David K. Jin

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 α4β1), 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.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo−/−) or TPO receptor–deficient (Mpl−/−) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo−/− and Mpl−/− mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4+ megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

PML inhibits HIF-1α translation and neoangiogenesis through repression of mTOR

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1α (HIF-1α) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis.

Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization

Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell-derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.

Regulation of Vasculogenesis by Platelet-Mediated Recruitment of Bone Marrow–Derived Cells

Bone marrow–derived cells contribute to physiological and pathological vascular remodeling throughout ontogenesis and adult life. During tissue regeneration and tumor growth, the release of cytokines and chemokines mediates the recruitment of hematopoietic and endothelial progenitor cells that contribute to the assembly of neovessels. Current evidence implies that platelets contribute structurally and instructively to vascular remodeling. Platelets adhere almost immediately to exposed or activated endothelium, and they are major storage and delivery vehicles for pro- and antiangiogenic growth factors including VEGF-A and thrombospondin (TSP), and cytokines and chemokines, such as stromal-derived factor 1 (SDF-1). By site-specific deployment of these factors, platelets orchestrate the local angiogenic stimulus within a tissue and direct the recruitment and differentiation of circulating bone marrow–derived cells. These insights have profound clinical implications; inhibition of platelet-deployed growth factors or their receptors may be an effective strategy to block tumor growth, whereas activation of these pathways may be used to accelerate revascularization and tissue regeneration.

Fetal Stromal-Dependent Paracrine and Intracrine Vascular Endothelial Growth Factor-A/Vascular Endothelial Growth Factor Receptor-1 Signaling Promotes Proliferation and Motility of Human Primary Myeloma Cells

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.

Tumor vasculature address book: Identification of stage-specific tumor vessel zip codes by phage display

Using in vivo phage display technology with murine tumorigenesis models, two reports have identified peptide motifs that selectively home to distinct molecular vascular targets in a tumor type- and stage-specific manner (Hoffman et al., 2003 and Joyce et al., 2003 [this issue of Cancer Cell]). By probing the surface-protein repertoire of these unique vascular beds, a pioneering draft of a molecular roadmap to decipher the heterogeneity of the vascular system in the context of carcinogenic progression has been plotted. Analysis of these phage peptides that differentially home to dysplastic or invasive tumor vasculature will lay the foundation for identifying unique functional tumor vascular-specific motifs that could potentially be applied to targeted therapeutic and imaging modalities.

SURROGATE MARKERS PREDICT ANGIOGENIC POTENTIAL AND SURVIVAL IN PATIENTS WITH GLIOBLASTOMA MULTIFORME

OBJECTIVEThe neovascularization of malignant brain tumors is a poorly understood phenomenon. Radiographic and histological evidence of increased vascularity correlate with clinical grade of gliomas. However, a quantitative noninvasive assay to assess glioma vascularity and associated clinical aggressiveness has not been developed. Circulating endothelial progenitor cells are unique vascular precursors recruited from the bone marrow through the circulation to form new tumor blood vessels. These cells were measured in patients undergoing surgery for glioblastoma multiforme (GBM). We hypothesized that this might reflect the extent of tumor vascularity, predict prognosis, or be useful as an assay to assess response to antiangiogenesis therapies. In addition, we report on a novel in vitro assay to assess the proangiogenic activity within the plasma samples obtained from glioma patients. METHODSFifty-six patients with various-grade gliomas had peripheral venous blood collected at the time of surgery and at subsequent visits during the follow-up period. The blood was separated into plasma and cellular fractions. The plasma was utilized in a human umbilical vein endothelial cell-based angiogenic assay. The cellular fraction containing endothelial progenitor cells was isolated, and specific cellular phenotypes were immunologically separated and counted using flow cytometry. Pathological samples were reviewed at the time of initial resection, and each patients clinical course was monitored until the time of manuscript submission. RESULTSPlasma derived from peripheral blood of patients with GBM scored significantly higher on the functional angiogenic scale compared with plasma derived from patients with low-grade gliomas and from controls. In addition, all patients with GBM had measurable numbers of bone marrow-derived endothelial precursor cells coexpressing CD133 and vascular endothelial growth factor receptor 2 in their peripheral circulation at the time of tumor resection. These cells range from less than 0.1% to 1.6% of the entire circulating mononuclear white blood cell population, or approximately 200 000 cells in some patients. A statistically significant relationship was observed between the percentage of endothelial progenitor cells in the peripheral blood at the time of initial GBM resection and survival. CONCLUSIONThese studies suggest that plasma and circulating CD133+ vascular endothelial growth factor receptor 2+ proangiogenic cells are present in the peripheral blood of patients with glioma and can be used as a surrogate biomarker to measure tumor angiogenicity. These cells can be measured at the time of diagnosis and monitored in the postoperative period. These assays can be used to predict tumor aggressiveness. Also promising is their potential to identify patients with increased angiogenic activity who might respond maximally to antiangiogenesis therapies or to assess tumor response in patients using those therapies as the use of these adjuvant molecular modalities becomes more prevalent in neuro-oncology.

Adenovirus Vector E4 Gene Regulates Connexin 40 and 43 Expression in Endothelial Cells via PKA and PI3K Signal Pathways

Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4−, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intra-tracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways.