Abstract 3371: Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer
Authors: Trapani Francesca , Williamson Stuart , Metcalf Robert L. , Leong Hui Sun , Abbott Benjamin , Antonello Jenny , Hodgkinson Cassandra , Franklin Lynsey , Hendrix Mary J. , Seftor Richard E b , Seftor Elisabeth , Rothwell Dominic , Brady Ged , Miller Crispin , Blackhall Fiona H. , Simpson Kathryn L. , Dive Caroline 
Published: July 2016

Abstract
Background: Despite a good initial response to chemotherapy, most small cell lung cancer (SCLC) patients relapse with drug resistant disease. Targeting tumor vasculature in SCLC with anti-angiogenic drugs produced disappointing results, therefore angiogenesis-independent tumor vascularisation pathways warrant further investigation. Vasculogenic mimicry (VM) is the ability of tumor cells to mimic endothelial cells by trans-endothelial differentiation, characterised by increased expression of vascular markers including VE-Cadherin. We previously demonstrated that VM correlates with poor Overall Survival in Limited Stage SCLC patients and sought to phenotypically and genetically characterise VM vessels using SCLC Circulating Tumour Cells (CTCs) and CTC-Derived eXplant (CDX) models1 and to explore the functional significance of VE-Cadherin for VM formation in vitro and in vivo.Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in tumors from 10 CDX models. Laser Capture Microdissection (LCM) and Copy Number Alteration (CNA) analysis was performed on CDX regions with high and low levels of VM. VE-Cadherin expression in SCLC CTCs was evaluated following ISET microfiltration of patients’ blood and Immunofluorescence staining for DAPI, CD45, pan Cytokeratin and VE-Cadherin. VE-Cadherin function was evaluated in vitro by Matrigel network assay using H446 SCLC cells and H446 shRNA VE-Cadherin knockdown (KD) cell lines, and in vivo by growth as xenografts, further treated with Cisplatin.
Results: VM was present in CDX models and LCM followed by CNA analysis of VM vessels confirmed their SCLC origin. ISET microfiltration and immunofluorescent staining of CTCs from 37/38 SCLC patients revealed VE-Cadherin as a putative VM biomarker in SCLC CTCs. VE-Cadherin shRNA KD in VM competent H446 SCLC cells abrogated their ability to form VM networks in vitro and in vivo. Cisplatin treatment of mice bearing H446 VE-Cadherin KD tumors resulted in reduced cisplatin binding compared to parental H446 tumors.
Conclusions: VM is present in CDX models and co-localises with VE-Cadherin expression. CNA confirms that VM vessels originate from tumour, and these VM-enriched regions bear a unique chromosomal signature compared to the low-VM regions. VE-Cadherin is required for network formation in vitro and VE-Cadherin levels and VM vessel numbers are positively correlated in vivo. Moreover, levels of VM in tumors had significant impact on both tumour growth kinetics and cisplatin delivery which has implications for drug senstivity. Our future research will interrogate the VM signaling pathway in SCLC and its role in chemosensitivity and establish feasibility for therapeutic targeting.
Citation Format: Francesca Trapani, Stuart Williamson, Robert L. Metcalf, Hui Sun Leong, Benjamin Abbott, Jenny Antonello, Cassandra Hodgkinson, Lynsey Franklin, Mary J. Hendrix, Richard E b Seftor, Elisabeth Seftor, Dominic Rothwell, Ged Brady, Crispin Miller, Fiona H. Blackhall, Kathryn L. Simpson, Caroline Dive. Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer. [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 3371.