Canine Hemangiosarcomas Research Articles

Hemangiosarcoma Cells Promote Conserved Host-Derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors.

Adjuvant treatment of canine splenic hemangiosarcoma with autologous dendritic cell therapy - a prospective, randomized double-blind study

Canine splenic hemangiosarcomas (HSA) are malignant mesenchymal tumors with a high tendency for metastasis. Median survival times after splenectomy followed by adjuvant chemotherapy usually range between 5 and 8 months. The aim of this prospective randomized double-blinded study was to examine the efficacy of a commercially available dendritic cell therapy (PetBioCell) following splenectomy. In addition, possible side effects of this therapy were evaluated. Twenty-one dogs with histologically confirmed splenic HSA without metastasis (stages I or II) were included in the study. Ten dogs received the dendritic cell therapy, and 11 dogs received a placebo. Injections were administered according to the manufacturer's instructions monthly for the first 3 months and then every 3 months until death. Survival times and toxicoses of both groups were compared. Follow-up data were available for all 21 patients; the observation period ranging until euthanasia or metastasis-related death. One patient that had received the dendritic cell therapy was euthanized due to prostatitis and experienced the longest survival time (668 days). One dog in the placebo-group lived for 448 days after splenectomy. The median survival times in the dendritic cell therapy and the placebo group amounted to 74 and 126 days, respectively. There was no significant difference in tumor-free interval (t(18) = 1.4, p = 0.911) and survival times (t(19) = -0.094, p = 0.463) between the 2 groups. Toxicoses reported in both groups were mild and self-limiting. Immunotherapy using autologous, immature and unprimed dendritic cells according to the PetBioCell method failed to show efficacy on tumor-free interval and survival time in the presented dog population with splenic hemangiosarcoma.

Immunohistochemical evidence of NF-kB activation in canine lymphomas, histiocytic sarcomas, hemangiosarcomas, and mast cell tumors.

Increased or constitutive activation of nuclear factor kappa B (NF-kB) is a feature of many chronic disease processes, including cancer. While NF-kB overactivation has been documented extensively in human oncology, there is a relative paucity of data documenting the same phenomenon in veterinary medicine. To assess NF-kB activity, antibodies to p65 and p100/p52, which are components of NF-kB heterodimers, were first validated for specificity and canine cross-reactivity via Western blot and labeling of immortalized cell pellets. Then, nuclear labeling for these antibodies was assessed via QuPath software in over 200 tumor tissue samples (10 hemangiosarcomas, 94 histiocytic sarcomas, 71 lymphomas, and 28 mast cell tumors) and compared to immunolabeling in appropriate normal tissue counterparts. Greater than 70% of spontaneous canine tumors evaluated in this study had more nuclear p65 and p100/p52 immunoreactivity than was observed in comparable normal cell populations. Specifically, 144/204 (70.58%) of tumors evaluated had positive p65 nuclear labeling and 179/195 (91.79%) had positive p100/p52 nuclear labeling. Surprisingly, greater nuclear p100/p52 reactivity was associated with a longer progression-free survival (PFS) and overall survival (OS) in canine lymphomas. These results provide support and preliminary data to investigate the role of NF-kB signaling in different types of canine cancer.

Abstract 2660: Targeting PI3K pathway to reprogram the tumor immune niche in angiosarcoma

Abstract Angiosarcoma is an aggressive, albeit rare, cancer in humans. Angiosarcomas are vascular malignancies that can occur anywhere in the body, and their metastatic propensity is high. The vascular sarcomas consist of disorganized, malignant vessel-forming cells. Recurrent mutations in the PIK3CA gene have been identified in angiosarcomas, and activation of the PI3K pathway appears to establish angiogenic programs. Naturally occurring hemangiosarcoma in dogs shares clinical and pathological features with human angiosarcoma. Our previous work has revealed that canine hemangiosarcomas and human angiosarcomas share mutational and transcriptional signatures that activate convergent PI3K signaling pathways. Appropriately powered canine studies take a comparative genomics approach, informing our research into human angiosarcomas. The cause of most sporadic angiosarcomas is unknown. Moreover, no therapeutic targets have been identified to improve outcomes. This study hypothesizes that the activation of PIK3CA mutations contributes to the molecular programs that modulate the immune niche in angiosarcoma. We first induced PIK3CA hotspot mutations (H1047R) in canine hemangiosarcoma cell line DHSA-1426 using CRISPR/Cas9. We found that PI3K-alpha selective inhibitor BYL719 at 10uM decreased cell proliferation of PIK3CA mutant cells by 40-50% compared with that of wild-type cells. In comparison, no significant difference was observed between mutant and wild-type cells treated with pan PI3K inhibitor, ZSTK474. Our western blot data showed an increase in γH2AX, a marker of DNA damage, in mutant cells when treated with BYL719 in a dose-dependent manner. BYL719 inhibited the phosphorylation of AKT and ERK in PIK3CA mutant tumor cells. Our RNA-seq transcriptomic data analysis revealed that PIK3CA mutations altered gene regulation programs associated with lipid biosynthesis, including phosphatidylinositol, DNA replication, angiogenic and vascular functions, and immune regulation. These data suggest that the activation of the PI3K pathway is essential for tumor cell growth and that PI3K-alpha selective inhibitor interferes with PIK3CA mutation-induced pathways, including AKT and ERK, through DNA damage-dependent mechanism in angiosarcoma. Our ongoing work is to determine if PIK3CA mutant tumor cells have cell-autonomous capacity to govern hematopoietic progenitors and immune cells, potentially establishing the tumor immune niche. It also includes establishing single-cell gene expression profiles to identify distinct cell clusters between PIK3CA mutant and wild-type cells, along with chromatin accessibility landscape using ATAC-seq. Tumorigenic capacity and immune regulatory mechanisms of PIK3CA mutations will be determined in angiosarcoma xenograft models. Citation Format: Emma Kozurek, Zhiyan Silvia Liu, Hai Dang Nguyen, Jong Hyuk Kim. Targeting PI3K pathway to reprogram the tumor immune niche in angiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2660.

Selected Articles from This Issue

Angiosarcomas (AS) are rare, aggressive tumors arising from vascular tissues, and harbor extensive genomic events such as driver gene mutations and chromosomal translocations. However, recurrent fusion products and specific genomic events that promote disease progression have thus far proven elusive. In this study, Kim and colleagues employ RNA sequencing data from human angiosarcomas and canine hemangiosarcomas—which closely mirror human AS—and identify 25 novel fusion products among the human and canine samples. Though the fusions were themselves non-recurrent, they co-occured alongside recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS, suggesting that genomic instability induced by these mutations may play a role in the genomic complexity of the disease. Further, the novel fusions were uniformly associated with increased angiogenic signaling, indicating that diverse genomic events in AS likely converge on the activation of a specific transcriptional program. Taken together, the data nominate a host of new molecular targets for investigation and clarify a unifying role for diverse genomic events in the pathogenesis of AS.Extracellular vesicles (EV) have emerged as a key mediator of intercellular signaling and have been linked to the pathogenesis of cancer. EV are loaded with different “cargo” depending on their source, and the characterization of these cargoes is important to understanding and contextualizing their role. Here, Ikeda and colleagues use proteomics to quantitatively probe the differences between EV from cancerous versus normal colon. Employing an ex vivo method for harvesting tissue-exudative EV (Te-EV) from patient samples, the authors find that the amino acid transporter CAT1 is enriched in Te-EV from colorectal cancer (CRC). CAT1-positive EV were found at higher abundance in the plasma of CRC patients and were shown to mediate CRC pathogenesis: by modulating the metabolism of arginine and nitric oxide in endothelial cells, CAT1-positive EV from CRC tissues promoted vascular cell proliferation and tubule formation. These observations identify a key mechanism underlying angiogenesis in CRC and nominate CAT1-positive EV as a potential plasma biomarker for CRC development and progression.Oncogene signaling is a potent driver of tumorigenesis and progression, but cells inherently resist oncogenic transformation via feedback loops leading to cellular senescence. Understanding how oncogene-induced senescence (OIS) is subverted by tumors is critical to the development of new methods to clinically manage cancers driven by BRAF- and RAS-activating mutations. Here, Garnett and colleagues employ a genetic screen to identify mediators of OIS in the context of BRAF activation. The authors found that Amylin, a 37-amino acid peptide that promotes glycolysis, is a key molecular determinant of the response to oncogene signaling. Amylin-ablated cells underwent a series of metabolomic and epigenetic changes that circumvented the onset of senescence in response to BRAF signaling. However, exogenous supplementation of Amylin to the cell growth medium restored the ability of cells to enter a senescent state. These data identify Amylin as a key control node governing the response to oncogene signaling in cancer cells, and suggest that Amylin treatment may hold clinical benefit for patients with oncogene-driven cancers.Acute myeloid leukemia (AML) is a genetically diverse cancer with over ten different molecular subtypes characterized by specific genomic events. EGFR pathway signaling has also been described in AML cells, but most AML cells paradoxically do not express any EGFR family members on the cell surface. In this study, Kam and colleagues address this discrepancy and identify intracellular expression of truncated ERBB2 isoforms in AML cells and myelodysplastic syndrome, an AML precursor lesion. These ERBB2 isoforms—which are also known to be pathogenic in breast cancer—mediated active signaling via classical EGFR-related pathways and were sensitive to inhibition with ERBB2 tyrosine kinase inhibitors. While ERBB2 inhibition was effective as a single agent, combination of the ERBB2 inhibitor lapatinib with the BCL-2 inhibitor venetoclax resulted in marked synergism at all tested doses. These observations nominate ERBB2 inhibitors for further investigation to complement the growing arsenal of therapeutic options for AML patients.

Pathomorphological features and mast cell count in canine visceral hemangiosarcomas

Visceral hemangiosarcoma were analyzed at the Department of Veterinary Pathology of the Faculty of Veterinary Medicine, University of Zagreb, over a 5-year period. From a total of 52 tumor masses in a total of 31 dogs, histological growth patterns (cavernous, capillary or solid) and the amount of tumor supporting stroma were evaluated. Additionally, sections were stained with toluidine blue for the purpose of mast cell detection and their number was determined in the tumor parenchyma, tumor stroma and edges. The average age of the affected animals was 10 years, males predominated, and the tumors occurred most frequently in cross breeds, German Shepherd dogs and Labrador Retrievers. The highest number of visceral hemangiosarcoma was found in the spleen (27/52). The most common growth pattern of visceral hemangiosarcoma was solely cavernous with a mixture of cavernous and solid patterns in different proportions. Mast cells were found in 44/52 (84.6%) of the visceral hemangiosarcomas. A smaller number of mast cells were found in the tumor parenchyma, and higher number were found in the stroma and tumor edges. The number of mast cells in tumors was not significantly associated with the tumor growth pattern, but there was a positive correlation between MCC - tumor parenchyma and stroma (rs = 0.28, P<0.05), MCC - tumor stroma + edges (rs = 0.74, P<0.05) and MCC - tumor parenchyma and MCC- tumor stroma+edges (rs = 0.30, P<0.05) in all the examined tumors. In splenic tumors, there was only a significant positive correlation between MCC - stroma+edges (rs P = 0.68 P<0.05). These results suggest a higher mast cell count in tumors with more developed stromal components in canine visceral hemangiosarcomas, and certainly indicate the need for further research on their role and the factors they release in the development and progression of hemangiosarcomas.

Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. IMPLICATIONS: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

PIK3CA mutations matter for cancer in dogs

Cancer is a genetic disease that arises from the accumulation of genetic mutations within a cell. Mutations in PIK3CA gene are frequently observed in human solid cancers, and also appear to occur in canine tumors. Specifically, recurrent somatic PIK3CA variants identified in canine mammary tumors and hemangiosarcomas are comparable to human hotspot mutations such as H1047R. PIK3CA mutations found in canine tumors encode functional proteins that may alter downstream PI3K/Akt/mTOR signaling pathway. Therefore, PI3K inhibitors have potential in cancer therapy for dogs. This article concisely reviews the emerging evidence concerning the genetic and molecular properties of PIK3CA mutations to discuss future perspectives in veterinary and comparative oncology.

Immunohistochemical detection of p53 and pp53 Ser392 in canine hemangiomas and hemangiosarcomas located in the skin

Backgroundp53 protein is essential for the regulation of cell proliferation. Aberrant accumulation of it usually occurs in cutaneous malignancies. Mutant p53 is detected by immunohistochemistry because it is more stable than the wild-type p53. However, post-translational modifications of p53 in response to ultraviolet radiation are important mechanisms of wild-type p53 stabilization, leading to positive staining in the absence of mutation. The aims were: 1) to analyze the immunohistochemical expression of p53 and phospho-p53 Serine392 in canine skin endothelial tumours; and 2) to determine if any relationship exists between p53 and phospho-p53 Serine392 overexpression and cell proliferation.Resultsp53 and phospho-p53 Serine392 immunolabeling was examined in 40 canine cutaneous endothelial tumours (13 hemangiomas and 27 hemangiosarcomas). Their expression was associated with tumour size, hemangiosarcoma stage (dermal versus hypodermal), histological diagnosis and proliferative activity (mitotic count and Ki-67 index). Statistical analysis revealed a significant increase of p53 immunoreactivity in hemangiosarcomas (median, 74.61%; interquartile range [IQR], 66.97–82.98%) versus hemangiomas (median, 0%; IQR, 0–20.91%) (p < .001) and in well-differentiated hemangiosarcomas (median, 82.40%; IQR, 66.49–83.17%) versus hemangiomas (p = .002). Phospho-p53 Serine392 immunoreactivity was significantly higher in hemangiosarcomas (median, 53.80%; IQR, 0–69.50%) than in hemangiomas (median, 0%; IQR, 0.0%) (p < .001). Positive correlation of the overexpression of p53 and phospho-p53 Serine392 with mitotic count and Ki-67 index was found in the cutaneous vascular tumours (p < .001). The Ki-67 index of the hemangiomas (median, 0.50%; IQR, 0–2.80%) was significantly lower than that of the hemangiosarcomas (median, 34.85%; IQR, 23.88–42.33%) (p < .001), and that specifically of well-differentiated hemangiosarcomas (median, 24.60%; IQR, 15.45–39.35%) (p = .001). Immunolabeling of 18 visceral hemangiosarcomas showed that the p53 (median, 41.59%; IQR, 26.89–64.87%) and phospho-p53 Serine392 (median, 0%; IQR, 0–22.53%) indexes were significantly lower than those of skin (p = .001; p = .006, respectively).ConclusionsThe p53 and phospho-p53 Serine392overexpression together with high proliferative activity in hemangiosarcomas versus hemangiomas indicated that p53 might play a role in the acquisition of malignant phenotypes in cutaneous endothelial neoplasms in dogs. The Ki-67 index may be useful in distinguishing canine well-differentiated hemangiosarcomas from hemangiomas.

Expression of Hematopoietic Stem and Endothelial Cell Markers in Canine Hemangiosarcoma.

Several chemicals and pharmaceuticals increase the incidence of hemangiosarcomas (HSAs) in mice, but the relevance to humans is uncertain. Recently, canine HSAs were identified as a powerful tool for investigating the pathogenesis of human HSAs. To characterize the cellular phenotype of canine HSAs, we evaluated immunoreactivity and/or messenger RNA (mRNA) expression of markers for hematopoietic stem cells (HSCs), endothelial cells (ECs), a tumor suppressor protein, and a myeloid marker in canine HSAs. Neoplastic canine cells expressed EC markers and a myeloid marker, but expressed HSC markers less consistently. The canine tumor expression results were then compared to previously published immunoreactivity results for these markers in human and mouse HSAs. There are 2 noteworthy differences across species: (1) most human HSAs had HSC marker expression, indicating that they were comprised of tumor cells that were less differentiated than those in canine and mouse tumors; and (2) human and canine HSAs expressed a late-stage EC maturation marker, whereas mouse HSAs were negative, suggesting that human and canine tumors may retain greater differentiation potential than mouse tumors. These results indicate that HSA development is variable across species and that caution is necessary when discussing translation of carcinogenic risk from animal models to humans.

Abstract 5357: Mutational and transcriptomic profiling identify distinct angiogenic and inflammatory subtypes of angiosarcoma

Abstract Angiosarcoma is an aggressive, albeit rare cancer in humans. The cause of the vast majority of sporadic angiosarcomas is unknown, mortality is high, and no therapeutic targets have been identified to improve outcomes. Hemangiosarcoma (HSA) is a common cancer of dogs, and it shares histopathologic features with human angiosarcoma. In our previous work, canine HSAs were classified into angiogenic, inflammatory, and adipogenic subtypes based on transcriptional profiles. However, the genetic and molecular events that regulate transcriptional subtypes in angiosarcoma are not currently understood. Our goal was to use a comparative genomics approach to apply knowledge from appropriately powered canine studies to inform our research into human sarcomas. In this study, we identified recurrent mutations in RNASeq data from 93 HSAs and 16 nonmalignant controls, based on mutations first identified in exomes from 42 paired tumor and normal samples. In addition to identifying recurrent somatic mutations we also identified translocation fusions, allowing elucidation of oncogenic mechanisms for vascular endothelial growth factor receptors (VEGFR), phosphoinositide-3 kinase (PIK3) signaling pathways, and the p53 DNA damage repair pathway in canine HSA. Significantly, mutational signatures were associated with distinct molecular subtypes of canine hemangiosarcomas, and both the angiogenic and the inflammatory subtypes were apparent in RNASeq data from human angiosarcomas (n=14), suggesting that comparable etiologic mechanisms are operative in the canine and human disease. Our ongoing work seeks to understand how the molecular mechanisms give rise to molecular subtypes of angiosarcoma by defining the association between driver mutations, signaling pathway alterations and transcriptional patterns, which should allow us to identify rational therapeutic targets. Citation Format: Jong Hyuk Kim, Kate Megquier, Aaron L. Sarver, Rachael Thomas, Chao Wang, Ingegerd Elvers, Elinor Karlsson, Matthew Breen, Kerstin Lindblad-Toh, Jaime F. Modiano. Mutational and transcriptomic profiling identify distinct angiogenic and inflammatory subtypes of angiosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5357.

Identification of the two KIT isoforms and their expression status in canine hemangiosarcomas.

BackgroundKIT is a tyrosine kinase growth factor receptor. High expression of KIT has been found in several tumors including canine hemangiosarcoma (HSA). This study investigated the correlation of KIT expression and c-kit sequence mutations in canine HSAs and benign hemangiomas (HAs).ResultsImmunohistochemistry (IHC) staining confirmed KIT expression in 94.4 % (34/36) of HSAs that was significantly higher than 0 % in HAs (0/16). Sequencing the entire c-kit coding region of HSAs and normal canine cerebellums (NCCs) revealed GNSK-deletion in exon 9. As for exon 9 genotyping by TA-cloning strategy, GNSK-deletion c-kit accounted for 48.6 % (68/140) colonies amplified from12 KIT-positive HSAs, a significantly higher frequency than 14.1 % (9/64) of colonies amplified from six NCCs.ConclusionsDue to the distinct expression pattern revealed by IHC, KIT might be used to distinguish benign or malignant vascular endothelial tumors. Moreover, the high incidence of GNSK-deletion c-kit in canine HSAs implicates KIT isoforms as possibly participating in the tumorigenesis of canine HSAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0772-y) contains supplementary material, which is available to authorized users.

Beta Adrenergic Signaling: A Targetable Regulator of Angiosarcoma and Hemangiosarcoma.

Human angiosarcomas and canine hemangiosarcomas are highly aggressive cancers thought to arise from cells of vascular origin. The pathological features, morphological organization, and clinical behavior of canine hemangiosarcomas are virtually indistinct from those of human angiosarcomas. Overall survival with current standard-of-care approaches remains dismal for both humans and dogs, and each is likely to succumb to their disease within a short duration. While angiosarcomas in humans are extremely rare, limiting their study and treatment options, canine hemangiosarcomas occur frequently. Therefore, studies of these sarcomas in dogs can be used to advance treatment approaches for both patient groups. Emerging data suggest that angiosarcomas and hemangiosarcomas utilize beta adrenergic signaling to drive their progression by regulating the tumor cell niche and fine-tuning cellular responses within the tumor microenvironment. These discoveries indicate that inhibition of beta adrenergic signaling could serve as an Achilles heel for these tumors and emphasize the need to design therapeutic strategies that target tumor cell and stromal cell constituents. In this review, we summarize recent discoveries and present new hypotheses regarding the roles of beta adrenergic signaling in angiosarcomas and hemangiosarcomas. Because the use of beta adrenergic receptor antagonists is well established in human and veterinary medicine, beta blockade could provide an immediate adjunct therapy for treatment along with a tangible opportunity to improve upon the outcomes of both humans and dogs with these diseases.

Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas.

Evaluation of Mast Cells in Dermal versus Subcutaneous Hemangiomas and Hemangiosarcomas in Dogs

This study investigated the role of mast cells in canine cutaneous vascular tumors, and is the first such study to distinguish between tumors arising in the dermis versus the subcutis. Mast cell numbers in canine cutaneous hemangiomas (HA) and hemangiosarcomas (HSA) were evaluated to identify a relationship between mast cells, tumor type (HA, HSA), histologic location (dermis, subcutis) and tumor recurrence. One hundred and sixty-seven biopsies from 148 dogs were evaluated. Using only one biopsy from each dog, mast cell counts (MCC) for each tumor (n = 148) were obtained by averaging the number of mast cells counted in ten 400× fields. A significant difference in mean MCC was found only between tumor types, with HA having more mast cells than HSA (4.2 ± 4.2 vs. 2.2 ± 2.6; p < 0.001). No significant difference in mean MCC existed between tumors that recurred and those that did not. There was no difference in recurrence rate between tumor type or histologic location. Our results indicate that benign HA contain more mast cells than malignant endothelial cell tumors, regardless of histologic location; whether this is a cause or effect relationship remains to be determined.

Claudin-5 protein is a new differential marker for histopathological differential diagnosis of canine hemangiosarcoma.

Claudin-5 protein is an endothel-specific claudin, present in tight junctions. To evaluate its usefulness as a differential diagnostic marker of canine hemangiosarcomas, the expression of claudin-5 molecule was studied in different canine tumours of vascular origin. Ninety two canine neoplastic tissue samples obtained from necropsies and biopsy specimens were routinely processed and stained immunhistochemically for claudin-5. The neoplastic endothelial cells of canine hemangiosarcomas, hemangiomas, and lymphangiomas showed a strong membrane immunoreactivity for claudin-5, but the other investigated canine malignant and benign tumours, including fibrosarcomas, myxo-, leiomyo-, cardiac rhabdomyo-, neurofibro-, synovial-, osteo-, and chondrosarcomas, spindle cell melanomas, hemangio-pericytomas, benign fibroblast proliferations, and leiomyomas were negative for this endothelial marker. In these non-vascular canine tumours intense immunostaining was detected in the endothelial cells of the incorporated intratumoural vessels and neovasculature. The canine splenic hematomas induced by hemangiosarcomas were distinguished from splenic hematomas induced by non-neoplastic lesions by the means of claudin-5 protein. In hemangiosarcomas the percentage of positive neoplastic endothelial cells was higher, and stronger when using the claudin-5 molecule compared to CD31 and vWf. The results show that claudin-5 molecule can be used as a new differential marker, and could also be of a diagnostic value in the differential diagnosis of canine hemangiosarcomas from sarcomas of other origin with hemorrhages or increased vascularization. Claudin-5 could help to reveal neoplastic proliferation of endothelial cells causing splenic hematomas and differentiate these tumours from non-vascular neoplastic splenic lesion. The immunohistochemical detection of the claudin-5 protein had a higher sensitivity than CD31, and vWf antigen in case of canine hemangiosarcomas.

Activation of matrix metalloproteinase (MMP)-2 by membrane type 1-MMP and abnormal immunolocalization of the basement membrane components laminin and type IV collagen in canine spontaneous hemangiosarcomas

We performed immunohistochemical investigation of the basement membrane (BM) components, namely, type IV collagen and laminin, in 83 canine hemangiosarcomas (HSAs), 22 hemangiomas, and some granulation tissues (GTs). Additionally, we analyzed the expression and activities of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP (MT1-MMP) using the same samples by immunohistochemistry and gelatin zymography to investigate whether MMPs were associated with the BM degradation. In immunohistochemistry for the BM components, many HSAs showed discontinuous linear/negative immunoreactivity in the BM (type IV collagen: 49.4%/14.5%, laminin: 60.3%/10.8%, respectively). In contrast, almost all hemangiomas showed continuous staining in the BM (type IV collagen: 90.9%, laminin: 95.5%, respectively). Interestingly, positive cytoplasmic immunoreactivity for type IV collagen and laminin was observed in 97.6% and 91.6% HSA, respectively. Although MMP-9 immunoreactivity wasn't detected in neoplastic and active angiogenic endothelial cells (ECs), MMP-2 was detected in all ECs of GTs and in neoplastic cells of both vascular tumors. A strong immunoreactivity for MT1-MMP was observed in active angiogenic ECs in GTs and in neoplastic ECs in HSAs. However, almost all hemangiomas showed weak/negative immunoreactivity. In gelatin zymography, significantly strong activity of active MMP-2 was observed in HSAs, similar to that in active angiogenesis in GTs; however, weak/no activity of active MMP-2 was detected in hemangiomas. In canine HSA, neoplastic cells had active MMP-2, possibly activated by MT1-MMP, and discontinuous status of BM might be associated with activity of active MMP-2.

Immunohistochemical analysis of ocular hemangiomas and hemangiosarcomas in dogs

To determine if molecular markers typically associated with ultraviolet exposure could be detected in canine ocular hemangiomas (HA) and hemangiosarcomas (HSA). Paraffin-embedded samples of canine ocular HA (n = 6) and HSA (n = 6) were examined for the presence of p53, p21, p16, cyclin D, PCNA, pAkt, telomerase, and estrogen receptor (ER)-alpha using immunohistochemistry. p53 and cyclin D protein were not detected in any of the canine HA or HSA samples. The majority of the HA and HSA were negative for both p21 and telomerase. pAkt immunoreactivity was absent in one HA, one HSA, but was present in five HA and five HSA. All of the HA or HSA samples were strongly positive for p16 and PCNA. ERalpha was expressed in all of the samples examined; there was more intense staining in the HSA samples compared to the HA samples. Results from this study describe the protein expression, via immunohistochemistry, that might be altered in UV exposure in HA and HAS formation. p53 may not play an important role in tumor development; rather, in the tumors examined, expression of cell cycle regulators independent of the p53 pathway appear central in HA and HSA formation and progression. In addition, this study finds that ERalpha may be involved in promoting the invasive behavior associated with HSA.

Phenotypic Characterization of Normal and Neoplastic Canine Endothelial Cells by Lectin Histochemistry

Cell surface glycoconjugate expression of endothelial cells in canine cutaneous hemangiomas and hemangiosarcomas was compared to normal cutaneous endothelial cells using eight different lectins (with and without neuraminidase pretreatment) in an indirect immunoperoxidase technique. Direct comparison of lectin binding pattern of neoplastic endothelial cells with adjacent normal endothelial cells revealed minor changes in the binding intensity of several lectins (enhanced: Wheat germ agglutinin [WGA]; reduced: Griffonia simplicifolia-I [GS-I], Ricinus communis agglutinin-I [RCA-I], Soybean agglutinin after neuraminidase pretreatment [Neu-SBA], and Wheat germ agglutinin after neuraminidase treatment [Neu-WGA]). Neoplastic endothelial cells in some tumors exhibited varying binding of Ulex europaeus agglutinin-I (UEA-I; not binding to normal canine endothelial cells) and no Soybean agglutinin (SBA) binding (variably binding to normal endothelial cells in small cutaneous vessels). Lectin binding of neoplastic cells was rather heterogenous within one tumor compared to the uniform binding pattern of normal endothelial cells. These lectin binding studies demonstrate the phenotypic heterogeneity of neoplastic endothelial cells, indicating changes of cell surface glycosylation during neoplastic transformation.