Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma.

BACKGROUND: Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. METHODS: In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. RESULTS: Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient's exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. CONCLUSIONS: The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma.

BACKGROUND: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. RESULTS: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. CONCLUSIONS: The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma.

BACKGROUND: Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay. Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC. RESULTS: Dogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease. CONCLUSIONS: Plasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection.

Characterization of five newly derived canine osteosarcoma cell lines.

BACKGROUND: Canine 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. RESULTS: Alkaline 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. CONCLUSIONS: Further 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.

Removal of hemangiosarcoma cells from canine blood with a cell salvage system and leukocyte reduction filter.

OBJECTIVE: To determine the ability of an intraoperative cell salvage (IOCS) system and a leukocyte reduction filter (LRF) to remove hemangiosarcoma (HSA) cells from canine blood. STUDY DESIGN: Cultured HSA cells were added to canine blood to simulate intraoperative hemorrhage and address hemoabdomen from ruptured splenic HSA. The blood/HSA cell mixture was processed through an IOCS, followed by LRF processing. SAMPLE POPULATION: Whole blood from 3 healthy dogs combined with cultured HSA cells. METHODS: The ability of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), multiparameter flow cytometry, and cytologic examination to detect 50 HSA cells per milliliter of culture media was confirmed. RT-PCR, multiparameter flow cytometry, and cytologic examination were used to determine the presence of cultured HSA cells at 4 points during processing. RESULTS: HSA cells were found in all control samples and in all samples after IOCS but prior to LRF processing with all 3 cell detection methods. HSA cells were not found after IOCS/LRF processing with all 3 cell detection methods. CONCLUSION: IOCS combined with LRF processing is able to remove cultured HSA cells from canine blood. The addition of LRF to IOCS may allow application of IOCS in dogs with HSA. CLINICAL SIGNIFICANCE: A combination of IOCS and LRF processing may provide an alternative to allogeneic blood transfusion in dogs with hemoabdomen due to HSA.

Cellular Transcriptomics of Carboplatin Resistance in a Metastatic Canine Osteosarcoma Cell Line.

Osteosarcoma prognosis has remained unchanged for the past three decades. In both humans and canines, treatment is limited to excision, radiation, and chemotherapy. Chemoresistance is the primary cause of treatment failure, and the trajectory of tumor evolution while under selective pressure from treatment is thought to be the major contributing factor in both species. We sought to understand the nature of platinum-based chemotherapy resistance by investigating cells that were subjected to repeated treatment and recovery cycles with increased carboplatin concentrations. Three HMPOS-derived cell lines, two resistant and one naïve, underwent single-cell RNA sequencing to examine transcriptomic perturbation and identify pathways leading to resistance and phenotypic changes. We identified the mechanisms of acquired chemoresistance and inferred the induced cellular trajectory that evolved with repeated exposure. The gene expression patterns indicated that acquired chemoresistance was strongly associated with a process similar to epithelial-mesenchymal transition (EMT), a phenomenon associated with the acquisition of migratory and invasive properties associated with metastatic disease. We conclude that the observed trajectory of tumor adaptability is directly correlated with chemoresistance and the phase of the EMT-like phenotype is directly affected by the level of chemoresistance. We infer that the EMT-like phenotype is a critical component of tumor evolution under treatment pressure and is vital to understanding the mechanisms of chemoresistance and to improving osteosarcoma prognosis.

Monitoring plasma nucleosome concentrations to measure disease response and progression in dogs with hematopoietic malignancies.

BACKGROUND: Hematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. METHODS: Circulating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. RESULTS: Plasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. CONCLUSIONS: Plasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

Feasibility and safety of whole lung irradiation in the treatment of canine appendicular osteosarcoma.

Whole lung irradiation (WLI) has been used successfully in humans as an adjuvant treatment for osteosarcoma. The aim of this study is to describe the feasibility and safety of WLI in dogs with appendicular osteosarcoma. Twelve client-owned dogs with appendicular osteosarcoma that had successfully completed amputation and four doses of carboplatin without evidence of gross metastasis were enrolled in this prospective clinical trial. Ten once-daily fractions of 1.75 Gy were administered to the planning target volume encompassing the lungs. Overall, WLI was well tolerated in these patients. No dogs developed symptoms of pneumonitis or pulmonary fibrosis. Haematopoietic toxicity evaluated during radiation therapy was found to be mild. The median disease free interval for WLI treated dogs was not significantly different than the median DFI for a group of historic control dogs (376 days for WLI treated dogs versus 304.5 days for control dogs; p = 0.5461). Although no significant improvement in outcome was observed with this study, WLI appears to be safe in dogs and warrants further investigation to characterize the efficacy and toxicity.

Anti-tumor effects of cryptotanshinone (C19H20O3) in human osteosarcoma cell lines.

Osteosarcoma is the most prevalent malignant bone tumor and occurs most commonly in the adolescent and young adult population. Despite the recent advances in surgeries and chemotherapy, the overall survival in patients with resectable metastases is around 20%. This challenge in osteosarcoma is often attributed to the drastic differences in the tumorigenic profiles and mutations among patients. With diverse mutations and multiple oncogenes, it is necessary to identify the therapies that can attack various mutations and simultaneously have minor side-effects. In this paper, we constructed the osteosarcoma pathway from literature and modeled it using ordinary differential equations. We then simulated this network for every possible gene mutation and their combinations and ranked different drug combinations based on their efficacy to drive a mutated osteosarcoma network towards cell death. Our theoretical results predict that drug combinations with Cryptotanshinone (C19H20O3), a traditional Chinese herb derivative, have the best overall performance. Specifically, Cryptotanshinone in combination with Temsirolimus inhibit the JAK/STAT, MAPK/ERK, and PI3K/Akt/mTOR pathways and induce cell death in tumor cells. We corroborated our theoretical predictions using wet-lab experiments on SaOS2, 143B, G292, and HU03N1 human osteosarcoma cell lines, thereby demonstrating the potency of Cryptotanshinone in fighting osteosarcoma.

In Silico Modeling of the Induction of Apoptosis by Cryptotanshinone in Osteosarcoma Cell Lines.

Osteosarcoma (OS) is the most common primary malignant bone tumor of both children and pet canines. Its characteristic genomic instability and complexity coupled with the dearth of knowledge about its etiology has made improvement in the current treatment difficult. We use the existing literature about the biological pathways active in OS and combine it with the current research involving natural compounds to identify new targets and design more effective drug therapies. The key components of these pathways are modeled as a Boolean network with multiple inputs and multiple outputs. The combinatorial circuit is employed to theoretically predict the efficacies of various drugs in combination with Cryptotanshinone. We show that the action of the herbal drug, Cryptotanshinone on OS cell lines induces apoptosis by increasing sensitivity to TNF-related apoptosis-inducing ligand (TRAIL) through its multi-pronged action on STAT3, DRP1 and DR5. The Boolean framework is used to detect additional drug intervention points in the pathway that could amplify the action of Cryptotanshinone.

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer.

INTRODUCTION: Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer. MATERIALS AND METHODS: Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results. RESULTS: Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma. CONCLUSIONS: When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use.

Assessing Combinational Drug Efficacy in Cancer Cells by Using Image-based Dynamic Response Analysis.

The landscape of translational research has been shifting toward drug combination therapies. Pairing of drugs allows for more types of drug interaction with cells. In order to accurately and comprehensively assess combinational drug efficacy, analytical methods capable of recognizing these alternative reactions will be required to prioritize those drug candidates having better chances of delivering appreciable therapeutic benefits. Traditional efficacy measures are primarily based on the "extent" of drug inhibition, which is the percentage of cells being killed after drug exposure. Here, we introduce a second dimension of evaluation criterion, speed of killing, based on a live cell imaging assay. This dynamic response trajectory approach takes advantage of both "extent" and "speed" information and uncovers synergisms that would otherwise be missed, while also generating hypotheses regarding important mechanistic modes of drug action.

Toward immunotherapy with redirected T cells in a large animal model: ex vivo activation, expansion, and genetic modification of canine T cells.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown promising antitumor activity in early phase clinical studies, especially for hematological malignancies. However, most preclinical models do not reliably mimic human disease. We reasoned that developing an adoptive T-cell therapy approach for spontaneous osteosarcoma (OS) occurring in dogs would more closely reproduce the condition in human cancer. To generate CAR-expressing canine T cells, we developed expansion and transduction protocols that allow for the generation of sufficient numbers of CAR-expressing canine T cells for future clinical studies in dogs within 2 weeks of ex vivo culture. To evaluate the functionality of CAR-expressing canine T cells, we targeted HER2(+) OS. We demonstrate that canine OS is positive for HER2, and that canine T cells expressing a HER2-specific CAR with human-derived transmembrane and CD28.ζ signaling domains recognize and kill HER2(+) canine OS cell lines in an antigen-dependent manner. To reduce the potential immunogenicity of the CAR, we evaluated a CAR with canine-derived transmembrane and signaling domains, and found no functional difference between human and canine CARs. Hence, we have successfully developed a strategy to generate CAR-expressing canine T cells for future preclinical studies in dogs. Testing T-cell therapies in an immunocompetent, outbred animal model may improve our ability to predict their safety and efficacy before conducting studies in humans.