Human Angiosarcoma Research Articles

Lysosomal drug sequestration as a mechanism of drug resistance in vascular sarcoma cells marked by high CSF-1R expression.

BackgroundHuman angiosarcoma and canine hemangiosarcoma are thought to arise from vascular tissue or vascular forming cells based upon their histological appearance. However, recent evidence indicates a hematopoietic or angioblastic cell of origin for these tumors. In support of this idea, we previously identified an endothelial-myeloid progenitor cell population with high expression of endothelial cell markers and the myeloid cell marker, colony stimulating factor 1 receptor (CSF-1R). Here, we further characterized these cells to better understand how their cellular characteristics may impact current therapeutic applications.MethodsWe performed cell enrichment studies from canine hemangiosarcoma and human angiosarcoma cell lines to generate cell populations with high or low CSF-1R expression. We then utilized flow cytometry, side population and cell viability assays, and fluorescence based approaches to elucidate drug resistance mechanisms and to determine the expression of hematopoietic and endothelial progenitor cell markers.ResultsWe demonstrated that cells with high CSF-1R expression enriched from hemangiosarcoma and angiosarcoma cell lines are more drug resistant than cells with little or no CSF-1R expression. We determined that the increased drug resistance may be due to increased ABC transporter expression in hemangiosarcoma and increased drug sequestration within cellular lysosomes in both hemangiosarcoma and angiosarcoma.ConclusionsWe identified drug sequestration within cellular lysosomes as a shared drug resistance mechanism in human and canine vascular sarcomas marked by high CSF-1R expression. Taken together, our results demonstrate that studies in highly prevalent canine hemangiosarcoma may be especially relevant to understanding and addressing drug resistance mechanisms in both the canine and human forms of this disease.

Vascular-targeted agents for the treatment of angiosarcoma

Angiosarcomas are rare, aggressive vascular tumours known to express vascular endothelial growth factor (VEGF), a key pro-angiogenic growth factor. The aim of this study was to determine the potential effects of vascular-targeted agents for the treatment of angiosarcoma, using two human cutaneous angiosarcoma cell lines (ASM and ISO-HAS), and human dermal microvascular endothelial cells (HuDMECs) for comparison. Protein arrays were used to assess the expression of angiogenesis-related proteins, and potential drug targets were assessed by ELISA and Western blotting. Response to vascular-targeted agents, including bevacizumab an anti-VEGF antibody, axitinib a VEGF-receptor tyrosine kinase inhibitor, everolimus an mTOR inhibitor, selumetinib a MEK inhibitor and vadimezan a vascular-disrupting agent were compared in functional in vitro cellular assays, including viability, differentiation and migration assays. ASM and ISO-HAS cells expressed a broad range of pro-angiogenic growth factors. ASM and ISO-HAS VEGF expression was significantly increased (p=0.029) compared with HuDMECs. Striking responses were seen to vadimezan with an IC50 of 90 and 150μg/ml for ASM and ISO-HAS cells, respectively. Selumetinib inhibited ASM with an IC50 of 1,750ng/ml, but was not effective in ISO-HAS. Everolimus reduced both ASM and ISO-HAS viable cell counts by 20% (p<0.001). Minimal responses were observed to bevacizumab and axitinib in assays with ASM and ISO-HAS cells. Further studies are warranted to investigate mTOR inhibitors, MEK inhibitors and vascular-disrupting agents for the treatment of angiosarcoma.

Pharmacologic Inhibition of MEK Signaling Prevents Growth of Canine Hemangiosarcoma

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal-regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease.

Protooncogene TCL1b functions as an Akt kinase co-activator that exhibits oncogenic potency in vivo

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human neoplastic diseases.

Expression of angiopoietin-TIE system components in angiosarcoma

Angiosarcoma is an aggressive malignancy of endothelial differentiation. Potential roles of the endothelial angiopoietin-tunica interna endothelial cell kinase (ANGPT-TIE) system in angiosarcoma diagnosis, pathogenesis, prognosis and treatment are undefined. To examine the expression and prognostic significance of angiopoietin-1, angiopoietin-2, TIE1 and TEK (TIE2) proteins in angiosarcoma, we immunohistochemically evaluated clinically annotated human angiosarcoma samples. Correlations of protein expression with overall survival and pathological features were explored. The cohort included 51 patients diagnosed with angiosarcoma at the age of 30–86 years (median 67). The 5-year overall survival was 45% with a median of 26 months. Moderate to strong expression of angiopoietin-1, TIE1 and TEK (TIE2) was identified in the majority of angiosarcomas and moderate to strong expression of angiopoietin-2 was observed in 42% of angiosarcomas. Increased angiopoietin-1 expression correlated with improved survival. Non-significant trends toward longer survival were also observed with increased TIE1 and TEK (TIE2) expression. Increased expression of angiopoietin-2, TIE1 and TEK (TIE2) was associated with vasoformative architecture. No differences in expression of these proteins were observed when patients were segregated by age, gender, presence or absence of metastases at diagnosis, primary tumor location, radiation association or the presence of necrosis. We conclude that components of the ANGPT-TIE system are commonly expressed in angiosarcomas. Reduced expression of these proteins is associated with non-vasoformative and clinically more aggressive lesions.

A Novel Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits Tumor Growth

Secreted frizzled-related protein 2 (SFRP2) is overexpressed in human angiosarcoma and breast cancer and stimulates angiogenesis via activation of the calcineurin/NFATc3 pathway. There are conflicting reports in the literature as to whether SFRP2 is an antagonist or agonist of β-catenin. The aims of these studies were to assess the effects of SFRP2 antagonism on tumor growth and Wnt-signaling and to evaluate whether SFRP2 is a viable therapeutic target. The antiangiogenic and antitumor properties of SFRP2 monoclonal antibody (mAb) were assessed using in vitro proliferation, migration, tube formation assays, and in vivo angiosarcoma and triple-negative breast cancer models. Wnt-signaling was assessed in endothelial and tumor cells treated with SFRP2 mAb using Western blotting. Pharmacokinetic and biodistribution data were generated in tumor-bearing and nontumor-bearing mice. SFRP2 mAb was shown to induce antitumor and antiangiogenic effects in vitro and inhibit activation of β-catenin and nuclear factor of activated T-cells c3 (NFATc3) in endothelial and tumor cells. Treatment of SVR angiosarcoma allografts in nude mice with the SFRP2 mAb decreased tumor volume by 58% compared with control (P = 0.004). Treatment of MDA-MB-231 breast carcinoma xenografts with SFRP2 mAb decreased tumor volume by 52% (P = 0.03) compared with control, whereas bevacizumab did not significantly reduce tumor volume. Pharmacokinetic studies show the antibody is long circulating in the blood and preferentially accumulates in SFRP2-positive tumors. In conclusion, antagonizing SFRP2 inhibits activation of β-catenin and NFATc3 in endothelial and tumor cells and is a novel therapeutic approach for inhibiting angiosarcoma and triple-negative breast cancer.

Abstract 2499: Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma.

Abstract People suffering from uncommon diseases often do not benefit from the advances in medical research due to lack of research. One such disease is angiosarcoma, a highly aggressive cancer originating from blood vessels with risk factors including toxic exposure, radiotherapy and lymphedema. Angiosarcoma in dogs (commonly called hemangiosarcoma) is a very similar disease with much higher prevalence, comprising up to 7% of all canine malignancies. For both species, an accurate diagnostic method is needed for more favorable outcome. We developed a biomarker discovery pipeline consisting of a high-throughput biomarker discovery platform and a database for data mining and statistical analysis for candidate identification. The platform screens for differential glycosylation of serum glycoproteins as potential biomarkers utilizing 20 individual lectins followed by direct coupling to tandem mass spectrometry. The database facilitates the identification of differential glycosylation and produces a ranked list of potential biomarker candidates. Using this pipeline, sera from 10 dogs with hemangiosarcoma with 10 age and sex-matched control sera were processed and analyzed. Candidates from the database were ranked based on its ability to classify normal and cancer. The ranked list of candidates for this screen composed of glycoproteins involved in cell migration, tumor immune response, and glycoproteins reported to be altered in human and canine angiosarcoma and other cancer types. Some examples will be shown to illustrate the change in specific glycosylation structures associated with cancer as measured by binding to specific lectin(s) while binding to other lectins are not changed. These glycosylation-specific changes are potentially more specific diagnostic biomarkers than circulating proteins alone. We are currently validating the identified candidates and pursuing the development of clinical tests to improve diagnosis and clinical outcomes of angiosarcoma for both dogs and humans. Citation Format: Eunju Choi, David Chen, Kim-Anh Lê Cao, Helle Bielefeldt-Ohmann, Caroline A. O'Leary, Michelle M. Hill. Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2013-2499

Imatinib and Dasatinib Inhibit Hemangiosarcoma and Implicate PDGFR-β and Src in Tumor Growth

Hemangiosarcoma, a natural model of human angiosarcoma, is an aggressive vascular tumor diagnosed commonly in dogs. The documented expression of several receptor tyrosine kinases (RTKs) by these tumors makes them attractive targets for therapeutic intervention using tyrosine kinase inhibitors (TKIs). However, we possess limited knowledge of the effects of TKIs on hemangiosarcoma as well as other soft tissue sarcomas. We report here on the use of the TKIs imatinib and dasatinib in canine hemangiosarcoma and their effects on platelet-derived growth factor receptor β (PDGFR-β) and Src inhibition. Both TKIs reduced cell viability, but dasatinib was markedly more potent in this regard, mediating cytotoxic effects orders of magnitude greater than imatinib. Dasatinib also inhibited the phosphorylation of the shared PDGFR-β target at a concentration approximately 1000 times less than that needed by imatinib and effectively blocked Src phosphorylation. Both inhibitors augmented the response to doxorubicin, suggesting that clinical responses likely will be improved using both drugs in combination; however, dasatinib was significantly (P < .05) more effective in this context. Despite the higher concentrations needed in cell-based assays, imatinib significantly inhibited tumor growth (P < .05) in a tumor xenograft model, highlighting that disruption of PDGFR-β/PDGF signaling may be important in targeting the angiogenic nature of these tumors. Treatment of a dog with spontaneously occurring hemangiosarcoma established that clinically achievable doses of dasatinib may be realized in dogs and provides a means to investigate the effect of TKIs on soft tissue sarcomas in a large animal model.

Establishment of a novel experimental model of human angiosarcoma and a VEGF-targeting therapeutic experiment

BackgroundAngiosarcoma is one of the most life-threatening neoplasms with strong resistance to conventional chemotherapy/radiotherapy; consequently, alternative therapeutic agents are urgently required. One factor in delaying the therapy development is the limitation of experimental models. ObjectiveWe established a novel experimental angiosarcoma model. MethodsFrom surgically resected tissue, human AS cell line was established. Using xenograft of AS cell line, we performed therapeutic experiments with the anti-human VEGF Ab or the receptor tyrosine kinase inhibitor. ResultsFirst we generated an angiosarcoma cell line, HAMON (human angiosarcoma, monoclonal), which expresses CD31 and produces tumors in immunodeficient mice. HAMON expresses VEGFR2 and that exogenous VEGF leads to HAMON proliferation in vitro. Anti-human VEGF Ab bevacizumab treatment failed to suppress HAMON proliferation in vitro and in vivo. Furthermore, the receptor tyrosine kinase inhibitor sunitinib did not suppress HAMON proliferation in vitro. Similarly, in in vivo therapeutic experiments, even high doses of sunitinib failed to inhibit tumor growth. Finally, we checked whether compensatory activation of VEGF signaling occurred after sunitinib addition. VEGF protein secretion, VEGF mRNA synthesis and VEGFR2 phosphorylation all were unaffected in HAMON after sunitinib treatment. ConclusionA novel in vitro and in vivo experimental model of human angiosarcoma has been successfully established. With this model, we were able to perform therapeutic experiments. In addition, our angiosarcoma cell line, HAMON, is quite useful for identifying key molecules in angiosarcoma.

Ikk4a/Arf Inactivation with Activation of the NF-κB/IL-6 Pathway Is Sufficient to Drive the Development and Growth of Angiosarcoma

Although human angiosarcoma has been associated frequently with mutational inactivation of the tumor suppressor gene Ink4a/Arf, the underlying mechanisms have not been delineated. Here we report that malignant angiosarcoma is associated with high levels of RelA/NF-κB and IL-6 in contrast to normal vessels or benign hemagiomas. Studies of Ink4a/Arf deficient mice not only recapitulate genetic traits observed in human angiosarcoma, but also unveil a possible therapeutic link comprised of the NF-kB/IL-6/Stat3 signaling axis. In Ink4a/Arf(-/-) cells, NF-κB controlled Stat3 signaling by transcriptionally controlling the expression of IL-6, gp130, and Jak2. Further, IL-6 mediated Stat3 signaling through the sIL-6R. Inhibition of Ikkβ solely in myeloid cells was insufficient to block angiosarcoma development; in contrast, systemic inhibition of Ikkβ, IL-6, or Stat3 markedly inhibited angiosarcoma growth. Our findings offer clinical implications for targeting the NF-kB/IL-6/STAT3 pathway as a rational strategy to treat angiosarcoma.

Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression

Ochratoxin A (OTA) is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis.

Efficacy of Tie2 Receptor Antagonism in Angiosarcoma

Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease.

The antioxidant role of Prdx6 on acute lung injury

Canine haemangiosarcoma (HSA), like human angiosarcoma, is an uncommon malignant vascular endothelial cell tumour associated with a poor prognosis. The peroxiredoxin (PRDX) family of peroxidases, which comprises six members in mammals (PRDX1–6), might contribute to cancer cell survival in the face of oxidative stress as these proteins exhibit frequent upregulation in cancer cells. In this study, we investigated the expression levels of PRDX6 in spontaneously arising primary canine HSAs by immunohistochemical analysis, identifying marked expression of this protein. Both PRDX6 mRNA and protein were overexpressed in HSA cell lines compared with normal canine endothelial cells, although some variation was observed between the different HSA cell lines. Small interfering RNA-induced downregulation of PRDX6 promoted apoptosis in the HSA cell lines. The observation that PRDX6 suppression increased the cytotoxicity of these cells suggests that PRDX6 might play an important cytoprotective role.

Abstract 2929: MAP/MEK pathway inhibitors reduce cell viability of canine hemangiosarcoma primary cells

Abstract Angiosarcoma is a rare endothelial derived soft-tissue tumor with an estimated incidence of 0.2/100,000 persons/year. Current treatment regimes include surgical resection followed by chemotherapy. Even after treatment, reoccurrence is likely and usually fatal. The canine equivalent of angiosarcoma, hemangiosarcoma (HSA), is a relatively common canine endothelial neoplasm with an overall incidence of 24/100,000. In addition, canine HSA is more prevalent in specific breeds suggesting a genetic component. Similar to human angiosarcoma, HSA present as two basic forms: visceral (spleen or cardiac) and dermal. Furthermore, canine HSA occur spontaneously making canine HSA a relevant model system to study human angiosarcoma. Interestingly, MEK/MAPK signaling cascade is activated in Kaposi's Sarcoma, another human endothelial derived cancer. We hypothesize that the MEK/MAPK signaling cascade is vital for HSA and angiosarcoma growth and survival. We performed expression analysis supplemented by small molecule inhibitor studies utilizing canine primary cell isolates derived from visceral and dermal HSA. MAPK pathway expression signatures are elevated in HSA primary isolates compared to proliferating endothelial cells. Consistent with mRNA expression studies, ERK2 is constitutively phosphorylated in serum starved conditions. In addition, HSA cell viability is reduced by the MEK1/2 inhibitor CI-1040 compared to isolated canine endothelial cells. Other inhibitors targeting tyrosine kinase receptors (Sorafenib, EGFR Inhibitor, and SU11652) also decrease HSA cell viability. We conclude a network of tyrosine kinase receptors constitutively activate the MEK/MAPK signaling pathway in HSA. This work provides insights in the etiology and therapeutic potential for targeting both canine HSA and angiosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2929. doi:10.1158/1538-7445.AM2011-2929

Sporadic cutaneous angiosarcomas generally lack hypoxia-inducible factor 1α: a histologic and immunohistochemical study of 45 cases

Cutaneous angiosarcoma (AS) is a rare malignant neoplasm of dermis composed of infiltrating cells of endothelial phenotype with overall poor prognosis. Although autocrine stimulation by vascular endothelial growth factor secretion may play a role in the pathogenesis of angiosarcoma, its mechanism has not been fully established. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to hypoxia.. The stability of HIF can regulate key proteins in angiogenesis and the alpha-subunit has been found in epithelial tumors, only 1 case of human retroperitoneal angiosarcoma, and rare vascular proliferations and tumors in knockout mice. We wanted to observe the utility of HIF-1alpha as a marker or explanatory factor in AS. Cases coded as "angiosarcoma" of dermis were culled and re-reviewed for inclusion as AS, based on patient folder, slides, and obtained immunohistochemistry including CD31 and smooth muscle actin (SMA). Hypoxia-inducible factor-1alpha was performed on a subset of cases, with additional available material. Forty-five cases met the criteria for AS; there were 17% females and 83% males, with a mean age at presentation of 67 years (range, 27-88 years). Tumors presented most commonly in the skin of the scalp followed by the left lower leg, face, nose, lower arm, neck, thigh, eyelid, ear, and temple. Associated basal cell carcinoma was noted in 1 patient; no others had other neoplasms or unrelated surgeries. There was no history of other primary, lymphedema, radiation, breast-associated, or thorotrast-induced angiosarcoma. The tumors ranged in size from 0.4 up to 9.5 cm, with a mean size of 2.4 cm. Histopathologically, most tumors were vasoformative, with either solid architecture (n = 35) or papillary endothelial hyperplasia-like foci (n = 7). All cases demonstrated infiltrative growth pattern, cytologic atypia, and mitotic activity, including atypical forms. Surface ulceration was present in 44% and solar elastosis in the most evaluable cases. Epithelioid morphology was present in 29% (n = 13) cases. Mild to moderate lymphocytic inflammatory response was noted in 62% (n = 28) cases. CD31 highlighted malignant endothelial cells. SMA (for pericytes) was generally absent. Hypoxia-inducible factor 1alpha was focally positive in cytoplasm of 3 of 18 (17%) cases studied. Treatment and follow-up data were only available on 4 cases: 2 died of disease within 4 years, 2 others had known recurrence within 2 years. Cutaneous angiosarcoma is largely found on the scalp of older individuals. Requirement for diagnosis includes extravascular proliferation of atypical endothelial cells with mitotic activity in vasoformative, solid, and papillary patterns. Absence of SMA can prove extravascular extension of tumor, outside their normal vessel confines. Cutaneous angiosarcoma generally lacks HIF-1alpha expression. Accordingly, the hypoxic response pathway is not thought to be a documentable common mechanism of angiogenesis in this entity.

Identification of an aberrant cell line among human adipose tissue-derived stem cell isolates

Adipose tissue-derived stem cells (ADSC) are isolated from the stromal vascular fraction (SVF) of adipose tissue and considered an excellent cell source for regenerative medicine. During the isolation and propagation of several human ADSC cell lines, we observed the emergence of an unusual cell line designated HADSC-6. Although initially fibroblast-like as typical ADSC are, HADSC-6 cells became homogeneously cuboid in shape, had very little cytoplasm, and formed aggregates with capsule-like boundary. Proliferation assay showed that HADSC-6 grew much faster than typical HADSC cell lines, such as HADSC-20. Immunocytochemistry showed that HADSC-6 did not express endothelial markers CD31 and vWF, and matrigel tube formation assay showed that it was unable to form endothelial-like tube structures. However, LDL uptake, a reliable endothelial marker, was positively identified. Chromosomal analysis showed that HADSC-6 cells were hypertriploid, and soft agar colony formation assay showed that they were able to proliferate and form large colonies in an anchorage-independent manner. However, tumorigenicity test showed that HADSC-6 was unable to form tumors in athymic mice. RT-PCR analysis showed that both HADSC-6 and HADSC-20 expressed VEGF-A, VEGF-B, VEGF-D, and VEGFR1 but not VEGFR2 or VEGFR3. VEGF-C, however, was expressed at a high level in HADSC-20 but undetectable in HADSC-6. In the IGF system, IGF-1 was abundantly expressed in HADSC-20 but marginally detectable in HADSC-6, and IGF-1R was abundantly expressed in HADSC-6 but not detectable in HADSC-20. In the FGF system, bFGF was abundantly expressed in HADSC-20 but marginally detectable in HADSC-6, and FGFR1 was abundantly expressed in both. Taken together, these results suggested that HADSC-6 cells were spontaneously transformed from the endothelium; therefore, they were further compared to previously published data of four naturally occurring human angiosarcoma cell lines. The results showed that the established angiosarcoma cell lines exhibit considerable variations among themselves and HADSC-6 displayed most of these variable characteristics.

Interaction of sunitinib and paclitaxel on inhibition of human angiosarcomas

14629 Background: Angiosarcoma (AS) is a malignancy of vascular endothelial cells. AS respond to cytotoxic chemotherapy and in particular taxanes such as paclitaxel. However tumor resistance is a m...

Differential Expression of Decorin by Human Malignant and Benign Vascular Tumors

An increasing amount of evidence indicates that a small extracellular chondroitin/dermatan sulfate proteoglycan, decorin, is indirectly involved in angiogenesis. Given that angiogenesis is a sine qua non for tumor growth and progression, we attempted to examine whether human malignant vascular tumors differ from human benign vascular tumors in terms of their decorin expression and synthesis. CD31 immunostaining demonstrated that the human malignant vascular tumors Kaposi's sarcoma and angiosarcoma were filled with capillary-like structures, whereas in benign cavernous and capillary hemangiomas, blood vessels were not as abundantly present. By utilizing in situ hybridization and immunocytochemical assays for decorin, we showed that there was no detectable decorin mRNA expression or immunoreactivity within the tumor mass in the Kaposi's sarcoma or angiosarcoma group. Instead, decorin was expressed in the connective tissue stroma lining the sarcoma tissue. In contrast to sarcomas, in hemangiomas, decorin mRNA expression and immunoreactivity were observed also within the tumor mass, particularly in the connective tissue stroma surrounding the clusters of intratumoral blood vessels. Finally, distribution of type I collagen was found to be similar to that of decorin in these tumor tissues. Our findings can be explained with different states of angiogenesis in dissimilar growths. In sarcomas, angiogenesis is extremely powerful, whereas in hemangiomas, angiogenesis has ceased. Thus, decorin is likely to possess a suppressive effect on human tumor angiogenesis in vivo, as previously described by studies using different experimental models. Decorin certainly provides a usable biomarker for distinguishing between benign and malignant vascular tumors in patients.

Mutation of the PTEN gene in a human hepatic angiosarcoma

The phosphatase and tensin homolog (mutated in multiple advanced cancers 1) gene, or PTEN, encodes a lipid phosphatase that contains a PTPase domain and a C2 domain and plays a role as a tumor suppressor that negatively regulates the cell-survival signaling pathway initiated by phosphatidylinositol 3-kinase (PI3K). The PTEN protein inhibits angiogenesis, and somatic mutations of the PTEN gene are involved in canine hemangiosarcoma. We screened for mutations of the PTEN gene in two patients with human hepatic angiosarcoma to determine whether PTEN is involved in the pathogenesis of human hepatic angiosarcoma. In one patient, who suffered from breast cancer, pharyngeal cancer, and hepatic angiosarcoma, we found a single base substitution in exon 7 (640C>T) of the PTEN gene in both the hepatic angiosarcoma and normal tissues. This transition results in a germline nonsense mutation (Q214X). These findings indicate that analysis of PTEN gene mutations may be useful for characterization of the molecular event in hepatic angiosarcoma and cancer predisposition.

Galectin-3 as a Potential Therapeutic Target in Tumors Arising from Malignant Endothelia

Angiosarcoma (ASA) in humans, hemangiosarcoma (HSA) in dogs are deadly neoplastic diseases characterized by an aggressive growth of malignant cells with endothelial phenotype, widespread metastasis, poor response to chemotherapy. Galectin-3 (Gal-3), a p-galactoside-binding lectin implicated in tumor progression, metastasis, endothelial cell biology, angiogenesis, regulation of apoptosis, neoplastic cell response to cytotoxic drugs, has not been studied before in tumors arising from malignant endothelia. Here, we tested the hypothesis that Gal-3 could be widely expressed in human ASA, canine HSA, could play an important role in malignant endothelial cell biology. Immunohistochemical analysis demonstrated that 100% of the human ASA (10 of 10), canine HSA (17 of 17) samples analyzed expressed Gal-3. Two carbohydrate-based Gal-3 inhibitors, modified citrus pectin (MCP), lactulosyl-l-leucine (LL), caused a dose-dependent reduction of SVR murine ASA cell clonogenic survival through the inhibition of Gal-3 antiapoptotic function. Furthermore, both MCP, LL sensitized SVR cells to the cytotoxic drug doxorubicin to a degree sufficient to reduce the in vitro IC50 of doxorubicin by 10.7-fold, 3.64old, respectively. These results highlight the important role of Gal-3 in the biology of ASA, identify Gal-3 as a potential therapeutic target in tumors arising from malignant endothelial cells.