Current understanding of comparative pathology and prospective research approaches for canine hemangiosarcoma.

Hemangiosarcoma (HSA) is a malignant tumor originating from endothelial cells. HSA typically develops in dogs, but is rare in other animals, including humans. Although surgery and chemotherapy are conventional treatments for HSA, neither treatment can significantly improve patient prognosis. To develop novel and effective therapeutics, a deeper understanding of HSA pathogenesis must be acquired. However, the limited research tools for HSA have been unable to make a breakthrough; therefore, it is crucial to widely utilize or establish novel research tools such as patient-derived xenograft models, organoids, and chicken embryo xenograft models. The pathogenesis of the human counterpart of HSA, angiosarcoma (AS), also remains incompletely understood, preventing the extrapolation of findings from humans to dogs, unlike other diseases. In this review, we summarize the clinicopathological and morphological features of HSA, and then we discuss the current understanding of the molecular pathology of HSA. Finally, we highlight promising research tools that may accelerate HSA basic research toward developing novel therapeutics. We also briefly summarize AS to help researchers comprehend HSA from the perspective of comparative pathology.

Diverse genome-wide DNA methylation alterations in canine hepatocellular tumours.

BACKGROUND: Canine hepatocellular tumours (HCTs) are common primary liver tumours. However, the exact mechanisms of tumourigenesis remain unclear. Although some genetic mutations have been reported, DNA methylation alterations in canine HCT have not been well studied. OBJECTIVES: In this study, we aimed to analyse the DNA methylation status of canine HCT. METHODS: Tissues from 33 hepatocellular carcinomas, 3 hepatocellular adenomas, 1 nodular hyperplasia, 21 non-tumour livers from the patients and normal livers from 5 healthy dogs were used. We analysed the DNA methylation levels of 72,367 cytosine-guanine dinucleotides (CpG sites) in all 63 samples. RESULTS AND CONCLUSIONS: Although a large fraction of CpG sites that were highly methylated in the normal liver became hypomethylated in tumours from most patients, we also found some patients with less remarkable change or no change in DNA methylation. Hierarchical clustering analysis revealed that 32 of 37 tumour samples differed from normal livers, although the remaining 5 tumour livers fell into the same cluster as normal livers. In addition, the number of hypermethylated genes in tumour livers varied among tumour cases, suggesting various DNA methylation patterns in different tumour groups. However, patient and clinical parameters, such as age, were not associated with DNA methylation status. In conclusion, we found that HCTs undergo aberrant and diverse patterns of genome-wide DNA methylation compared with normal liver tissue, suggesting a complex epigenetic mechanism in canine HCT.

Transcriptome and proteome analysis of dogs with precursor targeted immune-mediated anemia treated with splenectomy.

Precursor-targeted immune-mediated anemia (PIMA) in dogs is characterized by persistent non-regenerative anemia and ineffective erythropoiesis, and it is suspected to be an immune-mediated disease. Most affected dogs respond to immunosuppressive therapies; however, some are resistant. In this study, we carried out splenectomy as an alternative therapy for refractory PIMA in dogs, and analyzed gene expression levels in the spleen of dogs with or without PIMA and in serum before and after splenectomy. A total of 1,385 genes were found to express differentially in the spleens from dogs with PIMA compared with healthy dogs by transcriptome analysis, of which 707 genes were up-regulated, including S100A12, S100A8, and S100A9 that are linked directly to the innate immune system and have been characterized as endogenous damage-associated molecular patterns. Furthermore, immunohistochemistry confirmed that S100A8/A9 protein expression levels were significantly higher in dogs with PIMA compared with those in healthy dogs. A total of 22 proteins were found to express differentially between the serum samples collected before and after splenectomy by proteome analysis, of which 12 proteins were up-regulated in the samples before. The lectin pathway of complement activation was identified by pathway analysis in pre-splenectomy samples. We speculated that S100A8/9 expression may be increased in the spleen of dogs with PIMA, resulting in activation of the lectin pathway before splenectomy. These findings further our understanding of the pathology and mechanisms of splenectomy for PIMA.

An experimental challenge model for Leishmania donovani in beagle dogs, showing a similar pattern of parasite burden in the peripheral blood and liver.

Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver.

Malignant oligoastrocytoma in the spinal cord of a cat.

A 12-year and 3-month spayed female mixed cat was presented with severe lumbar pain. Magnetic resonance imaging and postmortem examination revealed a swollen lesion in the spinal cord at L3 level. Histologic examination identified extensive neoplastic cell proliferation with massive necrosis in the tumor tissue. Two types of neoplastic cells were recognized. One type of neoplastic cells were large cells characterized by round to polygonal shape and abundant eosinophilic cytoplasm (referred to as "large cells"). The other neoplastic cells were small, densely proliferated, and had round to irregular shape and scant eosinophilic cytoplasm (referred to as "small cells"). Both types of cells were positive for oligodendrocyte transcription factor 2 and SRY-box transcription factor 10. Glial fibrillary acidic protein was positive in large cells but negative in most small cells. Digital analysis for Ki-67-stained tumor tissues found that total 21.1% ± 6.5% of tumor cells were positive for Ki-67. Based on these findings, we diagnosed malignant oligoastrocytoma in the spinal cord.

Manipulating histone acetylation leads to antitumor effects in hemangiosarcoma cells.

Canine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. HSA cell lines treated with SAHA and VPA upregulated inflammatory-related genes and attracted macrophage cell line RAW264 cells, which suggests that SAHA and VPA can affect immune responses. JQ1 stimulated autophagy and inhibited the cell cycle in HSA cell lines. Finally, we demonstrated that JQ1 suppressed HSA tumour cell proliferation in vivo although SAHA and VPA did not affect tumour growth. These results suggest that BETi can be alternative drugs for HSA treatment. Although further research is required, our study indicated that dysregulation of histone acetylation is likely to be involved in HSA malignancy.

Safety Assessment of Ultrasound-Assisted Intravesical Chemotherapy in Normal Dogs: A Pilot Study.

Intravesical chemotherapy after transurethral resection is a treatment option in patients with non-muscle invasive bladder cancer. The efficacy of intravesical chemotherapy is determined by the cellular uptake of intravesical drugs. Therefore, drug delivery technologies in the urinary bladder are promising tools for enhancing the efficacy of intravesical chemotherapy. Ultrasound-triggered microbubble cavitation may enhance the permeability of the urothelium, and thus may have potential as a drug delivery technology in the urinary bladder. Meanwhile, the enhanced permeability may increase systemic absorption of intravesical drugs, which may increase the adverse effects of the drug. The aim of this preliminary safety study was to assess the systemic absorption of an intravesical drug that was delivered by ultrasound-triggered microbubble cavitation in the urinary bladder of normal dogs. Pirarubicin, a derivative of doxorubicin, and an ultrasound contrast agent (Sonazoid) microbubbles were administered in the urinary bladder. Ultrasound (transmitting frequency 5 MHz; pulse duration 0.44 µsec; pulse repetition frequency 7.7 kHz; peak negative pressure -1.2 MPa) was exposed to the bladder using a diagnostic ultrasound probe (PLT-704SBT). The combination of ultrasound and microbubbles did not increase the plasma concentration of intravesical pirarubicin. In addition, hematoxylin and eosin staining showed that the combination of ultrasound and microbubble did not cause observable damages to the urothelium. Tissue pirarubicin concentration in the sonicated region was higher than that of the non-sonicated region in two of three dogs. The results of this pilot study demonstrate the safety of the combination of intravesical pirarubicin and ultrasound-triggered microbubble cavitation, that is, ultrasound-assisted intravesical chemotherapy.

CECR2 drives breast cancer metastasis by promoting NF-κB signaling and macrophage-mediated immune suppression.

Metastasis is the major cause of cancer-related deaths due to the lack of effective therapies. Emerging evidence suggests that certain epigenetic and transcriptional regulators drive cancer metastasis and could be targeted for metastasis treatment. To identify epigenetic regulators of breast cancer metastasis, we profiled the transcriptomes of matched pairs of primary breast tumors and metastases from human patients. We found that distant metastases are more immune inert with increased M2 macrophages compared to their matched primary tumors. The acetyl-lysine reader, cat eye syndrome chromosome region candidate 2 (CECR2), was the top up-regulated epigenetic regulator in metastases associated with an increased abundance of M2 macrophages and worse metastasis-free survival. CECR2 was required for breast cancer metastasis in multiple mouse models, with more profound effect in the immunocompetent setting. Mechanistically, the nuclear factor κB (NF-κB) family member v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) recruits CECR2 to increase chromatin accessibility and activate the expression of their target genes. These target genes include multiple metastasis-promoting genes, such as TNC, MMP2, and VEGFA, and cytokine genes CSF1 and CXCL1, which are critical for immunosuppression at metastatic sites. Consistent with these results, pharmacological inhibition of CECR2 bromodomain impeded NF-κB-mediated immune suppression by macrophages and inhibited breast cancer metastasis. These results reveal that targeting CECR2 may be a strategy to treat metastatic breast cancer.

Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages.

Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cell line for their usefulness as syngeneic models for canine HSA. Our results showed that the ISOS-1 cell line developed tumors with similar morphology to canine HSA. ISOS-1 cells highly expressed KDM2B and had similar KDM2B target expression patterns with canine HSA. Moreover, we determined that in both ISOS-1 and canine HSA tumors, macrophages were present as a major constituent of the tumor microenvironment. These macrophages were positive for CD204, an M2 macrophage marker, and express PD-L1, an immune checkpoint molecule. Canine HSA with macrophages expressing PD-L1 had a smaller number of T-cells in tumor tissues than tumors with PD-L1 negative macrophages. ISOS-1-conditioned medium could induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line and mouse peritoneal macrophages. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA.

Systemic mucoid degeneration of the arterial tunica intima in a young dog.

A 27-mo-old, spayed female mixed-breed dog was presented with left forelimb pain, which progressed to full thickness necrosis of the soft tissues of multiple limbs. Clinical imaging and postmortem examination suggested multiple large arterial thromboemboli. Histologic examination of vascular lesions revealed markedly thickened tunica intima with polypoid intraluminal projections, which partially to entirely occluded the arterial lumen. The expanded tunica intima was comprised of intimal accumulation of Alcian blue-positive matrix with scattered spindle-to-satellite cells. These cells were positive for von Willebrand factor and vimentin but negative for α-smooth muscle actin, suggesting endothelial origin. Deposition of the intimal mucoid matrix was observed in the elastic and muscular arteries associated with regional ischemic changes. Mucoid emboli, likely from fragmentation of proliferative intimal tissue, were identified in smaller vessels supplied by affected arteries. Based on these findings, we diagnosed systemic mucoid degeneration of the arterial tunica intima. Such systemic arterial degeneration characterized by deposition of mucoid matrix in the tunica intima has not been reported previously in dogs, to our knowledge, and should be distinguished from thromboembolism and other degenerative vascular diseases.

The expression of histone lysine demethylase 2B in canine hemangiosarcoma is associated with disease progression.

Canine hemangiosarcoma (HSA), a highly fatal mesenchymal tumour of dogs, originates from the endothelial cells lining of blood vessels. It is characterized by a short survival time with a mean survival time of only 4 months. Recently, we showed that histone lysine demethylase 2B (KDM2B) was highly expressed in canine HSA and was important in HSA tumour cell survival by positively regulating DNA repair mechanisms. KDM2B has been reported to be related to disease progression and patient survival in several human cancers. Thus, in this study, we studied the relationship of KDM2B expression levels with several patient clinical profiles to investigate the role of KDM2B in clinical HSA tumours. We analysed 37 canine HSA cases and found that KDM2B is highly expressed in stage 3 HSA compared to stage 1 HSA. High KDM2B expression was also found in male dogs compared to female dogs. No correlation was observed between KDM2B expression and age. Classifying HSA patients into high and low KDM2B expression groups revealed that the high KDM2B group showed shorter overall survival than the low KDM2B group. Based on these results, we suggest that KDM2B expression is associated with disease progression in HSA.

Peliosis Hepatis with Chylous Ascites in a Dog.

A 6-year-old spayed female Toy Poodle dog was referred to the Hokkaido University Veterinary Teaching Hospital for abdominal distension. Abdominocentesis yielded ascitic fluid that had a mildly increased total protein concentration and a 2.7-fold higher triglyceride concentration than plasma, and was interpreted as chylous ascites. The patient had an enlarged liver, which contained multiple, small, nodular masses and cyst-like structures. Microscopically, these lesions were multifocal dilated spaces containing lymphocytes, endothelial cells, fibrin and islands of hepatocytes. Increased α-smooth muscle actin-positive cells were observed in hepatic sinusoids. Based on these findings, we diagnosed peliosis hepatis with chylous ascites, which is likely to have been due to lymphangiectasia and disrupted hepatic sinusoids. Neither Bartonella spp DNA nor mutations in ACVRL1 and MTM1 genes were detected, although there was a 47-fold increase in hepatic ACVRL1 expression compared with age-matched control liver. To the authors' knowledge, this is the first report of chylous ascites resulting from peliosis hepatis in any species.

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma.

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we find that lysine-specific demethylase 2b (KDM2B) is highly expressed in HSA cell lines compared with normal canine endothelial cells. Silencing of KDM2B in HSA cells results in increased cell death in vitro compared with the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced KDM2B silencing in tumor xenografts results in decreased tumor sizes compared with the control. Furthermore, KDM2B is also highly expressed in clinical cases of HSA. We hypothesize that pharmacological KDM2B inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treat HSA cells with GSK-J4, a histone demethylase inhibitor, and find that GSK-J4 treatment also induces apoptosis and cell death. In addition, GSK-J4 treatment decreases tumor size. Therefore, we demonstrate that KDM2B acts as an oncogene in HSA by enhancing the DNA damage response. Moreover, we show that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody.

Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei-infected cell types in naturally infected horses.

Long interspersed nucleotide element-1 hypomethylation in canine malignant mucosal melanoma.

Canine malignant melanoma is a common cancer with a high mortality rate and is a clinically important disease. DNA methylation has been considered to be a potential tumorigenic mechanism through aberrant DNA methylation at promoter region which represses gene transcription. Global hypomethylation could also facilitate chromosome instability. There are few reports regarding DNA methylation in canine malignant melanoma; therefore, the purpose of this study was to examine DNA methylation status of long interspersed nucleotide element-1 (LINE-1) to be a surrogate marker of genome-wide methylation changes in this disease. We measured levels of DNA methylation of two adjacent cytosine-guanine sites on CpG island (CGI) at the putative promoter of canine LINE-1 sequence by bisulphite-pyrosequencing in 41 canine melanoma patient samples as well as six cell lines compared with normal mucosae. The survival rates were obtained from owners or medical records. We found DNA methylation levels of LINE-1 in normal mucosae were methylated. Interestingly, both melanoma cell lines and clinical melanoma samples showed remarkable hypomethylation. In addition, patients with lower LINE-1 methylation showed worse prognosis than those with higher LINE-1 methylation, though the difference did not reach statistical significance (P = .09). Here, we demonstrate that hypomethylation of LINE-1 is an epigenetically aberrant feature in canine melanoma with possible prognostic value.

Acquired Resistance to HER2-Targeted Therapies Creates Vulnerability to ATP Synthase Inhibition.

Acquired resistance to HER2-targeted therapies occurs frequently in HER2+ breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab + pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors in vivo. In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2-targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance. SIGNIFICANCE: These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.

High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines.

Canine hemangiosarcoma (HSA) is an aggressive malignant endothelial tumor in dogs and characterized by poor prognosis because of its high invasiveness, high metastatic potential, and poor responsiveness to anti-cancer drugs. Although doxorubicin-based chemotherapy is regularly conducted after surgical treatment, its effects on survival rates are limited. Acquisition of drug resistance is one of the causes of this problem, but the underlying mechanisms remain unclear. In the present study, we aimed to identify the drug-resistance mechanism in canine HSA by establishing doxorubicin-resistant (DR) HSA cell lines. HSA cell lines were exposed to doxorubicin at gradually increasing concentrations. When the cells were able to grow in the presence of a 16-fold higher doxorubicin concentration compared with the initial culture, they were designated DR-HSA cell lines. Characterization of these DR-HSA cell lines revealed higher drug efflux pump capacity compared with the parental cell lines. Furthermore, the DR-HSA cell lines did not show activation of the DNA damage response despite carrying high DNA damage burdens, meaning that apoptosis was not strongly induced. In conclusion, canine HSA cell lines acquired doxorubicin resistance by increasing their drug efflux pump capacity and decreasing the DNA damage response. This study provides useful findings to promote further research on the drug-resistance mechanisms in canine HSA.

Anti-arthritic effect of pentosan polysulfate in rats with collagen-induced arthritis.

Pentosan polysulfate (PPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of PPS on arthritic profiles based on clinical score, ankle size, histological changes, and activity of inflammatory mediators using collagen-induced arthritic rat are reported. Model of arthritis was developed in Sprague Dawley rats by intradermal injection of bovine type II collagen emulsified with incomplete Freund's adjuvant. The rats were randomly divided into four groups: normal control, arthritic control, arthritic rats treated with PPS (at dose level 20 µg/g) and arthritic rats treated with meloxicam (2 µg/g). The treatment was continued daily until the day 30. Arthritic biomarkers (cartilage oligomeric matrix protein and tartrate-resistant acid phosphatase 5b) in synovial fluid, expression of inflammatory mediators (interleukin-1ß, and tumor necrosis factor-α) and osteoclast marker genes (cathepsin K, tartrate-resistant acid phosphatase) in synovial membrane were measured. Daily administration of PPS to the arthritic rats significantly decreased the severity of arthritis by effectively suppressing the symptoms of arthritis and improving the functional recovery based on clinical score and histopathological evidence. Intriguingly, identical downregulation pattern of arthritis profiles, biological markers as well as relative mRNA levels of osteoclast markers and cytokines were monitored in arthritic rats treated with PPS. In conclusion, PPS exerted protective effects against collagen-induced arthritis in rats. The results suggest that PPS acts as an anti-inflammatory and anti-arthritic agent in decreasing the arthritic effects in collagen-induced arthritic rats.

Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells.

BACKGROUND: Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells which usually shows poor prognosis due to its high invasiveness, metastatic rate and severe hemorrhage from tumor ruptures. Since the pathogenesis of HSA is not yet complete, further understanding of its molecular basis is required. RESULTS: Here, we identified Notch2 signal as a key factor in maintaining canine HSA cancer stem cell (CSC)-like cells. We first cultured HSA cell lines in adherent serum-free condition and confirmed their CSC-like characteristics. Notch signal was upregulated in the CSC-like cells and Notch signal inhibition by a γ-secretase inhibitor significantly repressed their growth. Notch2, a Notch receptor, was highly expressed in the CSC-like cells. Constitutive activation of Notch2 increased clonogenicity and number of cells which were able to survive in serum-free condition. In contrast, inhibition of Notch2 activity showed opposite effects. These results suggest that Notch2 is an important factor for maintaining HSA CSC-like cells. Neoplastic cells in clinical cases also express Notch2 higher than endothelial cells in the normal blood vessels in the same slides. CONCLUSION: This study provides foundation for further stem cell research in HSA and can provide a way to develop effective treatments to CSCs of endothelial tumors.

Cellular atypia is negatively correlated with immunohistochemical reactivity of CD31 and vWF expression levels in canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) is one of the most common mesenchymal tumors in dogs. Its high metastatic and growth rates are usually associated with poor prognosis. Neoplastic cells of HSA can show various levels of cellular atypia in the same mass and may consist of various populations at different differentiated stages. Up to present, however, there is no report analyzing their differentiation states by comparing cellular atypia with differentiation-related protein expressions. To evaluate whether cellular atypia can be used as a differentiation marker in HSA, we analyzed correlation between cellular atypia and intensities of CD31 and von Willebrand Factor (vWF) staining in HSA cases. We also compared cellular atypia and expression levels of CD31 and vWF in each growth patterns. Our results show that cellular atypia was negatively correlated to CD31 and vWF expression levels but no significant correlation was found between growth patterns and cellular atypia or CD31 and vWF expression levels. Our study suggests that cellular atypia is useful for identifying differentiation levels in HSA cases. This study also provides useful information to determine differentiation levels of cell populations within HSA based only on morphological analysis, which will aid further HSA research such as identifying undifferentiation markers of endothelial cells or finding undifferentiated cell population in tissue sections.