Farbod Shojaei

Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells

Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.

Bv8 regulates myeloid-cell-dependent tumour angiogenesis

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models

Recent studies suggest that tumor-associated CD11b+Gr1+ myeloid cells contribute to refractoriness to antiangiogenic therapy with an anti-VEGF-A antibody. However, the mechanisms of peripheral mobilization and tumor-homing of CD11b+Gr1+ cells are unclear. Here, we show that, compared with other cytokines [granulocyte-macrophage colony stimulating factor (GM-CSF), stromal derived factor 1α, and placenta growth factor], G-CSF and the G-CSF-induced Bv8 protein have preferential expression in refractory tumors. Treatment of refractory tumors with the combination of anti-VEGF and anti-G-CSF (or anti-Bv8) reduced tumor growth compared with anti-VEGF-A monotherapy. Anti-G-CSF treatment dramatically suppressed circulating or tumor-associated CD11b+Gr1+ cells, reduced Bv8 levels, and affected the tumor vasculature. Conversely, G-CSF delivery to animals bearing anti-VEGF sensitive tumors resulted in reduced responsiveness to anti-VEGF-A treatment through induction of Bv8-dependent angiogenesis. We conclude that, at least in the models examined, G-CSF expression by tumor or stromal cells is a determinant of refractoriness to anti-VEGF-A treatment.

HGF/c-Met Acts as an Alternative Angiogenic Pathway in Sunitinib- Resistant Tumors

Molecular and cellular mechanisms underlying resistance/low responsiveness to antiangiogenic compounds are under extensive investigations. Both populations of tumor and stroma (nontumor compartment) seem to contribute in inherent/acquired resistance to antiangiogenic therapy. Here, investigating in vivo efficacy of sunitinib in experimental models resulted in the identification of tumors that were resistant/sensitive to the therapy. Analysis of tumor protein lysates indicated a greater concentration of hepatocyte growth factor (HGF) in resistant tumors than in sensitive ones. In addition, using flow cytometry, c-Met expression was found to be significantly higher in endothelial cells than in tumor cells, suggesting that HGF might target the vascular endothelial cells in resistant tumors. Combination of sunitinib and a selective c-Met inhibitor significantly inhibited tumor growth compared with sunitinib or c-Met inhibitor alone in resistant tumors. Histology and in vitro analyses suggested that combination treatment mainly targeted the vasculature in the resistant tumors. Conversely, systemic injection of HGF in the sensitive tumor models conferred resistance to sunitinib through maintenance of tumor angiogenesis. In conclusion, our study indicates a role for HGF/c-Met pathway in development of resistance to antiangiogenic therapy and suggests a potential strategy to circumvent resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitor in the clinic.

Role of Bv8 in neutrophil-dependent angiogenesis in a transgenic model of cancer progression

The secreted Bv8 protein has been recently characterized as a regulator of myeloid cell mobilization and a neutrophil-derived mediator of tumor angiogenesis in several xenografts, but its role in tumor progression in an endogenous setting was unknown. The rat insulin promoter (RIP)–T-antigen (Tag) is a well characterized transgenic mouse model of multistage pancreatic β-cell tumorigenesis. Also, the role of neutrophils in RIP-Tag angiogenic switching, as assessed by systemic ablation using anti-Gr1 antibodies at different stages of tumor progression, has been recently described. Here, we show that early treatment of RIP-Tag mice with anti-Bv8 antibodies resulted in a significant reduction in the number of angiogenic islets relative to control antibody-treated mice, implicating Bv8 in the angiogenic switch during neoplasia. Histological analysis showed a significant reduction in vascular surface areas in hyperplastic and angiogenic lesions in pancreatic islets from anti-Bv8-treated mice. Anti-Bv8 treatment also inhibited the mobilization and homing of CD11b+Gr1+ cells to the peripheral blood and the emerging neoplastic lesions. However, anti-Bv8 treatment had no effect on tumor vascularization or burden when initiated at later stages of tumor progression. The stage-dependent efficacy of anti-Bv8 treatment appears remarkably similar to that reported after neutrophil ablation, suggesting that Bv8 is an important mediator of neutrophil-dependent angiogenesis in this transgenic model. In summary, our studies verify a role for Bv8 in the mobilization and recruitment of myeloid cells and in the induction of tumor angiogenesis in the early stages of neoplastic progression.

Refractoriness to Antivascular Endothelial Growth Factor Treatment: Role of Myeloid Cells

CD11b+Gr1+ cells, which include neutrophils, macrophages, and myeloid-derived suppressor cells, have been shown to contribute to tumor angiogenesis. Recently, we found that accumulation of CD11b+Gr1+ in tumors renders them refractory to angiogenic blockade by vascular endothelial growth factor (VEGF) antibodies. This effect was traced to a pathway of CD11b+Gr1+-mediated angiogenesis that is, at least in part, driven by the secreted protein Bv8, which is up-regulated by the important myeloid growth factor granulocyte colony-stimulating factor (G-CSF). Thus, G-CSF may promote tumor angiogenesis through a Bv8-dependent pathway that bypasses VEGF and renders tumors refractory to anti-VEGF therapy.

Role of myeloid cells in tumor angiogenesis and growth

Cells of the innate immune system have a key role in maintaining homeostasis by providing the first line of defense against many pathogens. Innate immunity can also modulate the activity of acquired immunity by several mechanisms. However, subsets of myeloid cells can facilitate tumor growth, because these cells produce angiogenic factors and can also prevent the immune system from attacking tumor cells. Recent studies also emphasize the role of myeloid cells in mediating refractoriness to anti-VEGF treatments. This function of myeloid cells occurs through a proangiogenic pathway that is, at least in part, driven by the secreted protein Bv8. This review summarizes recent findings on the complex role of bone marrow-derived cells in tumor growth.

Role of the microenvironment in tumor growth and in refractoriness/resistance to anti-angiogenic therapies.

Angiogenesis is critical for growth of many tumor types and the development of anti-angiogenic agents opened a new era in cancer therapy. However, similar to other anti-cancer therapies, inherent/acquired resistance to anti-angiogenic drugs may occur in cancer patients leading to disease recurrence. Recent studies in several experimental models suggest that both tumor and non-tumor (stromal) cell types may be involved in the reduced responsiveness to the treatments. The current review focuses on the role of stromal cells in tumor growth and in refractoriness to anti-VEGF treatment.

Antiangiogenic therapy for cancer: an update.

The identification and characterization of several important regulators of angiogenesis, which led to Food and Drug Administration approval of the first antiangiogenic drugs, has opened a new era in cancer therapy. This article focuses on the clinical progress in targeting one of the major regulators of angiogenesis, vascular endothelial growth factor-A and also discusses some recent advances in the elucidation of potential cellular and molecular mechanisms underlying refractoriness or resistance to antiangiogenic therapies.

Antiangiogenesis to treat cancer and intraocular neovascular disorders.

Identification and characterization of several important regulators of angiogenesis, and FDA approval of the first antiangiogenic drugs, has opened a new era in the therapy of cancer and neovascular age-related macular degeneration. This brief review focuses on the progress in targeting one of the major regulators of angiogenesis, VEGF-A, and also discusses potential cellular and molecular mechanisms underlying resistance to antiangiogenic treatments.