The "p53 signature" is a benign secretory cell outgrowth in the distal fallopian tube that shares properties with ovarian serous cancer -including p53 mutations -and is a putative serous cancer precursor. We expanded the precursor definition to all secretory cell outgrowths (SCOUTs) of 30 or more cells and scored normal (N) and altered (A) expression of both p53 and PAX2, a gene down-regulated in ovarian and endometrial cancer. SCOUTs were identified by BCL2/p73 staining in tubes from women with serous carcinoma, inherited mutations in BRCA1 or BRCA2, and controls. SCOUTs were prevalent in both proximal and distal tube and significantly associated with serous carcinoma versus the others (p <0.001). Eighty-nine percent were PAX2 (A); 26% were PAX2 (A)/p53 (A) (p53 signatures). PAX2 (A)/p53 (N) SCOUTs were free of p53 mutations; however, 12 of 13 p53 signatures were PAX2 (A). A tubal carcinoma and contiguous SCOUT were p53 (A)/PAX2 (A) and shared the same p53 mutation. SCOUTs are discretely localized alterations commonly containing altered expression of multiple genes within histologically benign tubal epithelium. Geographic distribution in the tube varies by genotype and immunophenotype, from widespread (PAX2) to confinement to a specific area (fimbria) of shared prevalence (PAX2 and p53). This study reveals, for the first time, an entity (SCOUT) that is associated with serous cancer, expands the topography of altered PAX2 expression in the female genital tract mucosa and highlights another potential pathway disturbance involved in early serous carcinogenesis in the fallopian tube.

Summary Embryonal rhabdomyosarcoma (ERMS) is an aggressive pediatric sarcoma of muscle. Here, we show that ERMS-propagating potential is confined to myf5+ cells and can be visualized in live, fluorescent transgenic zebrafish. During early tumor growth, myf5+ ERMS cells reside adjacent normal muscle fibers. By late stage ERMS, myf5+ cells are reorganized into distinct regions separated from differentiated tumor cells. Time-lapse imaging of late stage ERMS revealed that myf5+ cells populate newly formed tumor only after seeding by highly migratory myogenin+ ERMS cells. Moreover, myogenin+ ERMS cells can enter the vasculature, whereas myf5+ ERMS-propagating cells do not. Our data suggests that non-tumor propagating cells likely have important supportive roles in cancer progression and facilitate metastasis.

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Selfrenewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/ β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/ β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced selfrenewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/ β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

We used a morpholino-based gene-targeting screen to identify a novel protein essential for vascular development using the zebrafish, Danio rerio. We show that syndecan-2, a cell-surface heparan sulfate proteoglycan, is essential for angiogenic sprouting during embryogenesis. The vascular function of syndecan-2 is likely conserved, as zebrafish and mouse syndecan-2 show similar expression patterns around major trunk vessels, and human syndecan-2 can restore angiogenic sprouting in syndecan-2 morphants. In contrast, forced expression of a truncated form of syndecan-2 results in embryos with defects in angiogenesis, indicating that the highly conserved cytoplasmic tail is important for the vascular function of syndecan-2. We further show that vascular endothelial growth factor (VEGF) and syndecan-2 genetically interact in vivo using both gain-of-function and loss-offunction studies in zebrafish. VEGF-mediated ectopic signaling is compromised in syndecan-2 morphants, and ectopic syndecan-2 potentiates ectopic VEGF signaling. Syndecan-2 as a novel angiogenic factor is a potential candidate for use in the development of angiogenesis-based therapies. IntroductionVascular formation is composed of 2 processes, vasculogenesis and angiogenesis. In vasculogenesis, angioblasts derived from the lateral mesoderm migrate and differentiate into endothelial cells that form the tubular structures of primary vasculature. In angiogenesis, new vessels sprout from pre-existing vasculature and are further remodeled to form mature blood vessels. Angiogenesis is an important process during development, various physiologic phenomena, and pathologic conditions such as tumor growth and diabetic retinopathy. 1 Growth factors such as vascular endothelial growth factor (VEGF), angiopoietins, and ephrins play key roles during angiogenesis. [1][2][3] Identification and characterization of novel angiogenic factors during vertebrate development may further the understanding of key molecular players in angiogenesis as well as hold promise for potential application in angiogenesis-based therapies. [4][5][6] Conservation of vascular development in vertebrates 7 and the capacity to effectively target this process using morpholinos 8 allowed us to embark on a morpholino-based screen for novel angiogenic factors using zebrafish as the model system. Morpholinos were designed based on 5Ј leader sequences from a collection of zebrafish expressed sequence tags (ESTs) with high homology to human, Drosophila, or Fugu gene. Bioinformatics methods to enrich for partial sequences of cell-surface or secreted proteins based on full-length reference protein data sets have been determined as previously described. 9 As part of this ongoing effort, we have identified syndecan-2 (syn-2), a cell-surface heparan sulfate proteoglycan, as a novel factor essential for angiogenic sprouting during vascular development.Previous studies have demonstrated the important roles played by heparan sulfate proteoglycans (HSPGs) in a variety of biologic processes such as early embryon...

Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.

We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-

Cellular angiofibroma is a mesenchymal neoplasm that is characterized by a bland spindle cell component, morphologically reminiscent of spindle cell lipoma, and thick-walled vessels. The tumor occurs equally in men and women and usually arises in the inguino-scrotal or vulvovaginal regions. An earlier study of 51 cases from our group showed that the tumor follows a benign course without any tendency for recurrence. In 1 case, an intralesional microscopic nodule of pleomorphic liposarcoma was observed. The biologic significance of atypia or sarcomatous transformation in cellular angiofibroma remains uncertain. In this study, we characterized clinicopathologic features in 13 cases of cellular angiofibroma with morphologic atypia or sarcomatous transformation. Thirteen cases with atypia or sarcomatous transformation among 154 usual cellular angiofibromas identified between 1993 and 2009 were retrieved from consultation files. There were 12 females and 1 male ranging in age from 39 to 71 years (median age, 46 y). Tumor size ranged from 1.2 to 7.5 cm. In 11 cases, the tumors occurred in the vulva. One case each occurred in the paratesticular and hip regions. Most tumors were located in subcutaneous tissue. There were 4 cases of cellular angiofibroma with atypia. Three showed severely atypical cells as scattered foci within the cellular angiofibroma. One case showed a discrete nodule of atypical cells. There were 9 cases of cellular angiofibroma with morphologic features of sarcomatous transformation. In each case, abrupt transition to a discrete sarcomatous component was seen. Of these 9 cases, the sarcomatous component in 2 cases showed features of pleomorphic liposarcoma with multivacuolated lipoblasts readily identified. Three of these 9 cases showed discrete nodule(s) closely resembling atypical lipomatous tumor within usual cellular angiofibroma. In the remaining 4 cases, the sarcomatous component was composed of pleomorphic spindle cells arranged in various patterns. By immunohistochemistry, atypical cells and sarcomatous areas showed either multifocal or more diffuse p16 expression compared with either scattered or negative expression in the conventional cellular angiofibroma. The 3 cases with atypical lipomatous tumor-like areas were negative for MDM-2 and CDK4. Follow-up information was available for 7 patients (range from 2 to 75 mo; median: 14 mo). Six patients did not develop recurrence or metastasis. One patient died of metastatic carcinoma of unknown primary site 27 months after the diagnosis of cellular angiofibroma with sarcomatous transformation. Cellular angiofibroma with atypia or morphologic sarcomatous transformation occurs predominantly in the subcutaneous tissue of the vulva and, as yet, shows no evident tendency to recur based on limited clinical follow-up available for 7 cases. The sarcomatous component can show variable features including atypical lipomatous tumor, pleomorphic liposarcoma, and pleomorphic sarcoma NOS. Overexpression of p16 in the atypical cells and sarcomatous component sugge...