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. 2009 Jan 1;113(1):254-63.
doi: 10.1182/blood-2008-02-140020. Epub 2008 Oct 3.

The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade

Jeffrey S Scehnet  1 , Eric J Ley, Valery Krasnoperov, Ren Liu, Parmeet K Manchanda, Eric Sjoberg, Anna P Kostecke, Sachin Gupta, S Ram Kumar, Parkash S Gill
Affiliations

The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade

Jeffrey S Scehnet et al. Blood. .

Abstract

Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.

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Figures

Figure 1
Figure 1
The Eph and Ephrin expression in KS cell lines KS-SLK and KS-IMM, lymphatic endothelial cells transformed with HHV-8 (LEC/HHV-8), and KS tumor biopsy. (A) The mRNA expression of 13 Eph receptors and 8 Ephrin ligands was determined with RT-PCR. HT29 colon cancer cell line and HUVEC were included as controls. (B) Western blot analysis to for protein expression of selected expressed Eph-Ephrins. β-actin expression was done to show comparable protein loading.
Figure 2
Figure 2
Expression and characterization of sEphB4 and sEphB4-HSA. (A) sEphB4-HSA was expressed in CHO cells and purified to near homogeneity and separated on SDS-PAGE (Coomassie staining) under reducing and nonreducing conditions. (B) Saturation binding kinetics of sEphB4 and sEphB4-HSA in a solid-phase ELISA. Interaction of increasing concentrations of sEphB4 or sEphB4-HSA with sEphrinB2-AP was determined in a solid-phase ELISA. Each point was determined in triplicate. Dissociation constants were calculated with the use of nonlinear regression and Graphpad Prism. (C) Systemic pharmacokinetics of sEphB4 and sEphB4-HSA administered intraperitoneally. Mice were injected with a 10 mg/kg dose of either sEphB4 or sEphB4-HSA administered intraperitoneally. Each point represents the average of 2 separate experiments. Error bars represent the SEM. (D) Pharmacokinetic and saturation binding constants of sEphB4 and sEphB4-HSA. (E) Tyrosine phosphorylation of EphB4 receptor in MCF7 cells in response to stimulation with EphrinB2-Fc (15 minutes) in the absence or presence of EphB4-derived soluble proteins.
Figure 3
Figure 3
sEphB4-HSA activity in KS migration, invasion, and survival. (A) KS-SLK cells were grown to confluence, scraped, and treated with varied concentrations of sEphB4-HSA. Cell migration in the clear zone was documented by photographs at various time points at 20× fields. (B) KS cell invasion in response to growth factors. Modified Boyden chamber assay was used to determine KS cell invasion across Matrigel-precoated inserts. Data are presented as number of invading cells plus or minus SE from duplicate wells in 2 experiments. (C) Cell viability assay. KS cells were grown in triplicate in the presence of increasing concentrations of sEphB4-HSA for 72 hours. Cell viability was assessed by MTT assay. The experiment was repeated twice with similar results. Photomicrographs in panels A and B were taken with a Nikon Coolpix 5000 camera (Nikon, Tokyo, Japan) and a Carl Zeiss Invertoskop microscope (Zeiss, Goettingen, Germany) with a 4×/0.12 NA objective and 10× eyepiece.
Figure 4
Figure 4
sEphB4-HSA inhibits KS tumor growth in a murine tumor xenograft model. (A) Mice (n = 8/group) were implanted with 2 × 106 KS-SLK or KS-IMM cells and treated with sEphB4-HSA, VEGF moAb, or combination sEphB4-HSA/VEGF moAb; tumor volumes were measured 2 times a week; and the data are presented as tumor volume. After 5 weeks, tumor volumes were as follows; sEphB4-HSA (KS-SLK = 15.9% of control, P < .001; KS-IMM = 37.2% of control, P < .001) or combination sEphB4-HSA plus VEGF moAb (KS-SLK = 12.1% of control, P < .001; KS-IMM = 12.6% reduction, P < .001). (B) Mice spleens were injected with 106 SLK cells and treated with sEphB4-HSA, VEGF moAb, or combination sEphB4 plus VEGF moAb. After 5 weeks, livers were harvested and examined for the number of tumor metastases (tumors/livers) and are as follows; control (8/11), VEGF moAb (2/5), sEphB4-HSA (2/5), and combination of sEphB4-HSA and VEGF moAb (0/5).
Figure 5
Figure 5
Analysis of vascular perfusion, vessel density, tumor cell proliferation, apoptosis and hypoxia. (A) Tumors were harvested at completion of the study and examined by hematoxylin and eosin staining. Just before harvest, mice were infused with RCA-Lectin and hypoxia probe. Nuclei were counterstained with DAPI. RCA-Lectin localized the perfused vessels, CD31 localized microvascular endothelial cells, and the merged picture shows perfusion of total vessels in the field. Quantitation was performed with the use of Bioquant Image Analysis (Bioquant, Nashville, TN). Proliferating cells within the tumor were assessed by immunohistochemical detection of Ki-67 protein and quantified as described. All values are expressed as mean plus or minus SEM. *P < .01 by Student t test. Ki-67 pictures were taken with Carl Zeiss Invertoskop microscope with a 4×/0.12 NA objective and 10× eyepiece. Photomicrographs were taken using a Nikon Coolpix 5000 camera and a Nikon Eclipse E400 microscope with a 10× eyepiece. Magnification was as 40×/0.75 NA objectives. (B) sEphB4-HSA–treated tumor and adjacent normal tissue vessel density and perfusion. Dotted line demarcates the skin showing autofluorescence. Vessels in the subcutaneous tissue (subcutis) and the margin of the tumor show perfusion. Deeper tumor tissue shows progressive decrease in the vessel density and even greater decrease in vessel perfusion. (C) Gene expression analysis of tumor tissues was performed by quantitative PCR for VEGF, VEGFR1, VEGFR2, PDGF-β, and Dll4. Gene expression levels were corrected for β-actin levels.
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