Abstract 3279: The effect of Erlotinib treatment on human angiogenesis in vitro
Authors: Milosavljevic Tanja , Juge Elise , Guidry Russ , Woltering Eugene 
Published: July 2016

Background: Angiogenesis is a process by which new capillaries develop from previously formed venules. Epidermal growth factor receptor (EGFR) is a member of ErbB family of receptor tyrosine kinases (TKs). The EGFR signaling pathway is activated in proliferation, angiogenesis, tumor growth and progression. Overexpression of EGFR in many solid human tumors associates with poor prognosis. One of the effective strategies to target Erb receptors is use of small molecule inhibitors of the TK domain, such as Erlotinib. We hypothesized that inhibiting EGFR signaling by Erlotinib treatment will decrease physiologic and pathologic angiogenesis in an in vitro model system.

Methods: Tissue was embedded in a three-dimensional fibrin-thrombin clot, and evaluated for neovessel growth as per our in vitro Human Angiogenesis Model (HAM). Physiologic angiogenesis was studied in two venous tissues, human placental vein (HPV) and inferior vena cava (IVC). Human liver neuroendocrine tumor (NET) tissue modeled pathologic angiogenesis. All tissues were treated with Erlotinib in a dose response manner (1 μM, 10 μM, 100 μM) and evaluated for three angiogenesis parameters: percent initiation (%I), angiogenic growth (AG), and overall angiogenic response (OAR). Concentrations of angiogenesis-relevant ligand-receptor pairs in supernatant were determined by Human Angiogenesis/Growth panel (Milliplex, EMD Millipore). Effect of Erlotinib treatment on EGF pathway genes expression in IVCs and liver NETs was analyzed by TaqMan EGF pathway array. Angiogenesis data was analyzed for significance using Z-test (Primer) and paired t-test (MedCalc).

Results: Selected dose of Erlotinib [10 μM] consistently achieved statistically significant inhibition of both AG (p&lt;0.0001) and OAR (p&lt;0.0001) in all three tissue types compared to the control. Percent inhibition of%I was below 30% for HPV, IVC and liver NETs. Angiogenic growth was inhibited by 45.65% in HPV (p = 0.035), 52.14% in IVC (p&lt;0.0001) and 47.59% in liver NETs (p = 0.0107) and OAR by 60% in HPV (p = 0.105*), 53.61% in IVC (p&lt;0.0001) and 52.27% in liver NET (p = 0.0168). Preliminary ligand-receptor pair concentrations in IVCs and liver NETs show opposing response to Erlotinib treatment. Out of 92 EGF pathway-related genes, only twelve exhibited significant expression level changes [≥2-fold] in both IVC and liver NET Erlotinib-treated samples. Among them, only five genes demonstrated the same direction of change in both tissue types. The CAV2, GAB1, RHOD genes were down-regulated; MUC1 and PIK3c2b genes were upregulated.

Conclusions: Erlotinib is an effective antiangiogenic kinase inhibitor in HPV, IVC and liver NETs in-vitro. It impedes physiologic and pathologic angiogenesis via regulation of neovessel growth rather than initiation of sprouts. Five EGF pathway related genes commonly affected in both physiologic and pathologic models by Erlotinib treatment may be essential modulators of human angiogenesis.

Citation Format: Tanja Milosavljevic, Elise Juge, Russ Guidry, Eugene Woltering. The effect of Erlotinib treatment on human angiogenesis in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3279.