Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Because prognosis can vary depending on tumour stage, precise diagnosis is vital. Circulating tumour cells (CTC) detach from primary and secondary tumour sites into the bloodstream. Changes in three-dimensional (3D) nuclear organization are associated with different types of cancer and were examined in this study in CTCs of high-risk prostate cancer patients. CTCs were isolated from 3mL of patient blood samples of 20 high-risk prostate cancer patients before treatment; after neoadjuvant androgen deprivation therapy (ADT) but before radiotherapy (RT); and after completing RT. Telomere-specific fluorescence in situ hybridization was performed on filters containing cells, 3D images of 30 CTCs per filter were analysed. Changes in telomere organization were observed post ADT and RT; patients fell into three groups depending on the change in CTC telomeric profiles in response to ADT. These groups displayed responses characteristic to each group upon delivery of RT. 3D nuclear telomere profiles in CTCs post-ADT may indicate both ADT response and predict RT response in high-risk prostate cancer. / October 2016

Yes / Circulating tumour cells (CTCs) have significant diagnostic potential as they can reflect both the presence and recurrence of a wide range of cancers. However, this potential continues to be limited by the lack of robust and accessible isolation technologies. An alternative to isolation might be their direct detection amongst other peripheral blood cells, although this would require markers that allow them to be distinguished from an exceptionally high background signal. This review assesses the potential role of HOX genes, a family of homeodomain containing transcription factors with key roles in both embryonic development and oncogenesis, as unique and possibly disease specific markers of CTCs.

Survival rates in cancers have improved vastly over the years. However, some survival rates remain extremely low, as is the case for ovarian and lung cancer. The lack of robust and reliable biomarkers is strongly reflected in the absence of pre-screening programs, and as such, most patients in these cancer types are diagnosed only in advanced stages, leaving few treatment options. Moreover, relapse and resistance to therapies adds to the complexities of treating these diseases, even in the era of targeted drug development. Research has shown the presence of cancer material, in the form of circulating cancer cells (CTCs) and genomic material in the blood of patients, opening the possibility of 'liquid biopsies'. Liquid biopsies allow sampling of the disease to provide phenotypic and genomic data on the cancer in real-time and on a routine basis. Moreover, they overcome obstacles currently faced by conventional tissue biopsies. In this work we evaluate the use of a novel CTC imaging flow-cytometry platform, and report the ability to characterise and quantify these cells in blood samples. Moreover, we report significantly higher levels of CTCs in cancer patients compared to controls, and found them to be associated with a poorer prognosis. In particular, in lung cancer we observe these findings even in the early stages, suggesting a potential diagnostic use for this assay. We detect a similar trend in when analysing the ctDNA and suggest the possibility of using this technique with a prognostic value in the advanced setting. We also report on the analysis of existing microarray data by use of unique gene regulatory networks to identify biomarkers of interest. RAD51AP1 was identified by this process. Clinical validation revealed an over-expression of this gene in both tissue and blood of ovarian and lung cancers. Moreover, the gene over-expression was associated with a poor overall survival. Functional analysis in vitro revealed silencing RAD51AP1 suppressed tumour growth, in addition, various tumorigenic proteins were down-regulated, whilst apoptotic and immune genes were up-regulated. These results suggest a role for RAD51AP1 as a potential therapeutic target. In this study, we also demonstrate the ability to further exploit tumour genomic material in the blood by means of RNAseq, cancer panels, and CNI scoring to identify novel markers, that play an important role in disease genesis and evolution. RNAseq analysis identified XIST as a gene up-regulated in the blood and tissue of lung cancers. The ovarian cancer panels revealed 2 unique gene signatures in the ovarian cancer patients. With the CNI analyses also highlighting chromosomal aberrations from plasma analysis of cancer patients. Collectively, the use of all these techniques and exploitation of available blood based biomarkers could see significant improvements to survival rates in these, currently devastating diseases.

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.