VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.     

Defective ability to self-renew in vitro of highly purified primitive haematopoietic cells

Stromal cells play a critical role in haematopoiesis, both in a permissive and, probably, in a directive manner. Study of the interactions between stromal cells and haematopoietic stem cells, however, is difficult to perform using whole bone marrow, in which stem cells are indistinguishable from precursor cells and maturing haematopoietic cells, and where stromal and haematopoietic cells co-exist in a heterogeneous mixture. We have purified primitive haematopoietic spleen colony-forming cells (CFU-S) using fluorescence-activated cell sorting (FACS) and produced CFU-S populations which approach 100% purity (ref. 6 and B.I.L. and E.S., in preparation). This cell population is devoid of significant stromal cells and mature haematopoietic cells. Here, we report that when purified CFU-S are seeded onto a stromal adherent layer in vitro, foci of haematopoietic cells develop within the stroma followed by production of a wave of maturing and mature progeny. However, self-renewal of CFU-S does not occur and haematopoietic activity rapidly declines, indicating that caution should be applied in the use of highly purified stem cells for human bone marrow transplantation.     

Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells

Long-term reconstitution of the lymphohaematopoietic cells of a mouse after lethal irradiation requires the transplantation of at least (5-10) x 10(3) bone marrow cells. Several cell-separation techniques based on cell-surface characteristics have been used in attempts to identify the pluripotent haematopoietic stem cells (PHSC), and have allowed the long-term engraftment of lethally irradiated mice with an enriched fraction of fewer than 200 marrow cells. But these techniques enrich not only for PHSC but also for haematopoietic progenitors, especially day-12 spleen colony-forming units (CFU-S). Although day-12 CFU-S have been postulated to be primitive multipotential haematopoietic progenitors, with day-8 CFU-S representing later, more committed progenitors, recent evidence suggests that neither of these CFU-S represents mouse PHSC. Here we report that counterflow centrifugal elutriation, which sorts cells on the basis of size and density, can separate PHSC from these less primitive progenitors. The fraction containing the largest cells was enriched for the granulocyte-macrophage colony-forming units (CFU-GM), but gave only transient, early engraftment and was therefore depleted of PHSC. The intermediate fraction was enriched for CFU-S, but depleted of CFU-GM. Despite being devoid of CFU-GM and CFU-S, the fraction consisting of only morphological lymphocytes gave sustained, albeit delayed, reconstitution of all lymphohaematopoietic cells, and was therefore enriched for PHSC. We conclude that there are two vital classes of engrafting cells: committed progenitors, which provide initial, unsustained engraftment, and PHSC, which produce delayed, but durable, engraftment. Therefore for late haematological reconstitution, PHSC must be transplanted with a distinguishable source of early engrafting cells, thereby allowing the lethally irradiated host to survive initial aplasia.     

Spleen colony-forming cell as common precursor for tissue mast cells and granulocytes

The haematopoietic stem cells which produce colonies in the spleen of irradiated mice (CFU-S) can differentiate into erythrocytes, granulocytes, megakaryocytes and B lymphocytes. Although mast cell precursors are known to be present in the bone marrow, spleen, fetal liver and peripheral blood of mice, the relationship between the mast cell precursor and CFU-S has remained unclear. We have now made use of mice of two mutant genotypes to determine whether or not the tissue mast cell is a progeny of CFU-S. Giant granules of beige (C57BL/6-bg/bg, Chediak-Higashi syndrome) mice can be used for identification of the origin of both tissue mast cells and granulocytes, and WBB6F1-W/Wv mice are useful recipients because they lack tissue mast cells owing to a defect in mast cell precursors. We injected the cells from a single spleen colony into each WBB6F1-W/Wv mouse and demonstrated directly that the tissue mast cell is a progeny of CFU-S.     

Fibronectin receptor structures in the VLA family of heterodimers

Multiple cell surface proteins of relative molecular mass 115,000-155,000 (Mr 115K-155K) have been implicated as receptors mediating adhesion to extracellular matrix proteins. But the organization and relatedness of these peptides has remained unclear. In separate studies, the 'very late antigens' VLA-1 (Mr 210K/130K) and VLA-2 (Mr 160K/130K) were initially characterized as surface heterodimers appearing 2-4 weeks after in vitro stimulation of human T cells. Three more VLA heterodimers have since been discovered, which, like VLA-1 and VLA-2, are each composed of unique alpha-subunits in association with a common 130K beta subunit. This paper shows that the common VLA beta-subunit is equivalent to subunits found in structures with known fibronectin and laminin receptor activity, and that VLA-3 and VLA-5 are similar or identical to these previously defined receptors for adhesion molecules. Antibody blocking studies confirmed that at least some of the widely distributed VLA proteins of previously unknown function are involved in cell adhesion to fibronectin and laminin. We suggest that the VLA family of receptors may provide cells with multiple independent substrate adhesion capabilities.     

Identification and characterization of an inhibitor of haemopoietic stem cell proliferation

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.     

Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule alpha 4 beta 1, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-alpha 4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with alpha 4 beta 1 integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.     

ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.     

Osteoblastic cells regulate the haematopoietic stem cell niche

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.     

Laminin receptor on platelets is the integrin VLA-6

Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family. Here we report that adhesion of platelets to laminin is inhibited by a rat monoclonal antibody against the integrin family member, VLA-6. This antibody does not affect platelet adhesion to fibrinogen, fibronectin or to type I and III collagen. Binding to laminin does not require platelet activation and is not inhibited by fibronectin and laminin cell-attachment peptides. Platelet adhesion to laminin is supported by Mn2+, Co2+ and Mg2+, but not by Ca2+, Zn2+ and Cu2+. This cation preference is distinct from that characteristic for other platelet-adhesive glycoproteins.     

Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 and p40 subunits. Analyses in vitro have defined IL-12 as an important factor for the differentiation of naive T cells into T-helper type 1 CD4+ lymphocytes secreting interferon-gamma (refs 1, 2). Similarly, numerous studies have concluded that IL-12 is essential for T-cell-dependent immune and inflammatory responses in vivo, primarily through the use of IL-12 p40 gene-targeted mice and neutralizing antibodies against p40. The cytokine IL-23, which comprises the p40 subunit of IL-12 but a different p19 subunit, is produced predominantly by macrophages and dendritic cells, and shows activity on memory T cells. Evidence from studies of IL-23 receptor expression and IL-23 overexpression in transgenic mice suggest, however, that IL-23 may also affect macrophage function directly. Here we show, by using gene-targeted mice lacking only IL-23 and cytokine replacement studies, that the perceived central role for IL-12 in autoimmune inflammation, specifically in the brain, has been misinterpreted and that IL-23, and not IL-12, is the critical factor in this response. In addition, we show that IL-23, unlike IL-12, acts more broadly as an end-stage effector cytokine through direct actions on macrophages.     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

Regulated expression and binding of three VLA (beta 1) integrin receptors on T cells

Regulated adhesion of T cells to extracellular matrix (ECM) proteins is likely to be essential in T cell migration. Constitutive binding of various other cell types to ECM components is mediated by members of the VLA (very late antigen) subfamily of integrins. We describe here the regulated binding of resting CD4+ human T cells to ECM through three VLA integrins: VLA-4 and VLA-5 binding to fibronectin (FN), and a novel pathway of VLA-6 binding to laminin (LN). Binding to ECM is regulated in two ways. First, unlike other VLA-mediated interactions, VLA binding activity of the T cells is rapidly and dramatically augmented with cell activation without change in level of expression of the VLA molecules. Second, binding is regulated with T-cell differentiation; memory T cells express three- to four-fold more VLA-4, VLA-5, and VLA-6 than do naive cells, and bind more efficiently through them to FN and LN.     

Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer.

An inherited deficiency of beta-glucuronidase in humans, mice and dogs causes mucopolysaccharidosis VII (Sly syndrome), a progressive degenerative disease that reduces lifespan (to an average of 5 months in mice) and results from lysosomal storage of undegraded glycosaminoglycans in the spleen, liver, kidney, cornea, brain and skeletal system. Bone marrow transplantation in mutant mice provides a source of normal enzyme ('cross-correction'), which substantially improves the clinical condition and extends the average lifespan to 18 months. Gene therapy by transfer of a beta-glucuronidase gene into mutant haematopoietic stem cells is an alternative approach, but it is not known whether the low expression of vector-transferred genes in vivo would be sufficiently effective. Here we show that retroviral vector-mediated transfer of the gene to mutant stem cells results in long-term expression of low levels of beta-glucuronidase which partially corrects the disease by reducing lysosomal storage in liver and spleen.     

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.     

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.     

Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity

The receptors for erythropoietin and other cytokines constitute a new superfamily. They have no tyrosine-kinase or other enzyme motif and their signal-transducing mechanism is unclear. Here we describe two classes of activating mutations in the erythropoietin receptor (EPOR). A single point mutation in the exoplasmic domain enables it to induce hormone-independent cell growth and tumorigenesis after expression in nontumorigenic, interleukin-3-dependent haematopoietic cells. A C-terminal truncation in the cytoplasmic domain of the EPOR renders the receptor hyperresponsive to erythropoietin, but is insufficient to induce hormone-independent growth or tumorigenicity. The activating point mutation retards intracellular transport and turnover of the receptor. These alterations in metabolism and tumorigenicity caused by the EPOR with activating point mutations are similar to those observed in erythropoietin-independent activation of the wild type EPOR by association with gp55, the Friend spleen focus-forming virus glycoprotein.     

Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor

During mammalian ontogeny, haematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, where haematopoiesis occurs throughout adulthood. Unique features of bone that contribute to a microenvironmental niche for stem cells might include the known high concentration of calcium ions at the HSC-enriched endosteal surface. Cells respond to extracellular ionic calcium concentrations through the seven-transmembrane-spanning calcium-sensing receptor (CaR), which we identified as being expressed on HSCs. Here we show that, through the CaR, the simple ionic mineral content of the niche may dictate the preferential localization of adult mammalian haematopoiesis in bone. Antenatal mice deficient in CaR had primitive haematopoietic cells in the circulation and spleen, whereas few were found in bone marrow. CaR-/- HSCs from fetal liver were normal in number, in proliferative and differentiative function, and in migration and homing to the bone marrow. Yet they were highly defective in localizing anatomically to the endosteal niche, behaviour that correlated with defective adhesion to the extracellular matrix protein, collagen I. CaR has a function in retaining HSCs in close physical proximity to the endosteal surface and the regulatory niche components associated with it.     

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed. We now describe a modified culture system in which the 'early' BFU-E develop into 'late' BFU-E in response to added erythropoietin. Further maturation of these cells into CFU-E and non-nucleated erythrocytes can be achieved by mechanical agitation of the long-term cultures or by transferring the cells into dishes which do not allow cell attachment to occur.