Tumor cell-derived placental growth factor sensitizes antiangiogenic and antitumor effects of anti-VEGF drugs.

The role of placental growth factor (PlGF) in modulation of tumor angiogenesis and tumor growth remains an enigma. Furthermore, anti-PlGF therapy in tumor angiogenesis and tumor growth remains controversial in preclinical tumor models. Here we show that in both human and mouse tumors, PlGF induced the formation of dilated and normalized vascular networks that were hypersensitive to anti-VEGF and anti-VEGFR-2 therapy, leading to dormancy of a substantial number of avascular tumors. Loss-of-function using plgf shRNA in a human choriocarcinoma significantly accelerated tumor growth rates and acquired resistance to anti-VEGF drugs, whereas gain-of-function of PlGF in a mouse tumor increased anti-VEGF sensitivity. Further, we show that VEGFR-2 and VEGFR-1 blocking antibodies displayed opposing effects on tumor angiogenesis. VEGFR-1 blockade and genetic deletion of the tyrosine kinase domain of VEGFR-1 resulted in enhanced tumor angiogenesis. These findings demonstrate that tumor-derived PlGF negatively modulates tumor angiogenesis and tumor growth and may potentially serve as a predictive marker of anti-VEGF cancer therapy.

Antiangiogenic agents significantly improve survival in tumor-bearing mice by increasing tolerance to chemotherapy-induced toxicity.

Chemotherapy-induced broad toxicities are the leading cause of the drug-induced mortality in cancer patients. Antiangiogenic drugs (ADs) in combination with chemotherapy are widely used as front-line therapy for the treatment of various human cancers. However, the beneficial mechanisms underlying combination therapy are poorly understood. Here we show that, in several murine tumor models, administration of sunitinib markedly reduced chemotherapy-induced bone marrow toxicity. Intriguingly, in a sequential treatment regimen, delivery of ADs followed by chemotherapy demonstrated superior survival benefits compared with simultaneous administration of two drugs. In murine tumor models, we show that VEGF increased chemotoxicity by synergistically suppressing bone marrow hematopoiesis with cytostatic drugs. These findings shed light on molecular mechanisms by which ADs in combination with chemotherapy produce survival benefits in cancer patients and provide conceptual information guiding future designs of clinical trials, current practice, and optimization of ADs for the treatment of cancer.

VEGFR1-mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy.

VEGF coordinates complex regulation of cellular regeneration and interactions between endothelial and perivascular cells; dysfunction of the VEGF signaling system leads to retinopathy. Here, we show that systemic delivery of VEGF and placental growth factor (PlGF) by protein implantation, tumors, and adenoviral vectors ablates pericytes from the mature retinal vasculature through the VEGF receptor 1 (VEGFR1)-mediated signaling pathway, leading to increased vascular leakage. In contrast, we demonstrate VEGF receptor 2 (VEGFR2) is primarily expressed in nonvascular photoreceptors and ganglion cells. Moreover, blockade of VEGFR1 but not VEGFR2 significantly restores pericyte saturation in mature retinal vessels. Our findings link VEGF and PlGF to cancer-associated retinopathy, reveal the molecular mechanisms of VEGFR1 ligand-mediated retinopathy, and define VEGFR1 as an important target of antiangiogenic therapy for treatment of retinopathy.

Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature.

Vascular functions of PlGF remain poorly understood and controversial. Here, we show that tumor cell-derived PlGF-1 and PlGF-2 displayed significant remodeling effects on the tumor vasculature, leading to a normalized vascular phenotype and improved functions against leakage. In two murine tumor models, that is, T241 fibrosarcoma and Lewis lung carcinoma, stable expression of PlGF-1 and PlGF-2 in tumor cells resulted in significant reduction of tumor microvascular density and branch formation. Markedly, the vasculature in PlGF-expressing tumors consisted of relatively large-diameter microvessels with substantial improvement of pericyte coverage. Similarly, PlGF-induced vascular normalization and remodeling were also observed in a spontaneous human choriocarcinoma that expressed endogenous PlGF. Our findings shed light on functions of PlGF as a vascular remodeling factor that normalizes the tumor vasculature and thus may have conceptual implications of cancer therapy.

Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arterial-lymphatic conduits in zebrafish and glass catfish.

The blood and lymphatic vasculatures are structurally and functionally coupled in controlling tissue perfusion, extracellular interstitial fluids, and immune surveillance. Little is known, however, about the molecular mechanisms that underlie the regulation of bloodlymphatic vessel connections and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish (Kryptopterus bicirrhis) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO-induced vascular relaxation, which led to blood perfusion into the lymphatic system. NO blockage almost completely abrogated hypoxia-induced ALC relaxation and lymphatic perfusion. These findings uncover mechanisms underlying hypoxia-induced oxygen compensation by perfusion of existing lymphatics in fish. Our results might also imply that the hypoxia-induced NO pathway contributes to development of progression of pathologies, including promotion of lymphatic metastasis by modulating arterial-lymphatic conduits, in the mammalian system.

FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue.

Adipogenesis is spatiotemporally coupled to angiogenesis throughout adult life, and the interplay between these two processes is communicated by multiple factors. Here we show that in a transgenic mouse model, increased expression of forkhead box C2 (FOXC2) in the adipose tissue affects angiogenesis, vascular patterning, and functions. White and brown adipose tissues contain a considerably high density of microvessels appearing as vascular plexuses, which show redistribution of vascular smooth muscle cells and pericytes. Dysfunction of these primitive vessels is reflected by impairment of skin wound healing. We further provide a mechanistic insight of the vascular phenotype by showing that FOXC2 controls Ang-2 expression by direct activation of its promoter in adipocytes. Remarkably, an Ang-2-specific antagonist almost completely reverses this vascular phenotype. Thus, the FOXC2-Ang-2 signaling system is crucial for controlling adipose vascular function, which is part of an adaptation to increased adipose tissue metabolism.

Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis.

Tumors produce multiple growth factors, but little is known about the interplay between various angiogenic factors in promoting tumor angiogenesis, growth, and metastasis. Here we show that 2 angiogenic factors frequently upregulated in tumors, PDGF-BB and FGF2, synergistically promote tumor angiogenesis and pulmonary metastasis. Simultaneous overexpression of PDGF-BB and FGF2 in murine fibrosarcomas led to the formation of high-density primitive vascular plexuses, which were poorly coated with pericytes and VSMCs. Surprisingly, overexpression of PDGF-BB alone in tumor cells resulted in dissociation of VSMCs from tumor vessels and decreased recruitment of pericytes. In the absence of FGF2, capillary ECs lacked response to PDGF-BB. However, FGF2 triggers PDGFR-alpha and -beta expression at the transcriptional level in ECs, which acquire hyperresponsiveness to PDGF-BB. Similarly, PDGF-BB-treated VSMCs become responsive to FGF2 stimulation via upregulation of FGF receptor 1 (FGFR1) promoter activity. These findings demonstrate that PDGF-BB and FGF2 reciprocally increase their EC and mural cell responses, leading to disorganized neovascularization and metastasis. Our data suggest that intervention of this non-VEGF reciprocal interaction loop for the tumor vasculature could be an important therapeutic target for the treatment of cancer and metastasis.

R Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways.

In a fast-growing malignant tissue, tumor blood vessels are exposed to multiple growth factors and cytokines. Although the role of individual factors and their signaling pathways in regulation of tumor neovascularization is relatively well-studied, little is known about complex interactions between these factors and their cooperative effects in promoting tumor angiogenesis and metastasis. Our recent studies show that quiescent vascular endothelial cells usually remaining silence to platelet-derived growth factor (PDGF)-BB stimulation acquire their hyperresponsiveness after stimulation with fibroblast growth factor (FGF)-2, which transcriptionally switches on PDGF receptor expression in the activated endothelial cells. Interestingly, PDGF-BB also transduces positive feedback signals to the FGF-2 signaling system by amplifying its receptor expression in vascular mural cells. These uncoordinated reciprocal interactions in the tumor environment lead to the formation of disorganized and primitive vasculatures that facilitate tumor growth and metastasis in mice. These findings provide an example of complex interaction between tumor angiogenic factors. Thus, therapeutic development of antiangiogenic agents for the treatment of cancer should be aimed to block multiple angiogenic signaling pathways and their interactive loops.

Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts.

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.

Mouse corneal lymphangiogenesis model.

This protocol describes a powerful in vivo method to quantitatively study the formation of new lymphatic vessels in the avascular cornea without interference of pre-existing lymphatics. Implantation of 100 ng of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, VEGF-C or fibroblast growth factor-2, together with slow-release polymers, into a surgically created micropocket in the mouse cornea elicits a robust lymphangiogenic response. Newly formed lymphatic vessels are detected by immunohistochemical staining of the flattened corneal tissue with lymphatic endothelial-specific markers such as lymphatic vessel endothelial hyaluronan receptor-1; less-specific markers such as vascular endothelial growth factor receptor 3 may also be used. Lymphatic vessel growth in relation to hemangiogenesis can be readily detected starting at day 5 or 6 after pellet implantation and persists for ∼14 d. This protocol offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function.

PDGF-BB modulates hematopoiesis and tumor angiogenesis by inducing erythropoietin production in stromal cells.

The platelet-derived growth factor (PDGF) signaling system contributes to tumor angiogenesis and vascular remodeling. Here we show in mouse tumor models that PDGF-BB induces erythropoietin (EPO) mRNA and protein expression by targeting stromal and perivascular cells that express PDGF receptor-ß (PDGFR-ß). Tumor-derived PDGF-BB promoted tumor growth, angiogenesis and extramedullary hematopoiesis at least in part through modulation of EPO expression. Moreover, adenoviral delivery of PDGF-BB to tumor-free mice increased both EPO production and erythropoiesis, as well as protecting from irradiation-induced anemia. At the molecular level, we show that the PDGF-BB-PDGFR-bß signaling system activates the EPO promoter, acting in part through transcriptional regulation by the transcription factor Atf3, possibly through its association with two additional transcription factors, c-Jun and Sp1. Our findings suggest that PDGF-BB-induced EPO promotes tumor growth through two mechanisms: first, paracrine stimulation of tumor angiogenesis by direct induction of endothelial cell proliferation, migration, sprouting and tube formation, and second, endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia.

Pathological angiogenesis facilitates tumor cell dissemination and metastasis.

Clinically detectable metastases represent an ultimate consequence of the metastatic cascade that consists of distinct processes including tumor cell invasion, dissemination, metastatic niche formation, and re-growth into a detectable metastatic mass. Although angiogenesis is known to promote tumor growth, its role in facilitating early events of the metastatic cascade remains poorly understood. We have recently developed a zebrafish tumor model that enables us to study involvement of pathological angiogenesis in tumor invasion, dissemination and metastasis. This non-invasive in vivo model allows detection of single malignant cell dissemination under both normoxia and hypoxia. Further, hypoxia-induced VEGF significantly facilitates tumor cell invasion and dissemination. These findings demonstrate that VEGF-induced pathological angiogenesis is essential for tumor dissemination and further corroborates potentially beneficial effects of clinically ongoing anti-VEGF drugs for the treatment of various malignancies.