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Tumours display considerable variation in the patterning and properties of angiogenic blood vessels, as well as in their responses to anti-angiogenic therapy. Angiogenic programming of neoplastic tissue is a multidimensional process regulated by cancer cells in concert with a variety of tumour-associated stromal cells and their bioactive products, which encompass cytokines and growth factors, the extracellular matrix and secreted microvesicles. In this Review, we discuss the extrinsic regulation of angiogenesis by the tumour microenvironment, highlighting potential vulnerabilities that could be targeted to improve the applicability and reach of anti-angiogenic cancer therapies.

Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies.

The effects of hyperthermia on the expression of p53, the apoptosis-associated genes Bax and Bcl-2, Notch and S100A4 have been studied in the HepG2 cell line and the HUT cell line derived from HepG2, adapted for growth in hyperthermic conditions. Hyperthermia inhibits cell proliferation and induces apoptosis. HepG2 and HUT cells differed in respect of anchorage to growth surface, degree of proliferation and apoptosis and expression of p53, Bax, Bcl-2, Notch, and S100A4 genes. The induction of apoptosis and the inhibition of cell proliferation occurred independently of p53, and independently also of involvement of the apoptosis family genes Bax and Bcl-2. We demonstrate novel and marked differences between transient heat shock and heat adaptation in respect of pathways of signaling and generation of phenotypic effects in vitro. Different signaling patterns have been identified here. Pathways of signaling by S100A4, by its interaction with and sequestration of p53, and by Notch also seem differentially operational in the induction of apoptosis, and both appear to be activated as alternative pathways in the context of hyperthermia signaling independently of p53.

Transplanted neural stem/precursor cells possess peculiar therapeutic plasticity and can simultaneously instruct several therapeutic mechanisms in addition to cell replacement. Here, we interrogated the therapeutic plasticity of neural stem/precursor cells after their focal implantation in the severely contused spinal cord. We injected syngeneic neural stem/precursor cells at the proximal and distal ends of the contused mouse spinal cord and analysed locomotor functions and relevant secondary pathological events in the mice, cell fate of transplanted neural stem/precursor cells, and gene expression and inflammatory cell infiltration at the injured site. We used two different doses of neural stem/precursor cells and two treatment schedules, either subacute (7 days) or early chronic (21 days) neural stem/precursor cell transplantation after the induction of experimental thoracic severe spinal cord injury. Only the subacute transplant of neural stem/precursor cells enhanced the recovery of locomotor functions of mice with spinal cord injury. Transplanted neural stem/precursor cells survived undifferentiated at the level of the peri-lesion environment and established contacts with endogenous phagocytes via cellular-junctional coupling. This was associated with significant modulation of the expression levels of important inflammatory cell transcripts in vivo. Transplanted neural stem/precursor cells skewed the inflammatory cell infiltrate at the injured site by reducing the proportion of 'classically-activated' (M1-like) macrophages, while promoting the healing of the injured cord. We here identify a precise window of opportunity for the treatment of complex spinal cord injuries with therapeutically plastic somatic stem cells, and suggest that neural stem/precursor cells have the ability to re-programme the local inflammatory cell microenvironment from a 'hostile' to an 'instructive' role, thus facilitating the healing or regeneration past the lesion.

Endometriosis affects women of reproductive age, causing infertility and pain. Although immune cells are recruited in endometriotic lesions, their role is unclear. Tie2-expressing macrophages (TEMs) have nonredundant functions in promoting angiogenesis and growth of experimental tumors. Here we show that human TEMs infiltrate areas surrounding newly formed endometriotic blood vessels. We set up an ad hoc mouse model in which TEMs, and not Tie2-expressing endothelial cells, are targeted. We transplanted in wild-type recipients bone marrow cells expressing a suicide gene (Herpes simplex virus type 1 thymidine kinase) under the Tie2 promoter/enhancer. TEMs infiltrated endometriotic lesions. TEM depletion by ganciclovir administration arrested the growth of established lesions, without toxicity. Lesion architecture was disrupted, with: i) loss of glandular organization, ii) reduced neovascularization, and iii) activation of caspase 3 in CD31(+) endothelial cells. Thus, TEMs are important for maintaining the viability of newly formed vessels and represent a potential therapeutic target in endometriosis.

During organ development and remodeling, macrophages support angiogenesis, not only by secreting proangiogenic growth factors and matrix-remodeling proteases, but also by physically interacting with the sprouting vasculature to assist the formation of complex vascular networks. Recent data further indicate that embryonic and tumor-associated macrophages express similar genetic programs, possibly suggesting convergent functions in organogenesis and tumorigenesis. In this article, we review the role of macrophages in development, tissue injury and regeneration, by focusing on the mechanisms used by subsets of these cells, such as the TIE2-expressing macrophages, to regulate angiogenesis and lymphangiogenesis in both fetal and post-natal life.

We previously showed that Tie2-expressing monocytes (TEMs) have nonredundant proangiogenic activity in tumors. Here, we compared the gene expression profile of tumor-infiltrating TEMs with that of tumor-associated macrophages (TAMs), spleen-derived Gr1+Cd11b+ neutrophils/myeloid-derived suppressor cells, circulating “inflammatory” and “resident” monocytes, and tumor-derived endothelial cells (ECs) by quantitative polymerase chain reaction–based gene arrays. TEMs sharply differed from ECs and Gr1+Cd11b+ cells but were highly related to TAMs. Nevertheless, several genes were differentially expressed between TEMs and TAMs, highlighting a TEM signature consistent with enhanced proangiogenic/tissue-remodeling activity and lower proinflammatory activity. We validated these findings in models of oncogenesis and transgenic mice expressing a microRNA-regulated Tie2-GFP reporter. Remarkably, resident monocytes and TEMs on one hand, and inflammatory monocytes and TAMs on the other hand, expressed coordinated gene expression profiles, suggesting that the 2 blood monocyte subsets are committed to distinct extravascular fates in the tumor microenvironment. We further showed that a prominent proportion of embryonic/fetal macrophages, which participate in tissue morphogenesis, expressed distinguishing TEM genes. It is tempting to speculate that Tie2+ embryonic/fetal macrophages, resident blood monocytes, and tumor-infiltrating TEMs represent distinct developmental stages of a TEM lineage committed to execute physiologic proangiogenic and tissue-remodeling programs, which can be coopted by tumors.

BackgroundMonocytes (CD14+ cells) expressing the receptor TIE2 are a highly angiogenic subset that are pivotal to neovascularisation in the tumour environment. We hypothesised that TIE2-expressing monocytes (TEMs) are also important in neovascularisation of ischaemic tissues.MethodsFlow cytometry was used to quantify circulating TEMs in 40 patients with critical limb ischaemia (20 age-matched and 20 healthy controls). RT-PCR was used to confirm TIE2 expression in FACS-sorted TEMs. ELISA was used to measure circulating levels of the TIE2 ligand angiopoietin 2 (ANG2). Mice were subjected to hindlimb ischaemia and TEMs quantified. In an additional study, haemopoietic stem/progenitor cells, isolated from Pgk-rtTA-miR-126T transgenic mice, were transduced ex vivo with a TRE-miR-Tie2-OFP lentiviral vector and used to reconstitute lethally irradiated mice. These mice were treated with alternate daily doses of doxycycline to silence Tie2 expression on TEMs, and hindlimb ischaemia (HLI) was induced. Conversely, bone-marrow-derived macrophages (BMDMs) were enforced to express Tie2 using a Pgk-Tie2 lentivirus and delivered into the ischaemic hindlimb. Recovery of ischaemia was measured with laser Doppler.FindingsFlow cytometry revealed a ten-fold higher number of circulating TEMs in patients with critical limb ischaemia than in matched controls (mean 3·52% [SE 0·28] vs 0·39 [0·09], p