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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.

Cell therapies offer unquestionable promises for the treatment, and in some cases even the cure, of complex diseases. As we start to see more of these therapies gaining market authorization, attention is turning to the bioprocesses used for their manufacture, in particular the challenge of gaining higher levels of process control to help regulate cell behavior, manage process variability, and deliver product of a consistent quality. Many processes already incorporate the measurement of key markers such as nutrient consumption, metabolite production, and cell concentration, but these are often performed off-line and only at set time points in the process. Having the ability to monitor these markers in real-time using in-line sensors would offer significant advantages, allowing faster decision-making and a finer level of process control. In this study, we use Raman spectroscopy as an in-line optical sensor for bioprocess monitoring of an autologous T-cell immunotherapy model produced in a stirred tank bioreactor system. Using reference datasets generated on a standard bioanalyzer, we develop chemometric models from the Raman spectra for glucose, glutamine, lactate, and ammonia. These chemometric models can accurately monitor donor-specific increases in nutrient consumption and metabolite production as the primary T-cell transition from a recovery phase and begin proliferating. Using a univariate modeling approach, we then show how changes in peak intensity within the Raman spectra can be correlated with cell concentration and viability. These models, which act as surrogate markers, can be used to monitor cell behavior including cell proliferation rates, proliferative capacity, and transition of the cells to a quiescent phenotype. Finally, using the univariate models, we also demonstrate how Raman spectroscopy can be applied for real-time monitoring. The ability to measure these key parameters using an in-line Raman optical sensor makes it possible to have immediate feedback on process performance. This could help significantly improve cell therapy bioprocessing by allowing proactive decision-making based on real-time process data. Going forward, these types of in-line sensors also open up opportunities to improve bioprocesses further through concepts such as adaptive manufacturing.

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.

Thousands of cancer patients are currently in clinical trials evaluating antiangiogenic therapy in the neoadjuvant setting, which is the treatment of localized primary tumors prior to surgical intervention. The rationale is that shrinking a tumor will improve surgical outcomes and minimize growth of occult micrometastatic disease-thus delaying post-surgical recurrence and improving survival. But approved VEGF pathway inhibitors have not been tested in clinically relevant neoadjuvant models that compare pre- and post-surgical treatment effects. Using mouse models of breast, kidney, and melanoma metastasis, we demonstrate that primary tumor responses to neoadjuvant VEGFR TKI treatment do not consistently correlate with improved post-surgical survival, with survival worsened in certain settings. Similar negative effects did not extend to protein-based VEGF pathway inhibitors and could be reversed with altered dose, surgical timing, and treatment duration, or when VEGFR TKIs are combined with metronomic 'anti-metastatic' chemotherapy regimens. These studies represent the first attempt to recapitulate the complex clinical parameters of neoadjuvant therapy in mice and identify a novel tool to compare systemic antiangiogenic treatment effects on localized and disseminated disease.

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.

A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2-expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10-fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two-fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2-negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral-based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients.

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

Rationale & Hypothesis: Combretastatin A-4-phosphate (CA-4-P), a tubulin-binding, vascular-disrupting agent (VDA), induces rapid and selective tumor vascular shutdown and secondary tumor cell death. However, acute revascularization contributes to treatment resistance. A group of myeloid cells in tumors, tumor associated macrophages (TAMs) are known to drive angiogenesis and tumor progression. A potently pro-angiogenic subpopulation of TAMs has also been identified – Tie2 expressing monocytes (TEMs). We hypothesize that TAMs and/or TEMs infiltrate CA-4-P-treated tumors and promote tumor recovery. Methodology, Results & Conclusions: Late-stage Polyoma Middle T (PyMT) spontaneous murine breast adenocarcinomas were excised from CA-4-P- and saline-treated mice. Significant tumor necrosis was evident 24h following a single injection of 50mg/kg CA-4-P (19.37 ± 4.60%) compared with saline controls (1.76 ± 0.34%). This was accompanied by tumor infiltration of F4/80+ TAMs (5.61 ± 0.46% of tumor volume w.r.t. 0.71± 0.08% in controls). Significant increases in the number of MMP-9 expressing macrophages, primarily TEMs, were evident following CA-4-P treatment (5.72 ± 0.34% compared with 2.66 ± 0.25% in controls). TEMs were constitutive expressers of hypoxia-regulated CXCR4, which interacts via CXCL12 as a monocyte/macrophage chemoattractant. AMD3100, a chemical antagonist of CXCR4, was employed in combination with CA-4-P to prevent TEM recruitment into drug-treated tumors. TEM numbers were significantly depleted, which corresponded to heightened vascular damage (27% tumor necrosis w.r.t. 8% in CA-4-P alone). These results were replicated in a subcutaneous N202 murine mammary carcinoma model following 3 daily doses of 50mg/kg CA-4-P. Tumor growth was also significantly reduced at 72h in the combined treatment group (mean tumor volume ∼200mm3 w.r.t. ∼300mm3 in CA-4-P alone). In addition, specific TEM depletion was achieved in the N202 carcinoma model via genetic manipulation and bone marrow transplantation. Tumor necrosis was increased significantly when CA-4-P was administered in combination with TEM depletion (22.45%), compared to saline controls (0.37%) and CA-4-P therapy alone (12.89%). This was attributed to a significant reduction in MMP-9 positive TEMs (0.08 ± 0.18% compared with 1.37 ± 0.21% in saline controls and 3.67± 0.11% in CA-4-P treated tumors). Collectively, these data suggest that TEMs protect against CA-4-P or promote recovery of tumors following CA-4-P treatment. Therefore, targeting TEMs, in combination with vascular-disrupting agents such as CA-4-P may provide a novel therapeutic strategy for solid tumors. A.F. Welford was funded by a Cancer Research UK PhD studentship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2011-2849