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Impaired tumor microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression

Dana M. Brantley-Sieders^*, Wei Bin Fang, Donna J. Hicks^*, Guanglei Zhuang, Yu Shyr^, and Jin Chen^*,,,,1

Department of Medicine,
^* Division of Rheumatology and Immunology,
Department of Cancer Biology,
Department of Biostatistics,
Vanderbilt-Ingram Cancer Center,
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

¹ Correspondence: Department of Medicine, Vanderbilt University School of Medicine, A-4323 MCN, 1161 21st Ave., South, Nashville, TN 37232-2363, USA. E-mail: jin.chen{at}vanderbilt.edu

SPECIFIC AIMS

We investigated the role of host EphA2-deficiency on tumor angiogenesis and progression in vivo. We wanted to determine whether deletion of EphA2 in tumor-bearing hosts, particularly within tumor endothelium, could modulate tumor neovascularization, tumor volume, and metastasis.

PRINCIPAL FINDINGS

1. EphA2-deficiency impairs tumor volume, microvascular density, and lung metastasis in vivo
EphA2-deficient mice were backcrossed onto a Balb/c genetic background for seven generations to produce genetically compatible female recipients for transplantation of Balb/c-derived, 4T1 mammary adenocarcinoma cells. Ephrin-A1-expressing 4T1 tumor cells were orthotopically transplanted into the mammary gland fat pads of EphA2-deficient females or wild-type and heterozygous littermate controls. Primary tumors and lungs were harvested 22 days post-transplantation for assessment of tumor volume and microvascular density, as well as the number of metastases. We observed significantly decreased tumor volume in EphA2-deficient recipients vs. wild-type controls (Fig. 1 A), with 3 of 15 EphA2-deficient recipients developing microscopic, nonpalpable tumors and 2 of 15 wild-type control animals dying before the endpoint. Microvascular density was also decreased in orthotopic tumors isolated from EphA2-deficient recipients relative to wild-type controls based on quantification of von Willebrand factor (vWF) expression (Fig. 1B ) or CD31 (data not shown) in tumor sections. Quantification of total lesions in cleared lungs by staining with hematoxylin revealed a significant decrease in EphA2-deficient tumor recipients relative to controls (Fig. 1C, D ).

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of host-EphA2-deficiency on orthotopic tumor angiogenesis and metastasis. 4T1 tumors were transplanted orthotopically into one inguinal mammary gland fat pad of Balb/c EphA2 +/+, +/–, or –/– female mice. Primary tumors and lungs were collected 22 days post-transplantation and tumor volume, microvascular density, and surface lung lesions wee scored. A) Tumor volume was significantly decreased in EphA2-deficient recipients. B) Tumor vascularity was scored in sections from EphA2 –/– vs. +/+ or +/– control recipients based on immunostaining for the vascular endothelial markers von Willebrand factor (vWF). Microvascular density (MVD) was quantified by calculating vWF pixel area in x10 fields. MVD was significantly decreased in tumors from EphA2 –/– recipients. C) Total metastatic lesions were counted in cleared lungs stained with hematoxylin, and a lower number of lesions was detected in EphA2 –/– recipients relative to controls. D) Cleared lungs from EphA2 +/+ or –/– recipients were stained with hematoxylin to visualize surface and internal metastatic lesions (arrows indicate surface lesions, arrowheads indicate internal lesions). Scale bar = 5 mm in upper panels. Lower panel shows higher magnification of the EphA2 +/+ recipient lung with an internal lesion (arrowhead) adjacent to a larger surface lesions (arrow). Scale bar = 50 µm lower panel.

2. EphA2-deficient endothelial cells fail to contribute to tumor vasculature and support tumor progression in vivo
To test whether the defects in tumor progression and metastasis in EphA2-deficient animals were endothelial cell intrinsic, we performed tumor cell/endothelial cell cotransplantation experiments using EphA2 +/+, +/–, or –/– murine pulmonary microvascular endothelial cells (MPMEC) expressing ß-galactosidase. Tumor-endothelial plugs were harvested 7 days post-transplantation, sectioned, and costained with X-gal and CD31 to identify donor endothelial cells. As shown in Fig. 2 A, B, there was a marked reduction in the number of LacZ+ EphA2-deficient endothelial cells relative to the number of LacZ+, EphA2 +/+ control endothelial cells in plugs. Decreased number of donor EphA2-deficient endothelial cells appears to be due to increased apoptosis, as there is a significant increase in LacZ+/TUNEL+ nuclei in EphA2–/– cells, compared with EphA2+/+ controls within the tumor plug (Fig. 2E, F ). Morphologically, exogenous LacZ+/CD31+ EphA2+/+ endothelial cells coalesced, forming networks of interconnected structures surrounding clusters of tumor cells. In contrast, EphA2-deficient donor endothelial cells remained isolated and failed to form interconnected networks surrounding tumor cells (Fig. 2C, D ).

View larger version (59K): [in this window] [in a new window]   Figure 2. Effect of EphA2-deficient endothelial cells on tumor volume on cotransplantation. 4T1 tumor cells were mixed with EphA2+/+, +/–, or –/– primary mouse pulmonary microvascular endothelial cells (MPMEC) in Matrigel and cotransplanted subcutaneously into Balb/c nude female mice. Tumors were collected 7 days post-transplantation. A–D) Tumor sections were costained with X-gal (blue) and anti-CD31 antibodies (brown) to visualize donor endothelium and counterstained with eosin to visualize tumor cells (pink). Arrowheads indicate exogenous endothelial cells; asterisks indicate tumor cell clusters. Scale bars: A, B) 0.5 mm; C, D) 50 µm. E–F) Exogenous endothelial cell apoptosis was significantly increased in tumors harboring EphA2 –/– MPMEC vs. tumors harboring EphA2 +/+ MPMEC, as assessed by LacZ/TUNEL costaining. Arrows indicate LacZ+/TUNEL+ endothelial cells. Scale bar = 20 µm. G) Tumor volume was significantly decreased for tumors harboring no exogenous endothelial cells or harboring EphA2 –/– MPMEC relative to tumors cotransplanted with EphA2 +/+ or +/– MPMEC. H) EphA2 +/+ or –/– MPMEC lysates were subjected to Western blot analyses to detect the expression of EphA2, EphA4, ephrin-A1, EphB4, and ephrin-B2. While the level of EphA4 and ephrin-A1 was not changed, both EphB4 and ephrin-B2 levels were increased in EphA2-deficient MPMECs.

Tumor volume was significantly increased in tumors harboring EphA2 +/+ or +/– endothelial cells relative to tumors harboring no exogenous endothelial cells (Fig. 2G) . Tumors containing EphA2-deficient MPMEC displayed a tumor volume comparable to tumors harboring no exogenous endothelial cells, significantly lower than tumors containing EphA2 +/+ or +/– MPMEC. Taken together, these data suggest that loss of EphA2 in endothelial cells contributes significantly to defects in tumor angiogenesis and progression observed in EphA2-deficient mice.

3. EphA2-deficient endothelial cells fail to migrate in response to tumor cells in vitro
In modified transwell coculture assays, we found that EphA2-deficient MPMEC displayed significantly reduced migration in response to 4T1 tumor cells in vitro relative to MPMEC derived from control littermates. This effect appears to depend on expression of ephrin ligand in tumor cells, since less malignant 67NR tumor cells that express relatively lower levels of ephrin-A1 induce significantly less MPMEC migration than high ephrin-A1-expressing 4T1 tumor cells (data shown online). Moreover, overexpression of a kinase-defective, dominant negative EphA2 construct in human dermal microvascular endothelial cells (HMEC) also significantly inhibited 4T1-induced endothelial cell migration (data shown online). Figure 3 outlines a diagrammatic representation of our findings.

View larger version (29K): [in this window] [in a new window]   Figure 3. Model for EphA2 regulation of tumor angiogenesis and metastatic progression. A) Upon interaction with tumor cells expressing ephrin ligands, host endothelial EphA2 receptors are activated, phosphorylated, and initiate activation of phosphoinositide 3-kinase (PI3K) and Rac1 GTPase, which initiates endothelial cell chemotaxis, coalescence, and tumor neovascularization. This permits tumor cells to survive and metastasize to the lung. B) EphA2-deficient host endothelial cells display defective migration and coalescence in response to ephrin-expressing tumor cells. This leads to decreased tumor microvascular density, tumor cell survival, and lung metastasis in vivo.

CONCLUSIONS AND SIGNIFICANCE

While it has long been established that activation of oncogenic pathways and inhibition of tumor suppressor pathways facilitate tumor progression in a tumor cell-intrinsic manner, increasing evidence provides support for tumor cell-extrinsic, microenvironmental mechanisms of tumor progression. In particular, interaction between tumor cells and host endothelium is crucial for tumor growth and metastatic progression. In this study, we provide the first direct evidence that host EphA2 RTK is necessary for tumor progression. Targeted disruption of EphA2 impairs angiogenesis induced by ephrin-A1-expressing mouse mammary adenocarcinoma. Endothelial cells derived from EphA2-deficient animals display increased apoptosis and fail to incorporate into tumor vessels in vivo as efficiently as endothelial cells derived from wild-type control littermates, as well as failing to migrate in response to tumor cells in vitro. These data suggest that EphA2 is a major regulator of tumor angiogenesis. Host EphA2-deficiency impairs lung metastasis relative to controls, suggesting that EphA2 RTK functions within the tumor microenvironment to facilitate angiogenesis and metastatic progression of breast cancer.

The data presented in this study are consistent with previous studies in which soluble EphA-Fc receptors inhibited tumor survival, angiogenesis, and tumor-mediated endothelial cell migration, suggesting that EphA2 is a major target for this global EphA RTK inhibitor. While host EphA2-deficiency mimics the effects of soluble EphA-Fc receptor treatment on tumor angiogenesis and progression, the defects on tumor progression in EphA2-deficient mice are somewhat milder. This is likely due to the ability of soluble EphA-Fc receptors to inhibit EphA2 receptor function in tumor cells as well as in host stroma. In addition to tumor cell-intrinsic functions of EphA2 RTK, another possibility for the milder phenotype of tumor progression in EphA2-deficient hosts vs. tumor-bearing animals treated with soluble receptor involves the ability of soluble EphA receptors to inhibit signaling through multiple endogenous A class Eph receptors. Thus, the presence of EphA4 in EphA2-deficient hosts may partially compensate for the loss of EphA2. It would be of interest to determine whether EphA4-deficient animals, as well as animals lacking both EphA2 and EphA4, display defects in tumor angiogenesis and progression.

Cotransplantation of 4T1 tumor cells with normal endothelial cells increases tumor volume. As tumors are harvested 1 wk after transplantation, there is no blood flow yet in the assembled vessels formed by donor endothelial cells. Therefore, the fact that tumors grow better in the presence of the donor endothelial cells is not due to extra oxygen and nutrients supplied by new blood vessels. Rather, these data suggest that there could be paracrine signaling from donor endothelial cells to tumor cells to promote tumor growth.

The data presented in this study, along with other recently published reports, indicate that EphA2 plays a key role in breast tumor progression through both tumor and host-dependent mechanisms. Because this single factor influences tumor progression in tumor cells and host microenvironment, EphA2 RTK is an attractive target for the development of new treatments for breast cancer. Our EphA2-deficient model will be a valuable tool, since it may be used to assess development and progression of endogenous breast tumors in which EphA2 expression is eliminated in host and tumor simultaneously. These analyses, along with existing information on EphA2 function in tumor and host endothelium, could prove invaluable in developing new, targeted therapies for treatment of breast cancer.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4038fje;

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