Proteomic profiling of canine osteosarcoma patients

Background Canine osteosarcoma (OSA) is an aggressive primary bone tumor in dogs. Metastasis of canine OSA occurs mainly in the lungs through the hematogenous route. Identification of the proteins that are associated with metastasis using proteomic technologies is important not only for the discovery of OSA targets and signaling pathways associated with metastatic OSA but also to provide novel therapeutic targets. The objective of this study was to conduct a proteomic profiling analysis of canine OSA patients. We used mass spectrometry to examine the protein profiles.Results We found the overexpression of five proteins from OSA tissues—myosin (MYO7a), GTPase IMAP family member 5 isoform 1 (GIMAP5), PR domain 15 (PRDM15), A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), and interleukin receptor type 1 (IL1R1)—and one protein from the serum, rCG38920.Conclusions In this study, we identified numerous proteins in the tissue and serum of patients with malignant and benign OSA or non-OSA through a proteomic approach using mass spectrometry. These proteins may be useful for diagnosis and prognosis, including the prevention of misdiagnosis between malignant OSA and non-OSA as these have similar clinical symptoms in the early stages. In the future, further independent validation of these biomarkers using a greater number of patient samples and different techniques, including Western blot, is necessary to improve diagnostic power for differentiating malignant OSA patients from non-OSA patients.

3 cause of death. [1] Lymph nodes are involved less often (4.4-9%). [2,3] Despite advances in the methods of managing canine OSA, including limb amputation/sparing, chemotherapy, and palliative radiotherapy, prognosis remains poor because almost 90% of dogs will develop predominantly pulmonary metastasis. In these dogs, median survival times range from 3 months to 1 year, and less than 20% of dogs survive for more than 2 years following diagnosis. [4,5] Due to similarities in the biology and treatment of OSA in dogs and humans, canine OSA represents a valid and important tumor model. Increasingly, studies of OSA in pet dogs will become a standard component in the development process of novel therapies for human OSA. In addition, these studies will streamline the selection process to determine compounds that have a higher likelihood of success for the treatment of human patients.
The objective of this study was to conduct a proteomic profiling analysis of canine OSA patients. We used mass spectrometry to examine the protein profiles of serum and OSA tissues from patients.

Canine patient characteristics
A total of 25 canine patient samples were included in this serum and bone tissue proteome study. There were 10 canine OSA patients and a control group of 15 non-OSA orthopedic patients. The median age at the time of diagnosis of the OSA group and non-4 OSA group was 9 years. Male OSA patients were affected four times as often as females.
The males in the control group were affected three times as often as females. The breed distribution of the OSA group was as follows: 4 golden retrievers, 2 rottweilers, and 4 mixed breeds. The breed distribution of the control group was as follows: 4 golden retrievers, 5 mixed breeds, 1 poodle, 2 pomeranians, 2 shih tzus, and 1 French bulldog.
The median value of serum ALP of the OSA group and non-OSA group was 63 and 103.5 U/L, respectively.
The proteins were fractionated on a one-dimensional 12.5% SDS-PAGE mini slab gel and visualized using silver staining ( Figure 1). The band of interest was excised and digested. The peptides were analyzed using an HCTultra LC-MS, and the data were analyzed with DeCyder MS Differential Analysis Software and submitted to a database using the Mascot program. A Mascot search of the mammalian database resulted in a total number of 1,006 differentially expressed proteins among the control and canine OSA serum and tissues. interleukin-1 receptor type 1. Dynein was the only protein detected in the control serum, and rCG38920 was the only protein found in the OSA serum ( Table 1).

Distribution of the proteins in each functional class
Categorization of the most abundant proteins in each functional category using the Software Tool for Rapid Annotation of Proteins (STRAP) bioinformatics suite [15] revealed differences between the OSA proteome and the control proteome in canine patient samples. By biological process, the analysis showed that the majority of proteins were classified under cellular process (26.2%), regulation (26.1%), and "other" (14.7%); however, other proteins were mapped to the developmental process (8.8%), interaction with cells and organisms (6.8%), localization (6.6%), response to stimulus (4.4%), metabolic process (4.3%), immune system process (2.0%), and growth (0.1%) ( Figure 3).

Protein network analysis
To better understand the biological interaction of the identified proteins, we also constructed a protein-protein functional network using the online software Stitch 4.0(http://stitch.embl.de/) [16]. The network of differentially expressed protein-protein and protein-chemotherapeutic drugs, carboplatin and doxorubicin -the effective agents for adjuvant chemotherapy in dogs with OSA, were related to the apoptosis caspase signaling pathway, ezrin, and the proteins ADAMTS4 and IL1R1 in the canine OSA tissues and serum 6 is shown in Figure 6. The protein network analysis provided a clearer view of a complex framework of proteins that may be a result of the differences in canine malignant OSA and non-OSA patients.

Discussion
The findings of this study provided useful insight into OSA tumorigenesis and identified multiple signaling pathways to enrich our understanding of this cancer. Using an extraction method followed by SDS-PAGE, digestion, and mass spectrometry, we detected several proteins that are related to this tumor. We also evaluated the differential proteome in tissue and serum from the non-OSA bone controls and OSA patients.
We found the overexpression of five proteins in OSA tissues-myosin (MYO7a), GTPase IMAP family member 5 isoform 1 (GIMAP5), PR domain 15 (PRDM15), A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), and interleukin receptor type 1 (IL1R1) -and one protein in the serum: rCG38920. We found that ADAMTS4 and IL1R1 were involved in the ezrin and apoptosis caspase signaling pathways. High ezrin expression in canine tumors was associated with the early development of metastases and poor outcomes in pediatric OSA patients. [7,17] The complex protein networks indicated that carboplatin and doxorubicin (the effective agents for adjuvant chemotherapy in dogs with OSA) were related to the apoptosis caspase signaling pathway, ezrin, and the proteins ADAMTS4 and IL1R1. Therefore, these two proteins should be further evaluated as potential novel therapeutic target proteins for canine OSA. MYO7a, GIMAP5, and PRDM15 also were found to be differentially expressed between the OSA and control patients. MYO7a encodes for certain myosins, cytoskeletal-associated proteins that bind to actin filaments that are concentrated beneath the plasma membrane.
They are important for short-range transport during endocytosis and exocytosis and generate mechanical force for muscle contraction, cell migration, and cytokinesis. [18] In B-cell malignancy, possibly by inhibiting apoptosis. [26] GIMAP family member 6 is the biomarker identified for human prostate cancer. [27] PRDM15 is a putative histone methyltransferase, a class of enzymes frequently deregulated in human cancer, such as breast cancer, lung cancer, hepatoma, colorectal cancer, lymphoma, and osteosarcoma.
[28] The function of the only protein detected in the OSA serum, rCG38920, is still unknown. Validation of these identified proteins was necessary to determine if they can be useful as novel biomarkers for canine OSA in the future.

Conclusions
We

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for

In-gel digestion
After protein bands were excised, the gel plugs were dehydrated with 100% acetonitrile (ACN), reduced with 10mM DTT in 10mM ammonium bicarbonate at room temperature for 1 h, and alkylated at room temperature for 1 h in the dark in the presence of 100mM iodoacetamide in 10mM ammonium bicarbonate. After alkylation, the gel pieces were dehydrated twice with 100% ACN for 5 min. To perform in-gel digestion of proteins, 10 µl of trypsin solution (10 ng/µl trypsin in 50% ACN/10mM ammonium bicarbonate) was added to the gels, followed by incubation at room temperature for 20 min, and then 20 µl of 30% ACN was added to keep the gels immersed throughout digestion. The gels were incubated at 37 °C for a few hours or overnight. To extract peptide digestion products, 30 µl of 50% ACN in 0.1% formic acid was added to the gels, and then the gels were incubated at room temperature for 10 minutes in a shaker. Extracted peptides were collected and pooled together in the new tube. The pooled extracted peptides were dried by vacuum centrifuge and kept at -80 °C for further mass spectrometric analysis.   The protein-protein interaction networks. Interaction of the differentially expressed proteins in canine OSA tissues and serum predicted using the software