Identification of human brain tumour initiating cells

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.     

Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells

Glioblastoma is a highly angiogenetic malignancy, the neoformed vessels of which are thought to arise by sprouting of pre-existing brain capillaries. The recent demonstration that a population of glioblastoma stem-like cells (GSCs) maintains glioblastomas indicates that the progeny of these cells may not be confined to the neural lineage. Normal neural stem cells are able to differentiate into functional endothelial cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis through the release of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (refs 5-9). Here we show that a variable number (range 20-90%, mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumour cells, indicating that a significant portion of the vascular endothelium has a neoplastic origin. The vascular endothelium contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. In vitro culture of GSCs in endothelial conditions generated progeny with phenotypic and functional features of endothelial cells. Likewise, orthotopic or subcutaneous injection of GSCs in immunocompromised mice produced tumour xenografts, the vessels of which were primarily composed of human endothelial cells. Selective targeting of endothelial cells generated by GSCs in mouse xenografts resulted in tumour reduction and degeneration, indicating the functional relevance of the GSC-derived endothelial vessels. These findings describe a new mechanism for tumour vasculogenesis and may explain the presence of cancer-derived endothelial-like cells in several malignancies.     

Glioma stem cells promote radioresistance by preferential activation of the DNA damage response

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.     

A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula

It has been proposed that haematopoietic and endothelial cells share a common progenitor, termed the haemangioblast. This idea was initially conceived as a result of the observation that these two cell types develop in close proximity to each other within the embryo. Support for this hypothesis was provided by studies on single-cell-derived colonies that can produce both haematopoietic and endothelial cells in vitro. Although these data point towards the existence of a common progenitor for these two lineages, the presence of a bipotential progenitor cell has yet to be demonstrated in vivo. Through the construction of single-cell-resolution fate maps of the zebrafish late blastula and gastrula, we demonstrate that individual cells can give rise to both haematopoietic and endothelial cells. These bipotential progenitors arise along the entire extent of the ventral mesoderm and contribute solely to haematopoietic and endothelial cells. We also find that only a subset of haematopoietic and endothelial cells arise from haemangioblasts. The endothelial descendants of the haemangioblasts all clustered in a specific region of the axial vessels regardless of the location of their progenitors. Our results provide in vivo evidence supporting the existence of the haemangioblast and reveal distinct features of this cell population.     

In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment

The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.     

A clonogenic common myeloid progenitor that gives rise to all myeloid lineages

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.     

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.     

Generation of a prostate from a single adult stem cell

The existence of prostate stem cells (PSCs) was first postulated from the observation that normal prostate regeneration can occur after repeated cycles of androgen deprivation and replacement in rodents. Given the critical role of PSCs in maintaining prostate tissue integrity and their potential involvement in prostate tumorigenesis, it is important to define specific markers for normal PSCs. Several cell-surface markers have been reported to identify candidate PSCs, including stem cell antigen-1 (Sca-1, also known as Ly6a), CD133 (Prom1) and CD44 (refs 3-10). However, many non-PSCs in the mouse prostate also express these markers and thus identification of a more defined PSC population remains elusive. Here we identify CD117 (c-kit, stem cell factor receptor) as a new marker of a rare adult mouse PSC population, and demonstrate that a single stem cell defined by the phenotype Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) can generate a prostate after transplantation in vivo. CD117 expression is predominantly localized to the region of the mouse prostate proximal to the urethra and is upregulated after castration-induced prostate involution-two characteristics consistent with that of a PSC marker. CD117(+) PSCs can generate functional, secretion-producing prostates when transplanted in vivo. Moreover, CD117(+) PSCs have long-term self-renewal capacity, as evidenced by serial isolation and transplantation in vivo. Our data establish that single cells in the adult mouse prostate with multipotent, self-renewal capacity are defined by a Lin(-)Sca-1(+)CD133(+)CD44(+)CD117(+) phenotype.     

Identification and expansion of human colon-cancer-initiating cells

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.     

Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

The earliest thymic progenitors for T cells possess myeloid lineage potential

There exists controversy over the nature of haematopoietic progenitors of T cells. Most T cells develop in the thymus, but the lineage potential of thymus-colonizing progenitors is unknown. One approach to resolving this question is to determine the lineage potentials of the earliest thymic progenitors (ETPs). Previous work has shown that ETPs possess T and natural killer lymphoid potentials, and rare subsets of ETPs also possess B lymphoid potential, suggesting an origin from lymphoid-restricted progenitor cells. However, whether ETPs also possess myeloid potential is unknown. Here we show that nearly all ETPs in adult mice possess both T and myeloid potential in clonal assays. The existence of progenitors possessing T and myeloid potential within the thymus is incompatible with the current dominant model of haematopoiesis, in which T cells are proposed to arise from lymphoid-. Our results indicate that alternative models for lineage commitment during haematopoiesis must be considered.     

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.     

Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors

Haematopoiesis is maintained by a hierarchical system where haematopoietic stem cells (HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells. HSCs maintain themselves for the lifetime of the organism because of their ability to self-renew. However, multipotent progenitors lack the ability to self-renew, therefore their mitotic capacity and expansion potential are limited and they are destined to eventually stop proliferating after a finite number of cell divisions. The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood. Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1--p16Ink4a, p19Arf and Trp53 (triple mutant mice; p16Ink4a and p19Arf are alternative reading frames of the same gene (also called Cdkn2a) that encode different proteins)--have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase is associated with the acquisition of long-term reconstitution capacity by cells of the phenotype c-kit+Sca-1+Flt3+CD150-CD48-Lin-, which defines multipotent progenitors in wild-type mice. The pattern of triple mutant multipotent progenitor response to growth factors resembles that of wild-type multipotent progenitors but not wild-type HSCs. These results demonstrate that p16Ink4a/p19Arf and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. These pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.     

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.     

A human colon cancer cell capable of initiating tumour growth in immunodeficient mice

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.     

Progenitor cell maintenance requires numb and numblike during mouse neurogenesis

Neurons in most regions of the mammalian nervous system are generated over an extended period of time during development. Maintaining sufficient numbers of progenitors over the course of neurogenesis is essential to ensure that neural cells are produced in correct numbers and diverse types. The underlying molecular mechanisms, like those governing stem-cell self-renewal in general, remain poorly understood. We report here that mouse numb and numblike (Nbl), two highly conserved homologues of Drosophila numb, play redundant but critical roles in maintaining neural progenitor cells during embryogenesis, by allowing their progenies to choose progenitor over neuronal fates. In Nbl mutant embryos also conditionally mutant for mouse numb in the nervous system, early neurons emerge in the expected spatial and temporal pattern, but at the expense of progenitor cells, leading to a nearly complete depletion of dividing cells shortly after the onset of neurogenesis. Our findings show that a shared molecular mechanism, with mouse Numb and Nbl as key components, governs the self-renewal of all neural progenitor cells, regardless of their lineage or regional identities.