Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.

Increased evidence suggests that cancer-associated inflammation supports tumor growth and progression. We have previously shown that semaphorin 4D (Sema4D), a ligand produced by different cell types, is a proangiogenic molecule that acts by binding to its receptor, plexin B1, expressed on endothelial cells (Conrotto, P., D. Valdembri, S. Corso, G. Serini, L. Tamagnone, P.M. Comoglio, F. Bussolino, and S. Giordano. 2005. Blood. 105:4321–4329). The present work highlights the role of Sema4D produced by the tumor microenvironment on neoplastic angiogenesis. We show that in an environment lacking Sema4D, the ability of cancer cells to generate tumor masses and metastases is severely impaired. This condition can be explained by a defective vascularization inside the tumor. We demonstrate that tumor-associated macrophages (TAMs) are the main cells producing Sema4D within the tumor stroma and that their ability to produce Sema4D is critical for tumor angiogenesis and vessel maturation. This study helps to explain the protumoral role of inflammatory cells of the tumor stroma and leads to the identification of an angiogenic molecule that might be a novel therapeutic target.

The use of umbilical cord blood as a source of marrow repopulating cells for the treatment of pediatric malignancies has been established. Given the general availability, the ease of procurement, and progenitor content, cord blood is an attractive alternative to bone marrow or growth factor mobilized peripheral blood cells as a source of transplantable hematopoietic tissue. However, there is a major potential limitation to the widespread use of cord blood as a source of hematopoietic stem cells for marrow replacement and gene therapy. There may be enough hematopoietic stem cells to reconstitute children, but the ability to engraft an adult might require ex vivo manipulations. We describe an in vitro system in which the growth of cord blood CD34+ cells is sustained and greatly expanded for more than 6 months by the simple combination of two hematopoietic growth factors. Progenitors and cells belonging to all hematopoietic lineages are continuously and increasingly generated (the number of colony-forming unit–granulocyte-macrophage [CFU-GM] present at the end of 6 months of culture are well over 2,000,000-fold the CFU-GM present at the beginning of the culture). Very primitive hematopoietic progenitors, including long-term culture-initiating cells (LTC-ICs) and blast cell colony-forming units, are also greatly expanded (after 20 weeks of liquid culture, LTC-IC number is over 200,000-fold the initial number). The extremely prolonged maintenance and the massive expansion of these progenitors, which share many similarities with murine long-term repopulating cells, suggest that extensive renewal and little differentiation take place. This system might prove useful in diverse clinical settings involving treatment of grown-up children and adults with transplantation of normal or genetically manipulated hematopoietic stem cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

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Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and IntroductionDuring steady state conditions, adhesive interactions between the bone marrow (BM) stromal cells and primitive hematopoietic cells mostly result in stem cell retention, in a noncycling and nonmotile mode. However, low levels of progenitor cells are continuously released from the BM to the blood circulation as part of homeostasis. This process is dramatically amplified during emergency situations because of damage and cell death, as part of host defense and repair, in response to stress signals, including cytokines such as granulocyte colony-stimulating factor (G-CSF). Repetitive G-CSF stimulations are commonly used in the clinic, mimicking emergency situations to harvest stem and progenitor cells from the circulation for transplantation protocols. 1,2 The BM reservoir of immature and maturing leukocytes is dynamic, replenishing the blood with new cells on demand. These dynamic changes are achieved through a complex interplay between the immune and nervous systems, the bones and the BM microenvironment, involving cytokines, chemokines, proteolytic enzymes, and adhesion molecules. 3 In particular, oscillations in BM levels of stromal cell-derived factor 1 (SDF-1; transiently increased and subsequently degraded) and CXC chemokine receptor-4 (CXCR4) activation play a crucial role in promoting progenitor cell egress. 4,5 The cytokine hepatocyte growth factor (HGF) and its receptor c-Met control complex biologic programs known as "invasive growth" and tumor spreading. 6 Reactive oxygen species (ROS) are constantly generated during intracellular metabolism and in response to cytokines. Although excess ROS can cause oxidative damage to DNA, moderate levels have important roles in cell signaling, regulating different physiologic and pathologic cellular processes, including cell-cycle progression, migration, and invasion. 7 Finally, redox signaling has emerged as an important regulator of hematopoietic stem cell (HSC) self-renewal and lifespan. 8,9 The Forkhead Box, class O (FOXO) family of Forkhead transcription factors is a regulator of oxidative stress. 10 Loss of FOXO function in HSCs results in increased ROS levels, defective maintenance of quiescence, and reduced long-term repopulating ability. 11,12 FOXOs are a direct substrate of the protein kinase Akt, a mammalian target of rapamycin inhibition (mTOR) target, 13 which inactivates them by phosphorylation. 14 In this study, we demonstrate that c-Met expression levels on immature and maturing leukocytes in the BM reservoir are dynamic and dramatically increased when urgent requirements for enhanced leukocyte production and recruitment emerge. Moreover, full c-Me...

Objective: Conventional therapies are still unsuccessful in patients with carcinoma arising from the biliary tract. Somatic mutations of the epidermal growth factor receptor (EGFR) gene and the activation of its downstream pathways predict the sensitivity to small-molecule inhibitors in non^small cell lung carcinoma. Therefore, we analyzed EGFR mutations and related pathways in gallbladder and bile duct carcinomas to consider the possible application of these alternative therapeutic strategies. Experimental Design: Forty paraffin-embedded samples, including intrahepatic or extrahepatic cholangiocarcinoma and gallbladder carcinoma, were studied after tumor cell isolation by laser microdissection and sequencing of EGFR tyrosine kinase domain (exons 18-21). Activation of EGFR pathway was studied by evaluating phosphorylation of mitogen-activated protein kinase and Akt. Results: None of the 40 specimens had mutations in exon 18; one had one missense point mutation in exon19, two in exon 20, and three in exon 21. In addition, 36 of 40 specimens had the same silent mutation at codon 787 in exon 20, which was also found in peripheral blood cells from healthy donors. Tumor samples harboring EGFR mutation had phosphorylation of one or both downstream transducers analyzed. Conclusions: This is the first evidence of somatic mutations of the EGFR gene in bile duct carcinoma. Our findings suggest that a subgroup of patients with cholangiocarcinoma or gallbladder carcinoma exhibits somatic mutations of EGFR in the tyrosine kinase domain that can elicit cell signals sustaining survival and proliferation. These tumors might be further evaluated for their susceptibility to small-molecule inhibitor treatment.

IntroductionHematopoietic stem cells migrate during embryonic development from the fetal liver through the blood circulation, home to the bone marrow (BM) microenvironment, and repopulate it with immature and maturing blood cells of all lineages. Similarly, in clinical and experimental stem cell transplantation protocols, hematopoietic stem cells, which are infused into the blood circulation of patients and experimental animals, home and repopulate the BM. 1 The molecular mechanisms that regulate the homing and repopulation processes are crucial for stem cell function and development. [2][3][4][5] The CXC chemokine stromal cell-derived factor 1 (SDF-1) plays a major role in migration, proliferation, differentiation, and survival of many cell types including human and murine hematopoietic stem/progenitor cells. 6,7 SDF-1 is produced by multiple BM stromal cell types and by epithelial cells in many organs 8,9 and is highly expressed by human and murine BM endothelium. [10][11][12] CXCR4, the 7-transmembrane receptor of SDF-1, is widely expressed by a variety of hematopoietic cell types, neuronal cells, and different stromal cells. 13 SDF-1 is a chemotactic agent for human lymphoid, myeloid, and immature CD34 ϩ progenitor cells. 6,7,14,15 This chemokine induces integrin-dependent adhesion of CXCR4 ϩ human T lymphocytes 16 and immature CD34 ϩ CXCR4 ϩ cells 17 under shear flow and also mediates transendothelial migration of human progenitors. 18 In vivo cell migration and localization are also mediated by SDF-1/CXCR4 interactions. Murine T cells overexpressing human CXCR4 and CD4 accumulated in the BM of transgenic mice. 19 Prevention of CXCR4 expression by introducing SDF-1 intrakine blocked in vitro migration and in vivo dissemination of a T-cell hybridoma. 20 More important, mice reconstituted with progenitor cells expressing SDF-1-intrakine suffered impaired lymphoid and myeloid hematopoiesis, whereas transplantation of progenitors overexpressing SDF-1 led to increased myeloid and B-lymphoid hematopoiesis. 21 The key role of SDF-1 and CXCR4 in embryonic development was demonstrated by knockout studies in mice. The lack of either SDF-1 or its receptor in murine fetuses results in multiple lethal defects including impaired BM hematopoiesis. [22][23][24][25] Recently, Wright and colleagues have demonstrated that SDF-1 is the sole chemokine mediating in vitro migration of purified adult murine BM stem cells. 26 This important study suggests a major role for SDF-1/CXCR4 interactions also in adult murine stem cell migration and development.We demonstrated the essential role of SDF-1/CXCR4 interactions in both homing and high-level multilineage repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) 10,27,28 The antihuman CXCR4-neutralizing monoclonal antibody (mAb; clone 12G5) binds CXCR4 on the first and second extracellular domain as its ligand SDF-1, interfering with SDF-1 binding and signaling. 29,30 Coinjecting enriched human CD34 ϩ cells with neutralizing anti-CXCR4 mAb blocked homing and re...