Osteosarcoma PDX-Derived Cell Line Models for Preclinical Drug Evaluation Demonstrate Metastasis Inhibition by Dinaciclib through a Genome-Targeted Approach.

PURPOSE: Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, multiple models are needed to fully elucidate key aspects of disease biology and to recapitulate clinically relevant phenotypes. EXPERIMENTAL DESIGN: Matched patient samples, patient-derived xenografts (PDX), and PDX-derived cell lines were comprehensively evaluated using whole-genome sequencing and RNA sequencing. The in vivo metastatic phenotype of the PDX-derived cell lines was characterized in both an intravenous and an orthotopic murine model. As a proof-of-concept study, we tested the preclinical effectiveness of a cyclin-dependent kinase inhibitor on the growth of metastatic tumors in an orthotopic amputation model. RESULTS: PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication in a subset of cell lines. The cell lines were heterogeneous in their metastatic capacity, and heterogeneous tissue tropism was observed in both intravenous and orthotopic models. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden. CONCLUSIONS: The variation in metastasis predilection sites between osteosarcoma PDX-derived cell lines demonstrates their ability to recapitulate the spectrum of the disease observed in patients. We describe here a panel of new osteosarcoma PDX-derived cell lines that we believe will be of wide use to the osteosarcoma research community.

Development and characterization of new patient-derived xenograft (PDX) models of osteosarcoma with distinct metastatic capacities.

Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology, especially for highly aggressive cancers with a propensity for metastatic spread. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, a large panel of models is needed to fully elucidate key aspects of disease biology and to recapitulate clinically-relevant phenotypes. We describe the development and characterization of osteosarcoma patient-derived xenografts (PDXs) and a panel of PDX-derived cell lines. Matched patient samples, PDXs, and PDX-derived cell lines were comprehensively evaluated using whole genome sequencing and RNA sequencing. PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication (WGD) in a subset of cell lines. These cell line models were heterogeneous in their metastatic capacity and their tissue tropism as observed in both intravenous and orthotopic models. As proof-of-concept study, we used one of these models to test the preclinical effectiveness of a CDK inhibitor on the growth of metastatic tumors in an orthotopic amputation model. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden in this model.

Prognostic significance of the urokinase plasminogen activator system in tissue and serum of dogs with appendicular osteosarcoma.

Urokinase plasminogen activator (uPA) and its receptor uPAR promote cancer invasion and metastasis and are emerging therapeutic targets in both human and canine malignancies. While their clinical significance is well-characterized in multiple human tumor types, studies investigating their roles in osteosarcoma are lacking. The objectives of this study were to characterize serum and tissue uPA/uPAR expression in dogs with osteosarcoma and assess the prognostic significance. Serum samples and a tissue microarray of canine appendicular osteosarcoma were analyzed for uPA and uPAR expression by ELISA (n = 49) and immunohistochemistry (n = 38), respectively. Serum uPA activity was also measured by a chromogenic assay (n = 25). Survival analysis was performed by Kaplan-Meier survival analysis, log rank test, and Cox regression analysis. Serum uPA level was significantly higher in dogs with osteosarcoma than clinically healthy control dogs (median 1905 vs 1440 pg/ml, p = 0.008). The majority of canine osteosarcoma tissues expressed uPA (75.9%) or uPAR (77.6%), with 70.7% dual-positivity, indicating autocrine/paracrine activation of the pathway. Survival analysis revealed shorter progression free survival (PFS) in dogs with high serum uPA level in a discovery cohort (n = 29; median PFS 94 vs 266 days, p = 0.003) but not in a validation cohort (n = 23; median PFS 167 vs 490 days, p = 0.16). The difference was significant when both cohorts were combined (n = 49; median PFS 128 vs 266 days, p = 0.003). Serum uPAR and tissue uPA/uPAR levels were not prognostic. In Cox multivariate analysis, high serum uPA level and activity were both associated with poor prognosis, independent of serum ALP, tumor location, and peripheral lymphocyte/monocyte counts. These results indicate high utilization of the uPA pathway and association with disease progression in canine osteosarcoma. Further study involving prospective evaluation to confirm the prognostic significance is warranted. The high prevalence of tissue uPA and uPAR expression suggests the uPA system as a potential therapeutic target in canine osteosarcoma.

Genomic Complexity of Osteosarcoma and Its Implication for Preclinical and Clinical Targeted Therapies.

Osteosarcoma is a genomically complex disease characterized by few recurrent single-nucleotide mutations or in-frame fusions. In contrast, structural alterations, including copy number changes, chromothripsis, kataegis, loss of heterozygosity (LOH), and other large-scale genomic alterations, are frequent and widespread across the osteosarcoma genome. These observed structural alterations lead to activation of oncogenes and loss of tumor suppressors which together contribute to oncogenesis. To date, few targeted therapies for osteosarcoma have been identified. It is likely that effectiveness of targeted therapies will vary greatly in subsets of tumors with distinct key driver events. Model systems which can recapitulate the genetic heterogeneity of this disease are needed to test this hypothesis. One possible approach is to use patient-derived xenograft (PDX) models characterized with regards to their similarity to the human tumor samples from which they were derived. Here we review evidence pointing to the genomic complexity of osteosarcoma and how this is reflected in available model systems. We also review the current state of preclinical testing for targeted therapies using these models.

Cross-species genomic landscape comparison of human mucosal melanoma with canine oral and equine melanoma.

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma. Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites. Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53. We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa. A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B. Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity. Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis.

An evaluation of TAZ and YAP crosstalk with TGFß signalling in canine osteosarcoma suggests involvement of hippo signalling in disease progression.

BACKGROUND: Osteosarcoma (OSA) is the most common bone cancer in canines. Both transforming growth factor beta (TGFß) and Hippo pathway mediators have important roles in bone development, stemness, and cancer progression. The role of Hippo signalling effectors TAZ and YAP has never been addressed in canine OSA. Further, the cooperative role of TGFß and Hippo signalling has yet to be explored in osteosarcoma. To address these gaps, this study investigated the prognostic value of TAZ and YAP alone and in combination with pSmad2 (a marker of active TGFß signalling), as well as the involvement of a TGFß-Hippo signalling crosstalk in tumourigenic properties of OSA cells in vitro. An in-house trial tissue microarray (TMA) which contained 16 canine appendicular OSA cases undergoing standard care and accompanying follow-up was used to explore the prognostic role of TAZ, YAP and pSmad2. Published datasets were used to test associations between TAZ and YAP mRNA levels, metastasis, and disease recurrence. Small interfering RNAs specific to TAZ and YAP were utilized in vitro alone or in combination with TGFß treatment to determine their role in OSA viability, proliferation and migration. RESULTS: Patients with low levels of both YAP and pSmad2 when evaluated in combination had a significantly longer time to metastasis (log-rank test, p = 0.0058) and a longer overall survival (log rank test, p = 0.0002). No similar associations were found for TAZ and YAP mRNA levels. In vitro, TAZ knockdown significantly decreased cell viability, proliferation, and migration in metastatic cell lines, while YAP knockdown significantly decreased viability in three cell lines, and migration in two cell lines, derived from either primary tumours or their metastases. The impact of TGFß signaling activation on these effects was cell line-dependent. CONCLUSIONS: YAP and pSmad2 have potential prognostic value in canine appendicular osteosarcoma. Inhibiting YAP and TAZ function could lead to a decrease in viability, proliferation, and migratory capacity of canine OSA cells. Assessment of YAP and pSmad2 in larger patient cohorts in future studies are needed to further elucidate the role of TGFß-Hippo signalling crosstalk in canine OSA progression.

The autophagy inhibitor spautin-1, either alone or combined with doxorubicin, decreases cell survival and colony formation in canine appendicular osteosarcoma cells.

Dogs diagnosed with appendicular osteosarcoma typically succumb to metastatic disease within a year of diagnosis. The current standard of care for curative intent, amputation followed by adjuvant chemotherapy, increases survival time but chemoresistance is a major contributor to mortality. Unfortunately, the mechanisms driving the progression of metastatic disease and the development of chemoresistance are unknown. One theory is that autophagy may contribute to chemoresistance by providing neoplastic cells with a mechanism to survive chemotherapy treatment. Our objective was to evaluate the effect of combining an autophagy inhibitor with a standard chemotherapeutic drug on response to chemotherapy in canine appendicular osteosarcoma cells. We hypothesized that combining the autophagy inhibitor spautin-1 with doxorubicin treatment would enhance chemoresponsiveness. Using commercial (D17) and primary cell lines derived from 1° and 2° sites of osteosarcoma, we showed that this combination treatment enhances cell killing and inhibits colony formation. Our findings support the theory that autophagy contributes to chemoresistance in canine appendicular osteosarcoma and indicate that adding an autophagy inhibitor to the standard of care has the potential to improve outcome.

Evaluation of metronomic cyclophosphamide chemotherapy as maintenance treatment for dogs with appendicular osteosarcoma following limb amputation and carboplatin chemotherapy.

OBJECTIVE To determine the effectiveness of metronomic cyclophosphamide (MC) chemotherapy (primary treatment of interest) with adjuvant meloxicam administration as maintenance treatment for dogs with appendicular osteosarcoma following limb amputation and carboplatin chemotherapy. DESIGN Retrospective case series with nested cohort study. ANIMALS 39 dogs with a histologic diagnosis of appendicular osteosarcoma that underwent limb amputation and completed carboplatin chemotherapy from January 2011 through December 2015. PROCEDURES Dogs were grouped by whether carboplatin chemotherapy had been followed with or without MC chemotherapy (15 mg/m2, PO, q 24 h) and meloxicam (0.1 mg/kg [0.045 mg/lb], PO, q 24 h). The Breslow rank test was used to assess whether MC chemotherapy was associated with overall survival time (OST) and disease progression-free time (PFT) after limb amputation. RESULTS 19 dogs received carboplatin and MC chemotherapy, and 20 dogs received only carboplatin chemotherapy. No differences were identified between these groups regarding age, reproductive status, body weight, serum alkaline phosphatase activity, tumor location, or histologic grade or subtype of osteosarcoma. Median duration of MC chemotherapy for dogs in the carboplatin-MC group was 94 days (range, 7 to 586 days); this treatment was discontinued for 11 (58%) dogs when cystitis developed. Overall, 11 (28%) dogs survived to the time of analysis, for a median follow-up period of 450 days (range, 204 to 1,400 days). No difference in median PFT or OST was identified between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE Maintenance MC chemotherapy following limb amputation and completed carboplatin chemotherapy was associated with no increase in PFT or OST in dogs with appendicular osteosarcoma. Cystitis was common in MC-treated dogs, and prophylactic treatment such as furosemide administration could be considered to reduce the incidence of cystitis in such dogs.

Histologic Grade Does Not Predict Outcome in Dogs with Appendicular Osteosarcoma Receiving the Standard of Care.

Canine appendicular osteosarcoma is an aggressive bone neoplasm that imposes a short survival time. There are several published histologic grading systems for canine osteosarcoma but no universally accepted system. Location within the skeleton and therapy received are both correlated with survival time, but these factors were not always considered when the prognostic value of published grading systems was determined. Our objective was to compare 2 published histologic grading systems in a population of dogs with appendicular osteosarcoma treated with the standard of care for curative intent. Three evaluators graded 85 tumors using 2 histologic grading systems. The relationships between histologic grade as well as individual histologic features and outcome (survival time and disease-free interval) were evaluated using Kaplan-Meier survival functions and a univariate Cox proportional hazards model. Histologic grade, as assigned by any evaluator, did not correlate with outcome. Increased number of mitotic figures per 3 randomly selected 400× microscope fields, as assessed by 1 evaluator, was correlated with both survival time and disease-free interval; this was the only individual histologic feature that was significantly correlated with outcome for any evaluator. These findings cast doubt on the predictive value of routine histologic grading in dogs with appendicular osteosarcoma who receive amputation followed by adjuvant chemotherapy and highlight the need for better tools to predict outcome in canine appendicular osteosarcoma.

Bactericidal activity of tracheal antimicrobial peptide against respiratory pathogens of cattle.

Tracheal antimicrobial peptide (TAP) is a ß-defensin produced by mucosal epithelial cells of cattle. Although effective against several human pathogens, the activity of this bovine peptide against the bacterial pathogens that cause bovine respiratory disease have not been reported. This study compared the antibacterial effects of synthetic TAP against Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis. Bactericidal activity against M. bovis was not detected. In contrast, the Pasteurellaceae bacteria showed similar levels of susceptibility to that of Escherichia coli, with 0.125µg TAP inhibiting growth in a radial diffusion assay and minimum inhibitory concentrations of 1.56-6.25µg/ml in a bactericidal assay. Significant differences among isolates were not observed. Sequencing of exon 2 of the TAP gene from 23 cattle revealed a prevalent non-synonymous single nucleotide polymorphism (SNP) A137G, encoding either serine or asparagine at residue 20 of the mature peptide. The functional effect of this SNP was tested against M. haemolytica using synthetic peptides. The bactericidal effect of the asparagine-containing peptide was consistently higher than the serine-containing peptide. Bactericidal activities were similar for an acapsular mutant of M. haemolytica compared to the wild type. These findings indicate that the Pasteurellaceae bacteria that cause bovine respiratory disease are susceptible to killing by bovine TAP and appear not to have evolved resistance, whereas M. bovis appears to be resistant. A non-synonymous SNP was identified in the coding region of the TAP gene, and the corresponding peptides vary in their bactericidal activity against M. haemolytica.

Prevalence and genotype of Mycoplasma bovis in beef cattle after arrival at a feedlot.

OBJECTIVE: To determine the prevalence of Mycoplasma bovis infection in the lungs of cattle at various times after arrival at a feedlot, to measure the relationship between clinical disease status and the concentration and genotype of M bovis within the lungs, and to investigate changes in the genotype of M bovis over time. SAMPLE: Bronchoalveolar lavage fluid (BALF) from 328 healthy or pneumonic beef cattle and 20 M bovis isolates obtained from postmortem samples. PROCEDURES: The concentration of M bovis in BALF was determined via real-time PCR assays, and M bovis isolates from BALF were genotyped via amplified fragment length polymorphism (AFLP) analysis. RESULTS: Prevalence of M bovis in BALF was 1 of 60 (1.7%) at arrival to a feedlot and 26 of 36 (72.2%) and 36 of 42 (85.7%) at ≤ 15 days and 55 days after arrival, respectively. Neither the concentration nor the AFLP type of M bovis in BALF was correlated with clinical disease status. The M bovis AFLP type differed between early and later sampling periods in 14 of 17 cattle. CONCLUSIONS AND CLINICAL RELEVANCE: The findings implied spread of M bovis among calves and suggested that host factors and copathogens may determine disease outcomes in infected calves. Chronic pulmonary infection with M bovis may represent a dynamic situation of bacterial clearance and reinfection with strains of different AFLP type, rather than continuous infection with a single clone. These findings impact our understanding of why cattle with chronic pneumonia and polyarthritis syndrome inadequately respond to antimicrobial treatment.