Title: A High Affinity Method to Automate Circulating Tumour Cells Capture (CTCs) from Whole blood: a Revolutionary Technology in Cancer Detection and Monitoring
Funder: Department of Health (England)

Abstract: Hypothesis Antibody labelled magnetic particles can be used to enhance cancer diagnosis and target treatment by detecting circulating tumour cells in peripheral blood without prior sample processing. Project Summary Previous findings using the Autology antibody coated particles to remove ASCs and lymphocytes from primary tissues will be translated onto CTCs using cell surface markers against ectopic epithelial cells including EpCAM; a sentinel protein of a CTC phenotype. Initial research will test a range of commercial antibodies for EpCAM and other epithelial cell surface molecules to assess potential to work with the Autology capture particles to isolate epithelial cells. This will be refined using simple clean solutions of saline spiked with defined concentrations of cultured epithelial cells down to diagnostically appropriate numbers of 5-10cells/10 ml. Beads will be retained by gravity and/or magnetism and the presence of epithelial cells on the bead surface and capture kinetics measured using a variety of quantitative genomic and molecular biologic techniques. The viscous nature of blood combined with the very low target cell numbers requires a technological advance to overcome the limitations of standard magnetic particle capture. In response to this Prokyma developed the thin-film capture approach. Thin-film utilises a disposable chamber with larger, denser magnetic particles that flow through a defined geometry ensuring 100% particle retention due to an intimate contact between the magnetic field and the particles. The technology also includes an acoustic force step. After capture the magnetic field will be removed and beads re-suspended using acoustic force. Synergy between the dual forces of magnetic trapping and acoustic re-suspension allows automated and efficient washing, removing non-target cells and cell fragments trapped within inter-bead spaces, retaining CTCs. This removal of background will allow maximum resolution with subsequent analytical methods. Cell retention and depletion will be assessed using a combination of genomic and molecular strategies. Deep Sequencing allows for detection of mutations in cells but cannot resolve the difference if DNA from too many non-target cells is present. PCR is more sensitive and cost effective if the mutation sequence is known, however the number of possible variables are so large that multi-plex strategies with PCR are impractical. The project will assess if the cell capture presents sufficiently purified CTCs to detect mutations with the Ion-Torrent, permiting screening and enumerating by mutation specific PCR. Having confirmed thin film bead capture as a viable approach for CTC enumeration, prototype devices will be designed and manufactured to completely automate the diagnostic workflow. These devices will receive whole blood followed by a) mixing with capture beads, b) thin film capture, c) dual force washing and d) cell lysis for genomic analysis. The prototype will then be tested with banked clinical samples from known metastatic cancer patients to determine the impact of non CTC genomic and transcriptomic background. Samples from on-going clinical trials will allow the technology to be assessed for changing CTC numbers from patients undergoing therapy, allowing the clinical utility of the approach to be demonstrated. The deep sequencing information will allow extensive mutation analysis of multiple genes within the tumour cells, which combined with bioinformatics and immunohistochemistry can predict sentinel molecules at the cell surface which can be used to extract CTCs from blood. Additionally from a research perspective the Autology cell isolation system also possesses a novel pH release chemistry. Therefore, it is possible to release CTCs from the cell capture beads, in an identical phenotype to that found in peripheral circulation using a simple pH elevation to pH8.4. This enables primary cells to be removed and subject to in-vitro tissue culture for research purposes.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS