biologic significance of DMD loss was characterized using Co-IP, western blotting, Matrigel invasion and proliferation assays. Results: Our prior data found that canine OS recapitulates several genetic features of human OS including high structural complexity and translocation burden withTP53 (71%) the most frequently mutated gene. In addition to inactivating SETD2 mutations 42% of cases, we found that 50% of tumors possessed large somatic intragenic deletions and copy number loss of DMD, the gene encoding dystrophin. Building upon these data we found that truncated dystrophin isoforms (Dp260, Dp140, Dp71, Dp40) predominate in canine OS cell lines, with only Dp40 expressed in normal osteoblasts. Deletion of Dp71 in OS lines using CRISPR-Cas9 resulted in increased cell proliferation and markedly enhanced invasion through Matrigel. Consistent with the published literature, dystrophin and β-dystroglycan co-associated in the OS lines, and phosphorylation of β-dystroglycan was increased when dystrophin expression was downregulated. Lastly, dasatinib, a small molecule inhibitor of Src, inhibited β-dystroglycan phosphorylation and cell proliferation in the canine OS lines. Conclusions: These data provide important new information regarding the genomic profile of canine OS and suggest that aberrations in dystrophin expression may contribute to OS pathophysiology in dogs by promoting a metastatic phenotype. Future work will dissect exactly how altered dystrophin isoforms contribute to OS tumor biology with the goal of developing approaches for therapeutic intervention that target this pathway. Citation Format: Heather L. Gardner, Karthigayini Sivaprakasam, Natalia Briones, William Hendricks, Cheryl A. London. The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B026. doi:10.1158/1535-7163.TARG-19-B026

Abstract B081: Regulation and function of monocarboxylate transporters in osteosarcoma

Abstract Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. In both species, macroscopic metastasis exhibits inherent resistance to multiple agents (kinase inhibitors, chemotherapy, immunotherapy, among others). In both dogs and humans, the OS genomes exhibit low somatic mutational load and no clear molecular drivers. These data, along with a history of failed clinical trials suggest that contemporary approaches to therapeutic advancement (kinase inhibitors, immune checkpoint blockade) will likely have limited clinical impact, necessitating therapeutic innovation. Monocarboxylate transporters (MCTs) move lactic acid across the plasma membrane, providing a mechanism for tumor cells to meet their bioenergetic needs in a variety of limiting microenvironments. The objective of this work is credential MCTs (MCT1/MCT4) as therapeutic targets in OS and investigate mechanisms of synthetic lethality. Methods: STAT3, MCT1 and MCT4 expression and function were modulated in canine and human OS cell lines using a combination of CRISPR/cas9 techniques and pharmacologic agents (AZD3965, AstraZeneca; syrosingopine, Sigma Aldrich). Western blotting, luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to investigate the transcriptional regulation of MCT1/MCT4. The metabolic effects of STAT3 and MCT inhibition were investigated through measurement of lactate (Lactate-Glo, Promega) and respirometry (Seahorse XFe96 extracellular flux analyzer), while cell proliferation and invasion were evaluated using sulforhodamine B and Matrigel invasion assays. Results: MCT1/MCT4 and were found to be expressed in all OS cell lines, although at variable levels. Following knockdown of STAT3, MCT4 expression was downregulated in several OS cell lines. ChIP assays confirm enrichment of STAT3 and FOXM1 at the MCT4 promoter, and this was confirmed in STAT3 deficient OS lines that exhibited decreased MCT4-dependent luciferase reporter activity. The absence of MCT1 or MCT4 was associated with decreased basal and compensatory glycolysis and increased intracellular lactate. In addition, decreased proliferation and invasive capacity were noted concurrent with MCT loss. Dual MCT1/MCT4 inhibition exhibited synergy with doxorubicin, as determined by a combination index < 1. Conclusions: Loss of MCT1 or MCT4 function in OS cells decreases basal and compensatory glycolysis, cellular proliferation and invasive capacity. MCT4 is a direct transcriptional target of STAT3 and FOXM1, both of which exhibit constitutive activation in OS, supporting a link between MCT4/STAT3/FOXM1 and aerobic glycolysis. Despite promising results in vitro, rational drug combinations may be needed to maximize the effect of MCT1/4 inhibition. Together these studies will create a blueprint for clinical translational with the goal of optimizing strategies in dogs with OS prior to human trials. Citation Format: Heather L. Gardner, Joelle M. Fenger, Cheryl A. London. Regulation and function of monocarboxylate transporters in osteosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B081. doi:10.1158/1535-7163.TARG-19-B081

Vaccine strain Listeria monocytogenes abscess in a dog: a case report

BackgroundListeria monocytogenes is a promising therapeutic vaccine vector for cancer immunotherapy. Although highly attenuated, three cases of systemic listeriosis have been reported in people following treatment with Listeria-based therapeutic vaccines. This complication has thus far not been reported in canine patients.Case presentationA dog previously diagnosed with osteoblastic osteosarcoma was presented for care following administration of three doses of the Canine Osteosarcoma Vaccine-Live Listeria Vector. On routine staging chest radiographs, mild sternal lymphadenopathy and a right caudoventral thoracic mass effect were noted. Further evaluation of the mass effect with computed tomography and ultrasound revealed a cavitated mass associated with the 7th right rib. Aspirates of the mass cultured positive for Listeria monocytogenes. The mass and associated ribs were surgically removed. Histopathology was consistent with metastatic osteoblastic osteosarcoma. Treatment was continued with doxorubicin chemotherapy and at the time of publication, the dog was alive over 1 year following diagnosis with no evidence of further disease progression. Genotyping of the abscess-derived L. monocytogenes was consistent with the vaccine strain.ConclusionsThis case represents the first veterinary case to describe development of a Listeria abscess following administration of a Listeria-based therapeutic vaccine.

Aldoxorubicin-loaded nanofibers are cytotoxic for canine mammary carcinoma and osteosarcoma cell lines in vitro: A short communication

Chemotherapeutic drugs are given parenterally to treat various canine tumors. A limitation of parenteral administration is low drug penetration into the tumor, which reduces tumoricidal activity. Various drug carriers have been used to enhance tumor delivery, including albumin, liposomes and nanoparticles. A novel peptide-based nanofiber precursor (NFP) has been developed that is designed to take advantage of the leaky tumor neovasculature to promote drug delivery after parenteral administration. In this study, we loaded aldoxorubicin, an albumin-bound prodrug version of doxorubicin, onto NFP and tested the in vitro cytotoxicity in canine mammary carcinoma (CMT12, CMT25) and osteosarcoma (HMPOS, D-17, Abrams) cell lines. The half maximal inhibitory concentration (IC50) was determined with a luminescence-based cell viability assay. The IC50 for aldoxorubicin-loaded NFP was lower than free aldoxorubicin or doxorubicin in all cell lines, whereas non-drug loaded NFP had no cytotoxic effects. There were differences in IC50 between the osteosarcoma lines, with lower and higher IC50 for HMPOS and D-17 cells, respectively, with all drugs (aldoxorubicin-loaded NFP, free aldoxorubicin or free doxorubicin). Our results indicate that drug-loaded NFPs are cytotoxic for various canine mammary carcinoma and osteosarcoma cell lines in vitro and hold promise as a mechanism for enhancing delivery of chemotherapeutic agents to canine tumors.

In vitro Cytotoxicity and Pharmacokinetic Evaluation of Pharmacological Ascorbate in Dogs.

Background: High-dose, pharmacological ascorbate (P-AscH−) is preferentially cytotoxic to human cancer cells in vitro. Investigations on the efficacy of P-AscH− as an adjuvant treatment for multiple human cancers are on-going, but has yet to be formally investigated in dogs. The primary objectives of this study were to determine the pharmacokinetic (PK) profile of P-AscH− in healthy Beagle dogs and the effects of P-AscH− on canine osteosarcoma cells in vitro.Methods: Eight purpose-bred, healthy, spayed female Beagle dogs, between 20 and 21 months old, and 8–10 kg were administered two doses of P-AscH− (550 or 2,200 mg/kg) via intravenous infusion over 6 h, on separate days. Plasma ascorbate concentrations were measured at 12 time points during and after infusion for PK analysis using nonlinear mixed-effects (NLME) and non-compartmental analysis (NCA). Clonogenic assays were performed on 2 canine osteosarcoma cell lines (D-17 and OSCA-8) and canine primary dermal fibroblasts after exposure to high concentrations of ascorbate (75 pmoles/cell).Results: Plasma ascorbate levels in the dogs peaked at approximately 10 mM following 2,200 mg/kg and returned to baseline 6–8 h after dosing. Minor adverse effects were seen in two dogs. Ascorbate (75 pmoles/cell) significantly decreased survival in both the osteosarcoma cell lines (D-17 63% SD 0.010, P = 0.005; OSCA-8 50% SD 0.086, P = 0.026), compared to normal fibroblasts (27% SD 0.056).Conclusions: Pharmacological ascorbate is preferentially cytotoxic to canine-derived cancer cells. High levels of ascorbate can be safely administered to dogs. Further studies are needed to determine the effects of P-AscH− on canine patients.

Characterization of five newly derived canine osteosarcoma cell lines

BackgroundCanine and human osteosarcomas (OS) are notably similar and have a high rate of metastasis. There is a poor understanding of the tumor development process, predisposing causes, and varying levels of aggression among different cell lines. By characterizing newly developed canine osteosarcoma cell lines, treatments for people and pets can be developed. Of the seven subtypes of OS, three are represented in this group: osteoblastic (the most common), fibroblastic, and giant cell variant. To our knowledge, there are no other giant cell variant canine OS cell lines in the published literature and only one canine fibroblastic osteosarcoma cell line. Understanding the differences between the histologic subtypes in dogs will help to guide comparative research.ResultsAlkaline phosphatase expression was ubiquitous in all cell lines tested and invasiveness was variable between the cell lines tested. Invasiveness and oxidative damage were not correlated with in vivo growth rates, where TOT grew the fastest and had the higher percentage of mice with metastatic lesions. TOL was determined to be the most chemo-resistant during cisplatin chemotherapy while TOM was the most chemo-sensitive.ConclusionsFurther comparisons and studies using these cell lines may identify a variety of characteristics valuable for understanding the disease process and developing treatments for osteosarcoma in both species.Some of this data was presented as a poster by KMF at the August 5th, 2017 National Veterinary Scholars Program in Bethesda, MA.Characterization of 5 newly generated canine osteosarcoma cell lines.Kelli Franks, Tasha Miller, Heather Wilson-Robles.

Immune dysregulation and osteosarcoma: Staphylococcus aureus downregulates TGF-β and heightens the inflammatory signature in human and canine macrophages suppressed by osteosarcoma.

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti-tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co-cultured with osteosarcoma and S. aureus exhibited increased IFN-γ, TNF-α and IL-12p70 cytokine secretion, decreased TGF-β cytokine secretion and increased mRNA expression of TNF-α when compared with macrophages co-cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN-γ and TNF-α cytokine secretion, decreased TGF-β cytokine secretion, increased mRNA expression of TNF-α and increased surface receptor expression of CD80 when co-cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma-induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti-osteosarcoma macrophage response.