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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., Significance: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Background: Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis., Methods: Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis., Results: We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth., Conclusions: These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers., (© 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Angiosarcoma is a rare cancer of blood vessel-forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro , and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(-) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saha, Kim, Amaya, Witcher, Khammanivong, Korpela, Brown, Taylor, Bryan and Dickerson.)

Symptoms of depression are present in over half of all cancer patients, and selective serotonin reuptake inhibitor (SSRI) anti-depressant medications are prescribed to nearly a quarter of these individuals in order to cope with their disease. Previous studies have provided evidence that elevated serotonin (5-HT) and serotonin receptor levels may contribute to oncogenic progression, yet little is known regarding the mechanism by which this occurs. The data demonstrated that serotonin receptor mRNAs and proteins are expressed across diverse cancer types, and that serotonin stimulation of tumor cells activates oncogenic signaling mediators including components of the AKT, CREB, GSK3, and MAPK pathways. Selective pharmacological inhibition of the seven known classes of 5-HT receptors in sarcoma and breast cancer cells resulted in dose dependent decreases in tumor cell viability, activation of the p53 DNA damage pathway, suppression of MAPK activity, and significantly reduced tumor volume in an in ovo model. Based on a retrospective clinical analysis of 419 patients diagnosed with breast cancer, we discovered that use of SSRIs was associated with a 2.3-fold increase in tumor proliferation rates for late stage patients based on their Ki-67 index (P=0.03). These data provide evidence that serotonin signaling pathways, which treating oncologists often pharmacologically target to assist cancer patients to psychologically cope with their illness, activate signaling pathways known to promote tumor growth and survival.

Patients with metastatic angiosarcoma undergoing chemotherapy, radiation, and/or surgery experience a median progression free survival of less than 6 months and a median overall survival of less than 12 months. Given the aggressive nature of this cancer, angiosarcoma clinical responses to chemotherapy or targeted therapeutics are generally very poor. Inhibition of beta adrenergic receptor (β-AR) signaling has recently been shown to decrease angiosarcoma tumor cell viability, abrogate tumor growth in mouse models, and decrease proliferation rates in preclinical and clinical settings. In the current study we used cell and animal tumor models to show that β-AR antagonism abrogates mitogenic signaling and reduces angiosarcoma tumor cell viability, and these molecular alterations translated into patient tumors. We demonstrated that non-selective β-AR antagonists are superior to selective β-AR antagonists at inhibiting angiosarcoma cell viability. A prospective analysis of non- selective β-AR antagonists in a single arm clinical study of metastatic angiosarcoma patients revealed that incorporation of either propranolol or carvedilol into patients' treatment regimens leads to a median progression free and overall survival of 9 and 36 months, respectively. These data suggest that incorporation of non-selective β-AR antagonists into existing therapies against metastatic angiosarcoma can enhance clinical outcomes.

Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of β1-AR and β3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of β-blocker usage on tumor proliferation. Our analysis revealed that non-selective β-blockers, but not selective β-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of β-blockers were measured in a panel of breast cancer lines, demonstrating that mammary epithelial cells were resistant to propranolol, and that most breast cancer cell lines displayed dose dependent viability decreases following treatment. Selective β-blockers alone or in combination were not as effective as propranolol at reducing breast cancer cell proliferation. Molecular analysis revealed that propranolol treatment of the SK-BR-3 breast cancer line, which showed high sensitivity to beta blockade, led to a reduction in Ki67 protein expression, decreased phosphorylation of the mitogenic signaling regulators p44/42 MAPK, p38 MAPK, JNK, and CREB, increased phosphorylation of the cell survival/apoptosis regulators AKT, p53, and GSK3β. In conclusion, use of non-selective β-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC.

Hemangiosarcoma (HSA) is an aggressive and common cancer in dogs. While cutaneous masses are often treatable by tumor excision, visceral tumors are almost always incurable. Treatment advances for this disease have been limited due to a poor understanding of the overall tumor biology. Based upon its histological appearance, HSA has been presumed to originate from transformed endothelial cells; however, accumulating data now suggest a pluripotent bone marrow progenitor as the cell of origin for this disease. More recently, the identification of a novel subclassification of HSAs has provided a foundation to further our understanding of the cellular characteristics of HSA tumor cells, along with those of the cells comprising the tumor microenvironment. These discoveries hold promise for the development of new approaches to improve treatments for canine HSA, as well as to establish the utility of this disease as a spontaneous model to understand the pathogenesis and develop new treatments for vascular tumors of humans. In this review, we will provide a brief historical perspective and pathobiology of canine HSA, along with a focus on the recent advances in the molecular and cellular understanding of these tumors. In addition, future directions that should continue to improve our understanding of HSA pathogenesis will be discussed.

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.

Background: Bone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC). Intracellular BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) during cellular development. However, little is known about the role or regulation of BMP signaling in HNSCC CSCs., Methods: Two CSC-like populations, CD44(high)/BMI1(high) and CD44(high)/ALDH(high), were enriched from HNSCC cell lines and evaluated for the expression of SMURF1 by qRT-PCR, flow cytometry, and immunoblotting. The activation status of BMP signaling in these populations was determined by using immunoblotting to detect phosphorylated SMAD1/5/8 (pSMAD1/5/8) levels. Knockdown of SMURF1 transcripts by RNA interference was used to assess the role of SMURF1 in BMP signaling and CSC maintenance. Loss of CSC-like phenotypes following SMURF1 knockdown was determined by changes in CD44(high) levels, cellular differentiation, and reduction in colony formation., Results: Populations of enriched CSC-like cells displayed decreased levels of pSMAD1/5/8 and BMP signaling target gene ID1 while SMURF1, CD44, and BMI1 were highly expressed when compared to non-CSC populations. Stable knockdown of SMURF1 expression in CSC-like cells increased pSMAD1/5/8 protein levels, indicating the reactivation of BMP signaling pathways. Decreased expression of SMURF1 also promoted adipogenic differentiation and reduced colony formation in a three-dimensional culture assay, indicating loss of tumorigenic capacity. The role of SMURF1 and inhibition of BMP signaling in maintaining a CSC-like population was confirmed by the loss of a CD44(high) expressing subpopulation in SMURF1 knockdown cells., Conclusions: Our findings suggest that inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs, and that this inhibition is mediated by SMURF1. Targeting SMURF1 and restoring BMP signaling may offer a new therapeutic approach to promote differentiation and reduction of CSC populations leading to reduced drug resistance and disease recurrence.

Objectives: Human METCAM/MUC18 (huMETCAM/MUC18), a cell adhesion molecule, plays an important role in the progression of several epithelial cancers; however, its role in the progression of epithelial ovarian cancers is unknown. To initiate the study we determined expression of this protein in normal and cancerous ovarian tissues, cystadenomas, metastatic lesions, and ovarian cancer cell lines., Materials and Methods: Immunoblotting and immunohistochemical (IHC) methods were used to determine huMETCAM/MUC18 expression in lysates of frozen and formalin-fixed, paraffin-embedded tissue sections of normal human ovaries, and ovarian (benign) cystadenomas, carcinomas and metastatic lesions. We also determined expression levels of several downstream effectors of METCAM/MUC18 in these tissues., Results: HuMETCAM/MUC18 levels in ovarian carcinomas and metastatic lesions were significantly higher than in normal tissues and cystadenomas. IHC results showed that expression of huMETCAM/MUC18 in normal tissues and cystadenomas was mostly absent from epithelial cells, but in carcinomas and metastatic lesions it was localized to epithelial cells. In higher pathological grades of ovarian cancer and metastatic lesions, the percentage of cells stained in IHC was increased. Thirty percent of normal tissues weakly expressed the huMETCAM/MUC18 antigen, but 70% of cancer tissues and 100% of metastatic lesions expressed the antigen. Expression levels of several downstream effectors of huMETCAM/MUC18, Bcl2, PCNA and VEGF, were elevated in cancerous tissues, however, not that of Bax. The phospho-AKT/AKT ratio was elevated in metastatic lesions., Conclusion: Upexpression of huMETCAM/MUC18 may be a marker for the malignant potential of ovarian carcinomas. Progression of ovarian cancer may involve increased signaling in anti-apoptosis, proliferation, survival/proliferation pathway, and angiogenesis., (Copyright © 2014. Published by Elsevier B.V.)

Background: Human 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., Methods: We 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., Results: We 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., Conclusions: We 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.

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., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

Background: Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics., Methods: We report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated., Results: Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not increase docetaxel sensitivity (P > 0.05)., Conclusion: This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs.

Gene expression profiles of malignant tumors surgically removed from ovarian cancer patients pre-treated with chemotherapy (neo-adjuvant) prior to surgery group into two distinct clusters. One group clusters with carcinomas from patients not pre-treated with chemotherapy prior to surgery (C-L), while the other clusters with non-malignant adenomas (A-L). We show here that although the C-L cluster is preferentially associated with p53 loss-of-function (LOF) mutations, the C-L cluster cancer patients display a more favorable clinical response to chemotherapy as evidenced by enhanced long-term survivorships. Our results support a model whereby p53 mediated cell-cycle-arrest/DNA repair serves as a barrier to optimal chemotherapeutic treatment of ovarian and perhaps other carcinomas and suggest that inhibition of p53 during chemotherapy may enhance clinical outcome.

Background: Aberrant methylation of gene promoter regions has been linked to changes in gene expression in cancer development and progression. Genes associated with CpG islands (CGIs) are especially prone to methylation, but not all CGI-associated genes display changes in methylation patterns in cancers., Results: In order to identify genes subject to regulation by methylation, we conducted gene expression profile analyses of an ovarian cancer cell line (OVCAR-3) before and after treatment with the demethylating agent 5-aza-deoxycytidine (5-aza-dC). An overlapping subset of these genes was found to display significant differences in gene expression between normal ovarian surface epithelial cells and malignant cells isolated from ovarian carcinomas. While 40% of all human genes are associated with CGIs, > 94% of the overlapping subset of genes is associated with CGIs. The predicted change in methylation status of genes randomly selected from the overlapping subset was experimentally verified., Conclusion: We conclude that correlating genes that are upregulated in response to 5-aza-dC treatment of cancer cell lines with genes that are down-regulated in cancer cells may be a useful method to identify genes experiencing epigenetic-mediated changes in expression over cancer development.

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain.

We engineered a fusion protein, mrIL-12vp [mouse recombinant interleukin (IL)-12 linked to vascular peptide], linking the vascular homing peptide CDCRGDCFC (RGD-4C), a ligand for alphavbeta3 integrin, to mrIL-12 to target IL-12 directly to tumor neovasculature. The fusion protein stimulated IFN-gamma production in vitro and in vivo, indicating its biological activity was consistent with mrIL-12. Immunofluorescence techniques showed mrIL-12vp specifically bound to alphavbeta3 integrin-positive cells but not to alphavbeta3 integrin-negative cells. In corneal angiogenesis assays using BALB/c mice treated with either 0.5 microg/mouse/d of mrIL-12vp or mrIL-12 delivered by subcutaneous continuous infusion, mrIL-12vp inhibited corneal neovascularization by 67% compared with only a slight reduction (13%) in angiogenesis in the mrIL-12-treated animals (P = 0.008). IL-12 receptor knockout mice given mrIL-12vp showed a marked decrease in the area of corneal neovascularization compared with mice treated with mrIL-12. These results indicate that mrIL-12vp inhibits angiogenesis through IL-12-dependent and IL-12-independent mechanisms, and its augmented antiangiogenic activity may be due to suppression of endothelial cell signaling pathways by the RGD-4C portion of the fusion protein. Mice injected with NXS2 neuroblastoma cells and treated with mrIL-12vp showed significant suppression of tumor growth compared with mice treated with mrIL-12 (P = 0.03). Mice did not show signs of IL-12 toxicity when treated with mrIL-12vp, although hepatic necrosis was present in mrIL-12-treated mice. Localization of IL-12 to neovasculature significantly enhances the antiangiogenic effect, augments antitumor activity, and decreases toxicity of IL-12, offering a promising strategy for expanding development of IL-12 for treatment of cancer patients.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.

We established a canine hemangiosarcoma cell line derived from malignant endothelial cells comprising a spontaneous tumor in a dog to provide a renewable source of endothelial cells for studies of angiogenesis in malignancy. Pieces of the hemangiosarcoma biopsy were engrafted subcutaneously in a bg/nu/XID mouse allowing the tumor cells to expand in vivo. A cell line, SB-HSA, was derived from the xenograft. SB-HSA cells expressed vascular endothelial growth factor (VEGF) receptors 1 and 2, CD31, CD146, and alpha(v)beta(3) integrin, and produced several growth factors and cytokines, including VEGF, basic fibroblast growth factor, and interleukin (IL)-8 that are stimulatory to endothelial cell growth. These results indicated that the cells recapitulated features of mitotically activated endothelia. In vivo, SB-HSA cells stimulated robust angiogenic responses in mice and formed tumor masses composed of aberrant vascular channels in immunocompromised mice providing novel opportunities for investigating the effectiveness of antiangiogenic agents. Using this model, we determined that IL-12, a cytokine with both immunostimulatory and antiangiogenic effects, suppressed angiogenesis induced by, and tumor growth of, SB-HSA cells. The endothelial cell model we have described offers unique opportunities to pursue further investigations with IL-12, as well as other antiangiogenic approaches in cancer therapy.

Monoclonal antibody R24 recognizes ganglioside GD3 expressed on the cell surfaces of some tumor cells and on a subset of human T lymphocytes. Binding of R24 to these lymphocytes induces proliferation, cytokine production, and activation of intracellular signaling pathways. In the current report, we investigated expression of gangliosides by canine mononuclear immune cells and studied the ability of antiganglioside antibody to activate these cells using tumor cell killing as a measure of activation. A subset of canine monocytes, but not lymphocytes, was found to express gangliosides GD3 and GD2 as determined by the binding of monoclonal antibodies R24 and 14.G2a, respectively. Only R24 augmented the tumoricidal potential of fresh canine peripheral blood mononuclear cells (PBMCs) against tumor cell lines that did not express surface gangliosides GD3 or GD2. The augmenting effect of R24 on PBMC-mediated tumor cytotoxicity required cooperation between monocytes and lymphocytes because there was no enhancement of cytotoxicity mediated by R24 combined with either monocytes or lymphocytes individually. The enhancing effect of R24 on canine PBMC-mediated tumor cytotoxicity was blocked by anti-interleukin (IL)-12 neutralizing antibody, suggesting that R24 binding to monocytes triggered IL-12 release, contributing to the observed tumor killing effects. Reverse transcription-PCR confirmed that the binding of R24 to canine monocytes induced transcription of mRNA for canine IL-12. These data indicate that monocytes can be activated for tumoricidal responses through a membrane structure associated with ganglioside GD3 triggered by the binding of R24 and that the mechanism for enhanced cytotoxicity is due to the production and secretion of IL-12.