Development of a targeted diagnostic and therapeutic delivery system for cholangiocarcinoma

The current outcomes of peri-hilar cholangiocarcinoma (PH-CCA) are in general poor with no proven roles for any treatment modalities in improving survival apart from complete surgical extirpation. The dismal prognosis observed is contributed to by both delays in diagnosis and aggressive tumour biology. The advent of molecular targeting of solid cancers allows harnessing this property on microbubble and nanotechnology based platforms. Such a system provides for concurrent diagnosis and therapeutic effect (theragnosis), the feasibility of which is explored in this work. The expression of two candidate biomarkers with established roles in cancer progression, namely Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Matrix Metalloproteinase-9 (MMP9) were examined by Immunohistochemistry in 54 human samples of resected PH-CCA. These markers were then evaluated for their potential as molecular targets in theragnostic platforms in cholangiocarcinoma. Both NGAL and MMP9 were found to be abundantly expressed in both tumour tissue and control liver. Although higher NGAL expression was associated with nodal metastases, no prognostic impact was seen for either marker. NGAL expression was further interrogated in six cholangiocarcinoma cell lines by Western blotting, Immunofluorescence and ELISA techniques, which revealed localisation of NGAL to the cell membrane and its secretion, both indicating its suitability to a targeting platform. Nonetheless, its expression in normal liver tissue could lead to off-target effects. Concurrently, the expression of vascular ligands CD31, CD105 and Vascular Endothelial Growth Factor Receptor-2 were each assessed in the same cohort of tissue samples. CD105 expression was found to be independently predictive of poorer overall survival. Subsequent in vitro flow assay experiments using CD105 antibody conjugated microbubbles showed it bound murine SVR endothelial cells significantly more in comparison to isotype control microbubbles. Furthermore, evaluation of in vivo targeting with the aid of high frequency ultrasound demonstrated CD105-microbubbles bound to tumour endothelium significantly more than control microbubbles in a subcutaneous murine xenograft model of cholangiocarcinoma. This work therefore indicates NGAL is suited to a theragnostic approach in cholangiocarcinoma although larger data sets are required to explore its prognostic impact. CD105-microbubbles could be utilised in the delivery of such theragnostic particles to cholangiocarcinoma tissue, whilst in themselves providing a non-invasive index of tumour vascularity when imaged with ultrasound.