Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.

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.

Tumor-associated macrophages (TAMs) promote key processes in tumor progression, like angiogenesis, immunosuppression, invasion, and metastasis. Increasing studies have also shown that TAMs can either enhance or antagonize the antitumor efficacy of cytotoxic chemotherapy, cancer-cell targeting antibodies, and immunotherapeutic agents--depending on the type of treatment and tumor model. TAMs also drive reparative mechanisms in tumors after radiotherapy or treatment with vascular-targeting agents. Here, we discuss the biological significance and clinical implications of these findings, with an emphasis on novel approaches that effectively target TAMs to increase the efficacy of such therapies.

Tumor-infiltrating myeloid cells convey proangiogenic programs that counteract the efficacy of antiangiogenic therapy. Here, we show that blocking angiopoietin-2 (ANG2), a TIE2 ligand and angiogenic factor expressed by activated endothelial cells (ECs), regresses the tumor vasculature and inhibits progression of late-stage, metastatic MMTV-PyMT mammary carcinomas and RIP1-Tag2 pancreatic insulinomas. ANG2 blockade did not inhibit recruitment of MRC1(+) TIE2-expressing macrophages (TEMs) but impeded their upregulation of Tie2, association with blood vessels, and ability to restore angiogenesis in tumors. Conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis. Our findings support a model wherein the ANG2-TIE2 axis mediates cell-to-cell interactions between TEMs and ECs that are important for tumor angiogenesis and can be targeted to induce effective antitumor responses.

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Angiogenic tumor vessels are promising targets for the activity and the selective delivery of cancer therapeutics. The bone marrow contributes different cell types to the tumor stroma, including hematopoietic cells and, as recently suggested, vascular endothelial cells (ECs). Thus, transplantation of genetically modified bone marrow progenitors may represent a vehicle for the transport of gene therapy to tumors. We transduced bone marrow progenitors with lentiviral vectors expressing genes from transcription-regulatory elements of Tie2/Tek gene. When tumors were grown in the transplanted mice, the new vector marked a distinct hematopoietic population that 'homed' to the tumor and closely interacted with vascular ECs at the tumor periphery. These Tie2-expressing mononuclear (TEM) cells had a distinguishable phenotype and were present selectively at angiogenic sites. Unexpectedly, we did not find bone marrow-derived ECs in tumor vessels when we transplanted bone marrow progenitors constitutively expressing a marker gene from the Tie2 or ubiquitously active promoters. By delivering a 'suicide' gene, we selectively eliminated the TEM cells and achieved substantial inhibition of angiogenesis and slower tumor growth without systemic toxicity. Thus, TEM cells may account for the proangiogenic activity of bone marrow-derived cells in tumors, may represent a new target for drug development and may provide the means for selective gene delivery and targeted inhibition of tumor angiogenesis.

MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), have been implicated in tumor progression. We recently described a lineage of mouse monocytes characterized by expression of the Tie2 angiopoietin receptor and required for the vascularization and growth of several tumor models. Here, we report that TIE2 expression in human blood identifies a subset of monocytes distinct from classical inflammatory monocytes and comprised within the less abundant "resident" population. These TIE2-expressing monocytes (TEMs) accounted for 2% to 7% of blood mononuclear cells in healthy donors and were distinct from rare circulating endothelial cells and progenitors. In human cancer patients, TEMs were observed in the blood and, intriguingly, within the tumors, where they represented the main monocyte population distinct from TAMs. Conversely, TEMs were hardly detected in nonneoplastic tissues. In vitro, TEMs migrated toward angiopoietin-2, a TIE2 ligand released by activated endothelial cells and angiogenic vessels, suggesting a homing mechanism for TEMs to tumors. IntroductionHematopoietic cells of diverse lineages contribute to tumor progression. [1][2][3][4][5][6][7] Among these cells, tumor-associated macrophages (TAMs) play important roles in tumorigenesis. 3,[8][9][10] TAMs derive from circulating monocytes, which differentiate into macrophages upon homing to tumors. In tumors, TAMs play dichotomous functions. Although TAMs may exert direct antitumor activities, 11,12 increasing data indicate that they are skewed by the tumor microenvironment to a protumoral phenotype. 8,13 Indeed, TAMs can blunt antitumor immunity and stimulate angiogenesis, cell migration, invasion, and metastasis. 3,9 Whereas the divergent TAM functions (ie, antitumoral and protumoral activities) are thought to be contextually modulated by the tumor microenvironment, 8 emerging data suggest that distinct subsets of circulating monocytes exist that are committed to specific functions, including tissue remodeling and proangiogenic activity. [14][15][16] In this regard, we recently identified in mouse tumor models a subset of tumor-infiltrating monocytes characterized by the expression of the angiopoietin receptor Tie2/Tek, 15,17 a molecule previously known to be restricted to endothelial and hematopoietic stem cells. 18,19 In mice, Tie2-expressing monocytes (TEMs) home to tumors, where they are required for angiogenesis. Indeed, selective elimination of TEMs by a suicide gene strategy prevented angiogenesis and induced tumor regression. 15,17 In these tumor models, angiogenesis was inhibited despite the fact that TAM recruitment to tumors was not impaired, indicating that a specific subset of myeloid-lineage cells was primarily responsible for promoting angiogenesis. This concept has received support from other studies that reported proangiogenic activity of selected myeloid cell subsets in mouse tumors, including VEGFR-1 ϩ CD11b ϩ myeloid cells, Gr-1 ϩ CD11b ϩ myeloid suppressor cells, and CD11c ϩ MHC-II ϩ dendritic cell prec...