<div>Abstract<p><b>Purpose:</b> Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.</p><p><b>Experimental Design:</b> Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (<i>n</i> = 20) and in distinctive CHM (<i>n</i> = 38) and CC (<i>n</i> = 9) cohorts, (ii) an <i>in vitro</i> study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an <i>in vivo</i> study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.</p><p><b>Results:</b> Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.</p><p><b>Conclusions:</b> Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. <i>Clin Cancer Res; 23(22); 7130–40. ©2017 AACR</i>.</p></div>

<p>Supplemental Methods Figure S1: Reports clinical information's about normal pregnant women, patients with complete hydatiform moles or choriocarcinoma. Figure S2: Experimental procedure. Figure S3: Quantification of EG-VEGF PROKR1 and PROKR2 protein expression in CTL, Complete hydatiform mole (CHM) and choriocarcinoma (CC) placental section. Figure S4: EG-VEGF effect on JEG3 migration, in the absence or presence of PROKR1 or PROKR2 antagonists. Figure S5: JEG3-luc injected mice exhibited arrested gestation and disorganized vascularization. Figure S6: Antibody microarray analysis. Figure S7: Characterization of the angiogenic status of sera and placentas collected from CTL, CHM and CC patients. Figure S8: Antibody angiogenic microarray analysis of sera collected from CTL, CHM and CC patients.</p>

<div>Abstract<p><b>Purpose:</b> Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland–derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy.</p><p><b>Experimental Design:</b> Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control (<i>n</i> = 20) and in distinctive CHM (<i>n</i> = 38) and CC (<i>n</i> = 9) cohorts, (ii) an <i>in vitro</i> study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an <i>in vivo</i> study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice.</p><p><b>Results:</b> Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions.</p><p><b>Conclusions:</b> Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. <i>Clin Cancer Res; 23(22); 7130–40. ©2017 AACR</i>.</p></div>

<p>Supplemental Methods Figure S1: Reports clinical information's about normal pregnant women, patients with complete hydatiform moles or choriocarcinoma. Figure S2: Experimental procedure. Figure S3: Quantification of EG-VEGF PROKR1 and PROKR2 protein expression in CTL, Complete hydatiform mole (CHM) and choriocarcinoma (CC) placental section. Figure S4: EG-VEGF effect on JEG3 migration, in the absence or presence of PROKR1 or PROKR2 antagonists. Figure S5: JEG3-luc injected mice exhibited arrested gestation and disorganized vascularization. Figure S6: Antibody microarray analysis. Figure S7: Characterization of the angiogenic status of sera and placentas collected from CTL, CHM and CC patients. Figure S8: Antibody angiogenic microarray analysis of sera collected from CTL, CHM and CC patients.</p>

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