Insight into microRNAs' involvement in hematopoiesis: current standing point of findings.

Hematopoiesis is a complex process in which hematopoietic stem cells are differentiated into all mature blood cells (red blood cells, white blood cells, and platelets). Different microRNAs (miRNAs) involve in several steps of this process. Indeed, miRNAs are small single-stranded non-coding RNA molecules, which control gene expression by translational inhibition and mRNA destabilization. Previous studies have revealed that increased or decreased expression of some of these miRNAs by targeting several proto-oncogenes could inhibit or stimulate the myeloid and erythroid lineage commitment, proliferation, and differentiation. During the last decades, the development of molecular and bioinformatics techniques has led to a comprehensive understanding of the role of various miRNAs in hematopoiesis. The critical roles of miRNAs in cell processes such as the cell cycle, apoptosis, and differentiation have been confirmed as well. However, the main contribution of some miRNAs is still unclear. Therefore, it seems undeniable that future studies are required to focus on miRNA activities during various hematopoietic stages and hematological malignancy.

In vitro investigation of canine periodontal ligament-derived mesenchymal stem cells: A possibility of promising tool for periodontal regeneration.

Objectives: Recent investigations indicate that canine periodontal ligament-derived stem cells (cPDLSCs) may reveal a reliable strategy for repair of periodontal tissues via cell-based tissue engineering approaches. Due to limited research, this study aimed to demonstrate the phenotypic characterization of cPDLSc in comparison with canine bone marrow-derived mesenchymal stem cells (cBMSCs) in vitro. Methods: Mesenchymal stem cells (MSCs) were obtained from PDL and BM of five male adult Mongrel dogs. In vitro isolation and expansion as well as biologic characterization including colony unit formation (CFU), osteogenic and adipogenic differentiation, flow cytometric analysis of CD34 and CD44, and RT-PCR of alkaline phosphatase (ALP), osteocalcin (OCN), periostin (POSTN) and S100A4 were performed. Furthermore, electron microscopy analysis was done to complement the comparative research. Results: CFU assay revealed that colonies of cPDLSCs presented 70% confluency with a more finite lifespan than BM-MSCs, showing a significant increase in cPDLSCs. Both types of MSCs showed osteogenic and adipogenic phenotypic characterized with clusters of mineralized depositions and lipid vacuoles, respectively. Both types of MSCs expressed CD44 with limited expression of CD34. RT-PCR of cPDLSCs revealed that expression of ALP, POSTN, OCN and S100A4 genes were significantly higher than those of BMSCs. In addition, comparison of SEM and revealed that cPDLSCs expressed more extracellular collagen fibers. Conclusions: The current study indicated that cPDLSCs show potency as a novel cellular therapy for periodontal regeneration a large animal model.

Development of a MET-targeted single-chain antibody fragment as an anti-oncogene targeted therapy for breast cancer.

The usage of monoclonal antibodies (mAbs) and antibody fragments, as a matter associated with the biopharmaceutical industry, is increasingly growing. Harmonious with this concept, we designed an exclusive modeled single-chain variable fragment (scFv) against mesenchymal-epithelial transition (MET) oncoprotein. This scFv was newly developed from Onartuzumab sequence by gene cloning, and expression using bacterial host. Herein, we examined its preclinical efficacy for the reduction of tumor growth, invasiveness and angiogenesis in vitro and in vivo. Expressed anti-MET scFv demonstrated high binding capacity (48.8%) toward MET-overexpressing cancer cells. The IC50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 8.4 µg/ml whereas this value was measured as 47.8 µg/ml in MET-negative cell line BT-483. Similar concentrations could also effectively induce apoptosis in MDA-MB-435 cancer cells. Moreover, this antibody fragment could reduce migration and invasion in MDA-MB-435 cells. Grafted breast tumors in Balb/c mice showed significant tumor growth suppression as well as reduction of blood-supply in response to recombinant anti-MET treatment. Histopathology and immunohistochemical assessments revealed higher rate of response to therapy. In our study, we designed and synthetized a novel anti-MET scFv which could effectively suppress MET-overexpressing breast cancer tumors.

Systems biology and machine learning approaches identify drug targets in diabetic nephropathy.

Diabetic nephropathy (DN), the leading cause of end-stage renal disease, has become a massive global health burden. Despite considerable efforts, the underlying mechanisms have not yet been comprehensively understood. In this study, a systematic approach was utilized to identify the microRNA signature in DN and to introduce novel drug targets (DTs) in DN. Using microarray profiling followed by qPCR confirmation, 13 and 6 differentially expressed (DE) microRNAs were identified in the kidney cortex and medulla, respectively. The microRNA-target interaction networks for each anatomical compartment were constructed and central nodes were identified. Moreover, enrichment analysis was performed to identify key signaling pathways. To develop a strategy for DT prediction, the human proteome was annotated with 65 biochemical characteristics and 23 network topology parameters. Furthermore, all proteins targeted by at least one FDA-approved drug were identified. Next, mGMDH-AFS, a high-performance machine learning algorithm capable of tolerating massive imbalanced size of the classes, was developed to classify DT and non-DT proteins. The sensitivity, specificity, accuracy, and precision of the proposed method were 90%, 86%, 88%, and 89%, respectively. Moreover, it significantly outperformed the state-of-the-art (P-value ≤ 0.05) and showed very good diagnostic accuracy and high agreement between predicted and observed class labels. The cortex and medulla networks were then analyzed with this validated machine to identify potential DTs. Among the high-rank DT candidates are Egfr, Prkce, clic5, Kit, and Agtr1a which is a current well-known target in DN. In conclusion, a combination of experimental and computational approaches was exploited to provide a holistic insight into the disorder for introducing novel therapeutic targets.

Prognostic efficacy of the RTN1 gene in patients with diffuse large B-cell lymphoma.

Gene expression profiling has been vastly used to extract the genes that can predict the clinical outcome in patients with diverse cancers, including diffuse large B-cell lymphoma (DLBCL). With the aid of bioinformatics and computational analysis on gene expression data, various prognostic gene signatures for DLBCL have been recently developed. The major drawback of the previous signatures is their inability to correctly predict survival in external data sets. In other words, they are not reproducible in other datasets. Hence, in this study, we sought to determine the gene(s) that can reproducibly and robustly predict survival in patients with DLBCL. Gene expression data were extracted from 7 datasets containing 1636 patients (GSE10846 [n = 420], GSE31312 [n = 470], GSE11318 [n = 203], GSE32918 [n = 172], GSE4475 [n = 123], GSE69051 [n = 157], and GSE34171 [n = 91]). Genes significantly associated with overall survival were detected using the univariate Cox proportional hazards analysis with a P value < 0.001 and a false discovery rate (FDR) < 5%. Thereafter, significant genes common between all the datasets were extracted. Additionally, chromosomal aberrations in the corresponding region of the final common gene(s) were evaluated as copy number alterations using the single nucleotide polymorphism (SNP) data of 570 patients with DLBCL (GSE58718 [n = 242], GSE57277 [n = 148], and GSE34171 [n = 180]). Our results indicated that reticulon family gene 1 (RTN1) was the only gene that met our rigorous pipeline criteria and associated with a favorable clinical outcome in all the datasets (P < 0.001, FDR < 5%). In the multivariate Cox proportional hazards analysis, this gene remained independent of the routine international prognostic index components (i.e., age, stage, lactate dehydrogenase level, Eastern Cooperative Oncology Group [ECOG] performance status, and number of extranodal sites) (P < 0.0001). Furthermore, no significant chromosomal aberration was found in the RTN1 genomic region (14q23.1: Start 59,595,976/End 59,870,966).

Macrophage polarization by MSC-derived CXCL12 determines tumor growth.

BACKGROUND: Phenotypic and functional heterogeneity of macrophages is known to be the main reason for their ability to regulate inflammation and promote tumorigenesis. Mesenchymal stem cells (MSCs) are one of the principal cells commonly found in the tumor stromal niche, with capability of macrophage phenotypic switching. The objective of this study was to evaluate the role of C-X-C motif chemokine ligand 12 (CXCL12) produced by marrow-derived MSCs in the phenotypic and functional pattern of bone marrow-derived macrophages (BMDMs). METHODS: First, the CRISPR/Cas9 system was used for the CXCL12 gene knock-out in MSCs. Then, coculture systems were used to investigate the role of MSCsCXCL12-/- and MSCsCXCL12+/+ in determination of macrophage phenotype. To further analyze the role of the MSC-derived CXCL12 niche, cocultures of 4T1 mammary tumor cells and macrophages primed with MSCsCXCL12-/- or MSCsCXCL12+/+ as well as in-vivo limiting dilution assays were performed. RESULTS: Our results revealed that the expression of IL-4, IL-10, TGF-ß and CD206 as M2 markers was significantly increased in macrophages co-cultured with MSCsCXCL12+/+ , whereas the expression of IL-6, TNF-α and iNOS was conversely decreased. The number and size of multicellular tumor spheroids were remarkably higher when 4T1 cells were cocultured with MSCCXCL12+/+-induced M2 macrophages. We also found that the occurrence of tumors was significantly higher in coinjection of 4T1 cells with MSCCXCL12+/+-primed macrophages. Tumor initiating cells were significantly decreased after coinjection of 4T1 cells with macrophages pretreated with MSCsCXCL12-/-. CONCLUSIONS: In conclusion, our findings shed new light on the role of MSC-derived CXCL12 in macrophage phenotypic switching to M2, affecting their function in tumorigenesis.

Development of an RNA sequencing-based prognostic gene signature in multiple myeloma.

Several prognostic gene signatures have been developed to predict the clinical outcome in patients with multiple myeloma (MM). The most salient disadvantage of the previous signatures is their non-reproducibility in external datasets. Given the disadvantages and the superiority of RNA sequencing over microarrays in transcriptome profiling to produce more reliable outputs, we sought to develop a reproducible RNA sequencing-based prognostic gene signature for MM. Genes significantly associated with survival were detected in The Cancer Genome Atlas (TCGA) MM RNA sequencing dataset (MMRF-CoMMpass) (n = 412) through a strict pipeline containing four rigid filters. The reproducibility of the selected genes was checked in an independent dataset (GSE24080), containing 559 newly diagnosed patients with MM. The RNA sequencing-based prognostic signature was reconstructed based on the final genes in the training dataset (MMRF-CoMMpass) and externally validated in five independent datasets (i.e. GSE2658, GSE13624, GSE9782, GSE6477 and GSE57317), containing 1461 MM cases. The RNA sequencing-based signature was reconstructed using finally five reproducible genes: CCT2, CKS1B, PRKDC, NONO and UBE2A. This signature was able to robustly discriminate between low- and high-risk patients in both training and validation datasets (Ps ≤ 0·001). Our signature was also independent of and more powerful than the routine MM prognostic factors (i.e. ß2-microglobulin, albumin, age and sex) (Ps ≤ 0·01). Treatment regimens had no effect on RNA sequencing-based signature insofar as this signature succeeded in predicting the clinical outcome in various treatment groups (Ps ≤ 0·001).

Valproic acid restores the down-regulation of SDF-1 following kidney ischemia; experimental validation of a mathematical prediction.

BACKGROUND AND PURPOSE: Stromal-derived factor (SDF)-1, a chemokine recruiting leucocytes and stem cells, plays an essential role in tissue regeneration. In a previous study, we have unexpectedly found that the expression of this chemokine declines following kidney ischemia reperfusion (IR). To explain this observation, a mathematical model was constructed which proposed histone deacetylase (HDAC) as the main driver of SDF-1 down-regulation. To experimentally verify this prediction, the effect of valproic acid (VPA), a potent HDAC inhibitor, on the kinetics of kidney SDF-1 expression was here assessed. EXPERIMENTAL APPROACH: Adult mice were subjected to IR or sham operation and received VPA or vehicle. Next, SDF-1 expression as well as tissue repair indices were measured in a time course manner. FINDINGS / RESULTS: The transcriptional expressions of Sdf-1 alpha, beta, and gamma isoforms were noisy in the sham groups but the fluctuations disappeared following IR where a continuous declining trend was observed. VPA induced the over-expression of gamma, but not alpha and beta mRNA in IR mice which was accompanied with protein upregulation. Remarkably, VPA deteriorated kidney injury. CONCLUSION AND IMPLICATIONS: HDAC inhibition restores SDF-1 down-regulation following kidney IR. The present study is a classic example of the potential of computational modeling for the prediction of biomedical phenomena.

Impairment of endothelial progenitor cells function in patient with mustard gas intoxication.

Background: Sulfur mustard (SM), also known as mustard gas, was first widely used in the Iraq-Iran. After SM exposure, the most prominent clinical signs are the development of extensive non-healing skin wounds and pulmonary signs, persisting over long time. Since the most frequent complications in SM-intoxicated patients are respiratory and dermatologic lesions, and with respect to the important role of endothelial progenitor cells (EPCs) in the pathophysiology of these lesion, we conducted this study to recognize the potential effects of SM on biological features of EPCs in patients exposed with this gas.Methods: In this study, 30 patients with the history of SM exposure during the Iran-Iraq war (1984-1988), 27 patients with pulmonary signs with no history of SM exposure and 20 healthy participants were included. Cell population and function of EPCs were assessed 4 years post-exposure. For this purpose, circulating EPCs (cEPCs) were harvested and cultivated, then the biological features of these cells, including migratory, proliferative, and tubulogenic activities were analyzed. We also measured serum antioxidants levels and mRNA levels of some proangiogenic factors in EPCs from SM-intoxicated patients.Results: Our results showed lesser number of cEPCs in patients exposed with SM, which was associated with decreased proliferative, migratory, and tubulogenic activity of these cells. Also, we found the lesser serum activity of SOD, GPX and MDA in the SM group than in the healthy control group.Conclusions: SM exposure resulted in decreased proliferation and migration of EPCs, which was associated with decreased tubule formation and angiogenic factors.

Evaluating the effect of remote ischemic preconditioning on kidney ischemia-reperfusion injury.

BACKGROUND: Acute kidney injury is a high-risk complication in a variety of clinical situations mostly due to ischemia-reperfusion (IR) injuries. The novel idea of remote ischemic preconditioning (rIPC) was proposed to prevent serious ischemia sequels. To address the controversy of previous reports, the current study was performed to assess the effect of rIPC on kidney IR injury. MATERIALS AND METHODS: Male BALB/c mice were exposed to either rIPC or sham intervention, 24 h before kidney IR. In two independent sets of experiments, rIPC was accomplished by inducing three cycles of 5 min ischemia with 5 min reperfusion intervals through the ligation of the left external iliac artery or infrarenal abdominal aorta. Kidney IR injury was performed by left renal pedicle occlusion for 35 min and simultaneous right nephrectomy. After 48 h, mice were sacrificed for the assessment of kidney function and structure. RESULTS: According to the serum urea and creatinine, as well as histopathological measures, none of the exploited rIPC procedures could significantly protect against kidney IR injury. CONCLUSION: Based on our findings and the divergent results of previous animal and human studies, it can be concluded that the renoprotective effects of rIPC are minimal, if any, and are not robustly detectable.

Correction to: Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

After publication of this paper, the authors determined an error in Fig. 4. Below is the correct Fig. 4.

Comparative measurement of FeLV load in hemolymphatic tissues of cats with hematologic cytopenias.

BACKGROUND: Feline leukemia virus (FeLV) is a serious viral infection in cats. FeLV is found in some tissues, such as spleen, lymph nodes and epithelial tissues. However, there is controversy about the organ in which the virus can be reliably detected in infected cats. The purpose of this study was to determine the level of viral infection in hemolymphatic tissues, including blood, bone marrow and spleen by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). RESULTS: A total of 31 cats with clinical signs of FeLV infection associated with at least a single lineage hematologic cytopenia were included in this study. Peripheral blood, bone marrow and spleen samples were obtained from each cat. Complete blood counts, biochemical tests, and a rapid test to detect FeLV p27 antigen in blood samples of cats were performed. Of 31 cats, 9 had anemia alone, 4 had thrombocytopenia alone, 2 had neutropenia alone, 9 had bicytopenia of anemia and thrombocytopenia, 3 had bicytopenia of anemia and neutropenia, and 4 had pancytopenia. FeLV RNA was then detected by RT-qPCR in the whole blood, bone marrow and spleen. Viral RNA copy numbers were detected in all cats by RT-qPCR whereas 24 out of 31 cats were positive for the serum FeLV antigen. We detected a significantly greater number of viral RNA in the spleen compared with the whole blood and bone marrow. CONCLUSION: Spleen is a site where FeLV is most frequently detected in cats with hematologic cytopenias.

Transplantation of Mouse Induced Pluripotent Stem Cell-Derived Podocytes in a Mouse Model of Membranous Nephropathy Attenuates Proteinuria.

Injury to podocytes is a principle cause of initiation and progression of both immune and non-immune mediated glomerular diseases that result in proteinuria and decreased function of the kidney. Current advances in regenerative medicine shed light on the therapeutic potential of cell-based strategies for treatment of such disorders. Thus, there is hope that generation and transplantation of podocytes from induced pluripotent stem cells (iPSCs), could potentially be used as a curative treatment for glomerulonephritis caused by podocytes injury and loss. Despite several reports on the generation of iPSC-derived podocytes, there are rare reports about successful use of these cells in animal models. In this study, we first generated a model of anti-podocyte antibody-induced heavy proteinuria that resembled human membranous nephropathy and was characterized by the presence of sub-epithelial immune deposits and podocytes loss. Thereafter, we showed that transplantation of functional iPSC-derived podocytes following podocytes depletion results in recruitment of iPSC-derived podocytes within the damaged glomerulus, and leads to attenuation of proteinuria and histological alterations. These results provided evidence that application of iPSCs-derived renal cells could be a possible therapeutic strategy to favorably influence glomerular diseases outcomes.

Development of a Reproducible Prognostic Gene Signature to Predict the Clinical Outcome in Patients with Diffuse Large B-Cell Lymphoma.

Alongside various clinical prognostic factors for diffuse large B-cell lymphoma (DLBCL) such as the international prognostic index (IPI) components (ie, age, tumor stage, performance status, serum lactate dehydrogenase concentration, and number of extranodal sites), prognostic gene signatures have recently shown promising efficacy. However, previously developed signatures for DLBCL suffer from many major inadequacies such as lack of reproducibility in external datasets, high number of members (genes) in a signature, and inconsistent association with the survival time in various datasets. Accordingly, we sought to find a reproducible prognostic gene signature with a minimal number of genes. Seven datasets-namely GSE10856 (420 samples), GSE31312 (470 samples), GSE69051 (157 samples), GSE32918 (172 samples), GSE4475 (123 samples), GSE11318 (203 samples), and GSE34171 (91 samples)-were employed. The datasets were randomly categorized into training (1219 samples comprising GSE10856, GSE31312, GSE69051, and GSE32918) and validation (417 samples consisting of GSE4475, GSE11318, and GSE34171) groups. Through the univariate Cox proportional hazards analysis, common genes associated with the overall survival time with a P value less than 0.001 and a false discovery rate less than 5% were identified in 1219 patients included in the 4 training datasets. Thereafter, the common genes were entered into a multivariate Cox proportional hazards analysis encompassing the common genes and the international prognostic index (IPI) factors as covariates, and then only common genes with a significant level of difference (P < 0.01 and z-score >2 or <-2) were selected to reconstruct the prognostic signature. After the analyses, a 7-gene prognostic signature was developed, which efficiently predicted the survival time in the training dataset (Ps < 0.0001). Subsequently, this signature was tested in 3 validation datasets. Our signature was able to strongly predict clinical outcomes in the validation datasets (Ps < 0.0001). In the multivariate Cox analysis, our outcome predictor was independent of the routine IPI components in both training datasets (Ps < 0.0001). Furthermore, our outcome predictor was the most powerful independent prognostic variable (Ps < 0.0001). We developed a potential reproducible prognostic gene signature which was able to robustly discriminate low-risk patients with DLBCL from high-risk ones.

Regulation of plasticity and biological features of endothelial progenitor cells by MSC-derived SDF-1.

Bone marrow (BM) is a source of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs). MSCs provide a specific niche in the BM and biological features of EPCs may be changed with this niche. Stromal cell-derived factor 1 (SDF-1) secreted from primary BM-MSCs and biological features of this niche on EPC development are still yet to be understood. The aim of this study was to evaluate the role of SDF-1 produced by MSCs on EPC development. We applied the CRISPR/Cas9 system for the knock-out of the SDF-1 gene in BM-derived MSCs. BM-derived EPCs were then cocultured with MSCsSDF-1-/- or MSCsSDF-1+/+ to identify the role of MSC-derived SDF-1α on proliferation, migration and angiogenic activity of EPCs. Next, pre-expanded EPCs were harvested and co-transplanted with MSCsSDF-1-/- or MSCsSDF-1+/+ into sublethally irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed that proliferation, colony formation, migration and angiogenic activity of EPCs was significantly increased after coculture with MSCsSDF-1+/+. We also found that co-transplantation of EPCs with MSCsSDF-1+/+, in contrast to MSCsSDF-1-/-, into irradiated mice resulted in marrow repopulation and hematologic recovery, leading to improved survival of transplanted mice. In conclusions, MSC-derived SDF-1 niche plays an important role in the development of EPCs and this niche is essential for bone marrow repopulation by these cells and can enhance the efficiency of EPC therapy for ischemic diseases.

Improved angiogenic activity of endothelial progenitor cell in diabetic patients treated with insulin plus metformin.

Type 2 diabetes (T2DM) is associated with an increased vascular disease. Moreover, endothelial progenitor cell (EPC) function is impaired in diabetic patients. Decreased EPC number plays a critical role in reduced endothelial repair and development of the vascular disorder. To determine the effect of metformin and insulin plus metformin on functional activity of EPCs, 130 participants were divided into three groups (group 1: healthy control; group 2: metformin; group 3: insulin plus metformin). The concentration of EPCs in the circulation was first quantified. Thereafter, circulating EPCs (cEPCs) were harvested and the biological features of these cells including proliferative, clonogenicity, tubulogenic, and migratory properties were analyzed after expansion. The serum protein levels of some proangiogenic factors were also measured. Our results showed greater numbers of cEPCs in control and in diabetic patients treated with insulin plus metformin than in metformin-treated patients. Insulin plus metformin therapy was associated with augmented proliferative, clonogenicity, migratory, and tubulogenic activity of cEPCs in patients with T2DM. Increased serum concentrations of angiogenic factors were also observed in patients treated with insulin plus metformin. Western blot analysis showed increased protein levels of pTie-2/Tie2 and Pakt/AKT in cEPCs harvested from T2DM, treated with insulin metformin plus. This study showed that treatment with insulin plus metformin in diabetic patients is associated with increased mobilization of EPCs into the circulation, with potential beneficial effect in vascular protection in diabetic patients.

Distinct Tie2 tyrosine phosphorylation sites dictate phenotypic switching in endothelial progenitor cells.

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial and their progenitor cells, nominated as endothelial progenitor cells (EPCs). Tie2/Tek is a tyrosine kinase receptor expressed by endothelial cells that induces signal transduction pathways involved in endothelial biology. To address the potential importance of the various tyrosine residues of Tie2 in EPC development, we generated a series of Tie2 tyrosine mutated (Y1106F, Y1100F, and Y1111F) EPCs and then assess the biological features of these cells. Clonogenic, tubulogenic, proliferative, migratory, and functional properties of these cells were analyzed. Next, GFP-positive EPCs containing Tie2 tyrosine mutations were systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. We found that mutation in the Tie2 tyrosine 1106 residue directed EPCs toward a mature endothelial phenotype, which was associated with augmented tubulogenic and migratory properties, and increased phosphorylation of the active site (tyrosine 992) as well as increased vascular perfusion in the in vivo Matrigel plug assay. Moreover, transplantation of 1106 Tie2 mutant EPCs failed to reconstitute the bone marrow after myeloablation, whereas transplantation of EPCs with the 1100 or 1111 Tie2 tyrosine mutation resulted in bone marrow engraftment, leading to improved survival of recipient mice. Our findings demonstrate that the tyrosine 1106 residue in Tie2 plays a key role to maintain the stemness features of EPCs.

Aggressive chondroblastic osteosarcoma in a dog: A case report.

Canine osteosarcoma (OS) or osteogenic sarcoma is an aggressive tumor of the skeletal system, associated with a rapid progression and guarded prognosis. The osteosarcomas, mostly arise from the appendicular skeleton while axial OS (osteosarcoma of flat bones) are less reported in the majority of large breeds. This report describes complete para-clinical investigations of an aggressive chondroblastic OS involving facial flat bones with highly metastatic characterization in a large mix breed stray dog. Radiographic and computed tomography findings demonstrated an amorphous and active new bone formation, associated with the severe lytic areas in the left maxillary, orbital and zygomatic bones. Also, lots of nodular densities were distributed in all lung lobes. The cytological examination of the mass revealed individualized oval to spindle-shaped pleomorphic mesenchymal cells exhibiting many criteria of malignancy such as marked anisocytosis, anisokaryosis, prominent and multiple nucleoli. The punctate cytoplasmic vacuoles were obvious and bi-nucleated cells were frequently observed. These cells were seen in the background of an eosinophilic matrix. Histopathologic evaluation of the mass exhibited areas of osseous differentiation within the mass containing bony spicules and wavy bone formation along with the vast areas of cartilaginous differentiations with chondroblasts in lacunar spaces. Ultimately, chondroblastic OS with severe lung metastasis was confirmed and the animal was euthanized.