Proteomics-based discovery of novel vascular accessible markers in kidney cancer

Abstract

With worldwide 209 000 new cases and 102 000 deaths per year renal cell carcinoma accounts for 2-3% of all cancer incidences and shows the highest mortality rate among all genitourinary malignancies [1, 2]. Currently, surgical resection of the tumour bulk is the solely treatment strategy offering a possible long-term cure. Alternative treatments for metastasising disease targeting the mTOR and the VEGF pathways only result in tumour mass reduction while inducing severe side effects and resistances [3]. In order to improve patient survival novel, effective and well-tolerated therapeutics particularly for advanced stages are urgently needed. Thereby the antibody-based delivery of bioactive molecules directly into the tumour has proven to be a promising approach [4]. Importantly, the availability of vascular accessible tumour-associated biomarkers as target molecules is a prerequisite for the design of this innovative drug type [5]. In this thesis a proteomics based biomarker discovery approach for the identification of novel drug targets accessible from the vasculature in clear cell renal cell carcinoma is presented. Thereby, vascular accessible proteins with an increased expression in the primary kidney tumour as well as proteins specifically regulated in corresponding lung metastases were investigated. For the enrichment of vascular accessible proteins the in vivo biotinylation strategy was applied [6, 7]. This method relies on the terminal perfusion of tumour-bearing mice with a reactive ester derivate of biotin resulting in the covalent biotin labelling of vascular accessible proteins. However, the conventional in vivo perfusion technique only allows for the biotinylation of proteins accessible from the systemic circulation. In order to investigate the vascular accessible proteome of lung metastases a novel technique for the perfusion of the pulmonary circulation was developed and successfully applied. Subsequent to the in vivo perfusion, biotinylated proteins were captured on streptavidin sepharose followed by on resin alkylation, delipidation and tryptic digestion. Resulting modified peptides were separated by nano-UPLC and analysed by MALDI-MS and MALDI-MS/MS. Protein identification was based on the identification of the corresponding proteotypic peptides. Relative quantification of protein expression levels was carried out on representative subsets by comparative analysis of summed intensities of peptides contributing to the same protein. In the final dataset, mass spectrometric data deriving from seven patient-matched xenograft kidney tumour models, four spontaneous patient-matched xenograft lung metastasis models, healthy murine kidney and lung as well as three human renal primary cell lines were combined. In total, more than 3000 proteins were identified, whereof more than 80 % could be quantified. Comparative analyses of different technical replicates and various biological samples demonstrated the high reproducibility of the techniques applied in the presented study. As expected, variances between the different patient-matched xenograft models were significantly bigger than between biological and technical replicates within one model. The six biomarker candidates IGFBP3, Lcn2, Ltbp2, SLC16A1, TGFBI and TNXB were selected for further validation studies. Candidate proteins were validated based on both mRNA and protein levels using RT-qPCR and immunofluorescence, respectively. The results obtained in the initial proteomics discovery experiment were confirmed in the course of the validation study. The upregulation of several marker candidates in tumour tissue compared to healthy kidney was confirmed when analysing a small set of patient samples. Finally, a proof-of-principle biodistribution study investigating the vascular accessibility of marker 6 for a monoclonal antibody indicated marker 6 to be a potential drug target. However, marker 6 accessibility for intravenously injected antibodies requires optimization. In summary, this large scale proteomics study on vascular accessible proteins in clear cell renal cell carcinoma resulted in the identification of marker 6 as a possible target protein for novel antibody-based kidney cancer therapies.