Identification of novel vascular targets in lung cancer

Lung cancer is now the leading cause of cancer mortality in the UK, accounting for 24% of cancer deaths in men and 21% in women (Office for National Statistics, 2011). Worldwide rates vary markedly; overall lung cancer accounted for 18% of all cancer deaths in 2008 (Ferlay et al, 2010). Rates have been impacted little by the advances in diagnosis and treatment to date, and in some groups, such as British women (Office for National Statistics, 2011), they continue to rise. Lung cancer is divided into two broad categories – non-small cell (NSCLC) and small cell. More than 80% of lung cancers are NSCLC, which is comprised mostly of squamous cancers and adenocarcinomas. Key changes over the past 10 years in NSCLC include the reclassification of tumour types (Nair et al, 2011), use of biological markers to guide certain therapies, use of adjuvant therapy after selected complete resections and advances in selection and planning for surgery and radiotherapy – concepts that have been reviewed comprehensively elsewhere (Goldstraw et al, 2011). The most promise has been shown by CT-based screening, though concerns have been raised about the potential for multiple invasive tests and cost-effectiveness of the strategy (Bach et al, 2012). Since many patients present with advanced disease surgery is often impossible, hence there is a great need for novel therapeutic agents. This is particularly important as current first line regimes add little >2 months to the average survival (Goldstraw et al, 2011). In addition, many therapies are less beneficial (or indeed more harmful) in squamous tumours, such as the antifolate agent pemetrexed (Scagliotti et al, 2011), anti-vascular endothelial growth factor (VEGF) antibody bevacuzimab (Johnson et al, 2004) and tyrosine kinase inhibitor (TKI) gefitinib (Wang et al, 2012). Selection for the latter drug usually also involves testing for EGFR mutation (Wang et al, 2012), meaning that the pool of patients in whom each drug works is increasingly small. Therefore, the identification of novel biomarkers or therapeutic targets is a priority for lung cancer. A functional vasculature contributes to tumour progression and malignant cell metastasis. Endothelial cells lining the tumour vasculature are exposed to molecular factors and mechanical forces that are absent in healthy tissue. For example, the vasculature in solid tumours is often in a hypoxic environment (Dachs and Chaplin, 1998) and is exposed to elevated the levels of hypoxically induced angiogenic factors such as VEGF (Relf et al, 1997). Tumour vessels may also be leaky, tortuous, sometimes blind ended and have poor vascular smooth muscle and pericyte coverage (Baluk et al, 2005). As a result, the tumour endothelial transcriptome is markedly different from that in healthy tissue and provides a unique source for cancer target identification. In the last decade, attempts to identify tumour endothelial markers (TEMs) have included construction of SAGE libraries from freshly isolated endothelium (ST Croix et al, 2000), use of microarray platforms (Ho et al, 2003), proteomic analysis of freshly isolated endothelial cell membranes (Ho et al, 2003; Oh et al, 2004) as well as bioinformatics data mining (Huminiecki and Bicknell, 2000; Herbert et al, 2008). These efforts identified several targets including the EDB domain of fibronectin, a series of numbered TEMs, annexin A and recently CLEC14A reviewed in Meyer, 2010. Recent studies have shown that TEMs are often tissue dependent, and that endothelial transcriptomes have been documented for colon (Van Beijnum et al, 2006), breast (Bhati et al, 2008; Jones et al, 2012) and ovarian cancer (Sasaroli et al, 2011). Known TEMs are often weakly expressed in lung tumours (Mura et al, 2012) and this prompted us to investigate TEMs in the lung. For successful expression profiling it is essential to obtain a pure endothelial population. We demonstrate in this study that rapid tissue digestion followed by magnetic bead isolation yields pure endothelial isolates. Following expression profiling of endothelial isolates from NSCLC patient samples, novel cell-surface targets of lung tumour vasculature were identified, presenting attractive potential for developing targeted therapies for lung cancer.