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Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however,the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore,the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis,and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435,MDA-MB-231,MDA-MB-468,MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133,and for functional activity of aldehyde dehydrogenase (ALDH),an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation,colony-forming ability,adhesion,migration,invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells,ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P textless 0.05),colony formation (P textless 0.05),adhesion (P textless 0.001),migration (P textless 0.001) and invasion (P textless 0.001). Furthermore,following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice,ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P textless 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis. View Publication

BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor,thereby causing relapse and patient death. Ewing's sarcoma,the second most common form of bone tumor in adolescents and young adults,follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved,even for patients with metastatic disease,but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells,from both human cell lines and human xenografts grown in immune deficient mice,which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity,sphere-formation,and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro,but this can be overcome by the ATP binding cassette transport protein inhibitor,verapamil. Importantly,these cells are not resistant to YK-4-279,a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy. View Publication

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread,requires CD4 and coreceptor expression in target cells,and may also lead to syncytium formation and cell death. Here,we show that in addition to this classical coreceptor-mediated transmission,the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped,captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions,without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion,we describe a novel mechanism of HIV transmission,activated by the contact of infected and uninfected primary CD4 T cells,by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir. View Publication

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1),the AKT modulator and proto-oncogene,is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes,which further included the kinases LYN,SYK,ZAP70,and PKC. High TCL1 levels were also strongly associated with aggressive disease features,such as advanced clinical stage,higher white blood cell counts,and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie,shorter progression-free survival,P textless .001),regardless of therapy regimen,especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful. View Publication

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA,TRKB,TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR),we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF,respectively,efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover,activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication