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.

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

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.

Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux.

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 (ß-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 ß-ARs or by preventing the lysosomal accumulation of doxorubicin. ß-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of ß-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 ß-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 ß-ARs.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumour growth of feline oral squamous cell carcinoma.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

Emerging therapeutic approaches for canine sarcomas: Pushing the boundaries beyond the conventional.

Sarcomas represent a group of genomically chaotic, highly heterogenous tumours of mesenchymal origin with variable mutational load. Conventional therapy with surgery and radiation therapy is effective for managing small, low-grade sarcomas and remains the standard therapeutic approach. For advanced, high-grade, recurrent, or metastatic sarcomas, systemic chemotherapy provides minimal benefit, therefore, there is a drive to develop novel approaches. The discovery of "Coley's toxins" in the 19th century, and their use to stimulate the immune system supported the application of unconventional therapies for the treatment of sarcomas. While promising, this initial work was abandoned and treatment paradigm and disease course of sarcomas was largely unchanged for several decades. Exciting new therapies are currently changing treatment algorithms for advanced carcinomas and melanomas, and similar approaches are being applied to advance the field of sarcoma research. Recent discoveries in subtype-specific cancer biology and the identification of distinct molecular targets have led to the development of promising targeted strategies with remarkable potential to change the landscape of sarcoma therapy in dogs. The purpose of this review article is to describe the current standard of care and limitations as well as emerging approaches for sarcoma therapy that span many of the most active paradigms in oncologic research, including immunotherapies, checkpoint inhibitors, and drugs capable of cellular metabolic reprogramming.

5-HT serotonin receptors modulate mitogenic signaling and impact tumor cell viability.

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.

Propranolol and breast cancer-a work in progress.

The non-selective beta-blocker propranolol is a leading candidate for repurposing as a novel anti-cancer agent. Emerging evidence, including human data, suggests that there are multiple mechanisms of action particularly relevant to breast cancer. This editorial reviews a number of recent studies that show it has anti-metastatic activity that warrants clinical investigation, including investigation as a potential perioperative therapy in breast cancer.

Non-selective beta blockers inhibit angiosarcoma cell viability and increase progression free- and overall-survival in patients diagnosed with metastatic angiosarcoma.

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 (ß-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 ß-AR antagonism abrogates mitogenic signaling and reduces angiosarcoma tumor cell viability, and these molecular alterations translated into patient tumors. We demonstrated that non-selective ß-AR antagonists are superior to selective ß-AR antagonists at inhibiting angiosarcoma cell viability. A prospective analysis of non- selective ß-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 ß-AR antagonists into existing therapies against metastatic angiosarcoma can enhance clinical outcomes.

Use of non-selective ß-blockers is associated with decreased tumor proliferative indices in early stage breast cancer.

Previous studies suggest beta-adrenergic receptor (ß-AR) antagonists (ß-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 ß1-AR and ß3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of ß-blocker usage on tumor proliferation. Our analysis revealed that non-selective ß-blockers, but not selective ß-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 ß-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 ß-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 GSK3ß. In conclusion, use of non-selective ß-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.

Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC.

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.

Transcriptome profiling in oral cavity and esophagus tissues from (S)-N'-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis.

Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value < 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers. © 2016 Wiley Periodicals, Inc.

Growth Attenuation of Cutaneous Angiosarcoma With Propranolol-Mediated ß-Blockade.

IMPORTANCE: Patients with stage T2 multilesion angiosarcomas of the scalp and face that are larger than 10 cm demonstrate a 2-year survival rate of 0%. To our knowledge, major therapeutic advances against this disease have not been reported for decades. Preclinical data indicate that blocking ß-adrenergic signaling with propranolol hydrochloride disrupts angiosarcoma cell survival and xenograft angiosarcoma progression. OBSERVATIONS: A patient presented with a ß-adrenergic-positive multifocal stage T2 cutaneous angiosarcoma (≥20 cm) involving 80% of the scalp, left forehead, and left cheek, with no evidence of metastasis. The patient was immediately administered propranolol hydrochloride, 40 mg twice a day, as his workup progressed and treatment options were elucidated. Evaluation of the proliferative index of the tumor before and after only 1 week of propranolol monotherapy revealed a reduction in the proliferative index of the tumor by approximately 34%. A combination of propranolol hydrochloride, 40 mg 3 times a day, paclitaxel poliglumex, 2 mg/m2 infused weekly, and radiotherapy during the subsequent 8 months resulted in extensive tumor regression with no detectable metastases. CONCLUSIONS AND RELEVANCE: Our data suggest that ß-blockade alone substantially reduced angiosarcoma proliferation and, in combination with standard therapy, is effective for reducing the size of the tumor and preventing metastases. If successful, ß-blockade could be the first major advancement in the treatment of angiosarcoma in decades.

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.

Pathobiology of Hemangiosarcoma in Dogs: Research Advances and Future Perspectives.

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.

SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in head and neck squamous cell carcinoma.

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.

Frequent and increased expression of human METCAM/MUC18 in cancer tissues and metastatic lesions is associated with the clinical progression of human ovarian carcinoma.

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.

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

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.

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment.

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into "IL-8 high" and "IL-8 low" groups. Genome-wide gene expression profiling showed that samples in the "IL-8 high" tumor group were enriched for genes associated with a "reactive microenvironment," including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA.

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 αvß3 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.