The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorageindependent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in ~50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.

Synthesis of a radiolabeled diglyceride 3-[ 18 F]fluorodipalmitoyl-1,2-glycerol ( 18 F-fluorodipalmitin, [ 18 F]FDP) and its potential as a reagent for radiolabeling long-circulating liposomes were investigated. The incorporation of 18 F into the lipid molecule was accomplished by nucleophilic substitution of p-toluenesolfonyl moiety with a decay corrected yield of 43 ± 10% (n = 12). Radiolabeled long-circulating PEG-coated liposomes were prepared using a mixture of DPPC, cholesterol, DSPE-PEG2000 (61:30:9) and [ 18 F]FDP with a decay corrected yield of 70 ± 8% (n = 4). PET imaging and biodistribution studies were performed with free [ 18 Liposomes are vesicles composed of one or more concentric phospholipid bi-layers and such vesicles have been widely investigated as possible drug carriers [1,2]. Prolonged blood circulation of the liposomes is achieved with the addition of a polyethylene glycol (PEG) coating, which efficiently minimizes their removal by macrophages of the reticuloendothelial system [3][4][5][6]. Liposomes with various target-specific ligands attached to their surface are being investigated for targeted drug delivery [1,2,7]. Liposomes labeled with radioisotopes such as 99m Tc, 186 Re, 67 Ga, 111 In, and 18 F were previously employed to study the biodistribution of different types of liposomes in various animal models using scintigraphy, SPECT and PET. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods GeneralThe solvents and chemicals were purchased from Aldrich (Milwaukee, WI). The 1 H and 13 C NMR spectra were recorded using a Bruker Avance 500 spectrometer and the chemical shifts are reported relative to TMS. Analytical reversed-phase HPLC was performed using a Phenomenex Jupiter 5μ C4 300A column ( . The size distribution of liposomes was determined using a particle size analyzer Nanotrac NPA150 purchased from Microtrac Inc. (North Largo, FL). The lipid concentration was determined using a Phospholipids B kit purchased from Wako Chemicals, Inc. (Richmond, VA). Synthesis of 3-tosyl-1,2-dipalmitoyl glycerol (2)The precursor 2 was prepared according to the previously published procedure [32] from 1,2-dipalmitoyl-sn-glycerol (1) and 4-toluenesulfonyl chloride. The crude product was subsequently purified by recrystalization from hexane. 1 mL). The content of the vial was dissolved in anhydrous acetonitrile (0.35 mL) and transferred into a suspension of 2 (5.5 mg) in anhydrous acetonitrile (0.5 mL). The reaction mixture was heated to 100 ºC for 20 minutes and allowed to cool to room temperature for 5 min...

The CD34 ؉ CD38 ؊ phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34 ؉ cells, HSC content is difficult to measure since committed CD34 ؉ CD38 ؉ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/ severe combined immunodeficiency (SCID)-repopulating cells (SRCs) . IntroductionCell surface expression of the CD34 antigen is a reliable indicator of enrichment for hematopoietic progenitor and stem cells. 1,2 However, cells within the CD34 ϩ compartment are heterogeneous and include committed CD34 ϩ CD38 ϩ progenitors that lack stem cell activity. 3,4 The CD34 ϩ CD38 Ϫ fraction makes up 1% to 10% of the CD34 ϩ population and is highly enriched for both extended long-term culture-initiating cells (ELTC-ICs) and the most primitive assayable cells that are capable of repopulating nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (SCID-repopulating cells [SRCs]). [3][4][5][6][7] Using fluorescence-activated cell sorting to collect steady-state cord blood (CB) CD34 ϩ CD38 Ϫ cells, Bhatia et al 4 demonstrated a frequency of 1 SRC per 617 CB CD34 ϩ CD38 Ϫ cells and no detectable SRCs within the CD34 ϩ CD38 ϩ fraction. 4 Bhatia et al 8 also showed, via a limiting dilution analysis, that purified human CB CD34 ϩ CD38 Ϫ cells could be cultivated ex vivo with cytokines resulting in a 2-to 4-fold increase in SRCs at day 4 followed by loss of SRCs by day 9. A subsequent study by Glimm and Eaves 9 further demonstrated that self-renewal divisions occur within primitive CB-repopulating cells during 5-day cytokine suspension cultures. Other studies have suggested that human CB SRCs can be maintained ex vivo in liquid suspension cultures from 1 to 12 weeks. [10][11][12] Conversely, ex vivo culture of adult (bone marrow [BM], peripheral blood) sources of human CD34 ϩ stem cells with cytokines, with and without stroma, has been reproducibly associated with the loss of primitive repopulating cells over time. [13][14][15][16][17] Investigations into the proliferative capacity and SRC content of purified human BM CD34 ϩ CD38 Ϫ cells have been more limited in part due to a lack of culture conditions that support the maintenance or expansion of adult hematopoietic stem cells (HSCs). 8,18 Recent studies have indicated that expression of CD38 antigen on CD34 ϩ CD38 ϩ hematopoietic progenitors may be downmodulated during ex vivo culture with cytokines, calling into question the reliability of the CD34 ϩ CD38 Ϫ phenotype as an indicator of HSC content during or after culture. 19,20 As evidence that the CD34 ϩ CD38 Ϫ phenotype did not correlate with primitive stem cell content, Dorrell et al 19 demonstrated that surface expression of myeloid maturation antigens, CD33 and CD13, increased more than 2-fold on CB CD34 ϩ CD38 ...