Circulating tumour cells and EpCAM as tools for diagnostic and mechanistic studies of lung cancer

Abstract

Increasingly, lung cancer treatment depends on the molecular characteristics of the lung tumour and studying the mechanisms of potential lung cancer molecular markers is important in order to identify new, targeted treatment options. The cell surface protein, EpCAM is one potential therapeutic target. Traditionally considered an adhesion protein, recent studies in breast and colon cancer have shown that EpCAM also undergoes proteolytic cleavage. Its c-terminal tail is released, acting as nuclear signalling factor that may contribute to cancer progression. Lung Cancer is the leading cause of global cancer-related mortality, largely due to a tendency for late-stage diagnosis. Screening with CT scans suffers from high false positives and requires additional confirmatory diagnosis, using more invasive techniques. Less-invasive detection methods could potentially improve the ease of screening and detection, increasing the chances for early diagnosis. EpCAM is widely used as a target for isolating circulating tumour cells (CTCs) from the blood of cancer patients. This is problematic as EpCAM-positive CTCs only represent a sub- population of total CTCs. This thesis evaluates the clinical utility of EpCAM- independent CTCs detection for diagnosis, as well as the mechanisms of EpCAM cleavage, in the context of lung cancer. Two different EpCAM-independent CTC isolation systems were assessed. Both isolate CTCs from peripheral blood using a size- and deformability-exclusion strategy. The Clearbridge system comprises a microfluidic chip with permeable traps and the ScreenCell system utilizes a permeable membrane subjected to a vacuum pressure gradient. A combination of primary, matched normal and tumour lung tissue and lung cancer cell lines were used to investigate EpCAM cleavage activity in lung cancer. The ScreenCell system was both easier to use and produced better results than the Clearbridge system. EpCAM cleavage experiments showed that while it does occur in lung cancer, it differed from previously reported EpCAM cleavage behaviour in breast and colon cancer cell lines. The most interesting difference was the discovery of N-terminal cleavage in lung cancer, which has not been described in published literature. While both CTC-isolation systems have the potential to be used as a diagnostic blood test for lung cancer, low sensitivity and specificity values prohibit their use in the clinical setting presently. The discovery of EpCAM cleavage in lung cancer, especially N-terminal cleavage, is particularly relevant in the context of treatment strategies targeting extracellular domains of EpCAM, and EpCAM cleavage mechanisms and their effect on lung cancer progression warrants further exploration.