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.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit + stem/progenitors. In MMP-9 −/− mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.

Summary The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

About 50% of cancer patients receive radiation therapy. Here we investigated the hypothesis that tumor response to radiation is determined not only by tumor cell phenotype but also by microvascular sensitivity. MCA/129 fibrosarcomas and B16F1 melanomas grown in apoptosis-resistant acid sphingomyelinase (asmase)-deficient or Bax-deficient mice displayed markedly reduced baseline microvascular endothelial apoptosis and grew 200 to 400% faster than tumors on wild-type microvasculature. Thus, endothelial apoptosis is a homeostatic factor regulating angiogenesis-dependent tumor growth. Moreover, these tumors exhibited reduced endothelial apoptosis upon irradiation and, unlike tumors in wild-type mice, they were resistant to single-dose radiation up to 20 grays (Gy). These studies indicate that microvascular damage regulates tumor cell response to radiation at the clinically relevant dose range.

Emerging data suggest that a subset of circulating human CD34+ cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 ± 0.3% of CD34+ cells isolated from fetal liver (FL), 2 ± 0.5% from mobilized peripheral blood, and 1.4 ± 0.5% from cord blood were VEGFR-2+. In addition, most CD34+VEGFR-2+ cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34+ cells phenotypically identifies a unique population of CEPs. CD34+VEGFR-2+ cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34+VEGFR-2+ cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34+ cells that give rise to endothelial colonies, CD34+ cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2+ cells with VEGF and FGF-2 resulted in differentiation of AC133+VEGFR-2+ cells into adherent AC133−VEGFR-2+Ac-LDL+(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133+VEGFR-2+ cells. These data suggest that circulating CD34+ cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.

Emerging evidence suggests that bone marrow-derived endothelial, hematopoietic stem and progenitor cells contribute to tissue vascularization during both embryonic and postnatal physiological processes. Recent preclinical and pioneering clinical studies have shown that introduction of bone marrow-derived endothelial and hematopoietic progenitors can restore tissue vascularization after ischemic events in limbs, retina and myocardium. Corecruitment of angiocompetent hematopoietic cells delivering specific angiogenic factors facilitates incorporation of endothelial progenitor cells (EPCs) into newly sprouting blood vessels. Identification of cellular mediators and tissue-specific chemokines, which facilitate selective recruitment of bone marrow-derived stem and progenitor cells to specific organs, will open up new avenues of research to accelerate organ vascularization and regeneration. In addition, identification of factors that promote differentiation of the progenitor cells will permit functional incorporation into neo-vessels of specific tissues while diminishing potential toxicity to other organs. In this review, we discuss the clinical potential of vascular progenitor and stem cells to restore long-lasting organ vascularization and function.

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