The main cause of death in cancer patients is not the primary tumor but rather metastasis in distant organs, which is initiated by the spread of the CTCs through the blood stream. However, clinical decision-making in oncology is usually based on a one-time histopathologic analysis of the primary tumor. Metastases can evolve many years after primary tumor resection and harbor unique genomic alterations. Biopsy of metastases is an invasive procedure with side effects like pain and bleedings, and depending on the location of the tumor lesion impossible in a considerable fraction of patients. In addition, intra-patient heterogeneity of metastases at different sites makes this approach even more complicated, because the lesion accessible by the needle biopsy may not contain the most aggressive tumor clones that need to be eradicated by therapy. Thus, the capture and analysis of CTCs as “liquid biopsy” will become a valuable diagnostic tool, once a reliable, cost-effective method is established. This will benefit patients by better diagnostics and follow up with less invasive methods as well as reduced early death and the related personal grief and societal productivity losses. In view of the need for multiple sequential blood samples obtained from an individual patient for monitoring cancer therapy, the envisaged number of CTC tests will even exceed the cancer incidences described above. Moreover, the CTC capture antigens tested in this project are shared by the major cancer types in the EU (breast, prostate, colon and lung cancer), which indicates the broad applicability of the new CTC platform.
During the first ERC PoC grant CAPTURE-CTC the following milestones relevant for the future validation and commercialization of the CTC chip planned in this project were achieved:
Generation of a Micropatterned Microfluidic Capture Chip and Proof of its Capturing Capability: The capture principal of the micropatterned microfluidic chip has been successfully developed. The chip manufacturing procedure has been optimized to ensure a reliable and reproducible “production” of chips for all required tests in the course of the project and tested the developed cell capture principal with viable breast cancer cell line cells sensitized with biotinylated antibodies. Following cell immobilization, the flat surface of the glass slide allows for extraction of cells of interest by micromanipulation, while keeping cell morphology intact as crucial prerequisite for further characterization.
Improvement of Micropatterned Microfluidic Capture Chip Structure and Performance: The modular setup of the chip-manufacturing readily allows alterations in chip architecture, making it a very versatile platform. Multiple structural parameters were modified and compared in respect to cell-capture efficiency to elucidate an optimal Capture-CTC chip design. Conclusively, we determined an ideal chip structure consisting of a 50 µm streptavidin pattern combined with a herringbone chamber ceiling (125 µm total height, 100 x 50 x 75 groove dimensions) yielding cell recoveries of up to 96 %.
Capture of cancer cells in spiked blood samples from healthy donors: CTCs are extremely rare within a high background of healthy blood cells (1 CTC in 108 - 109 blood cells). An additional pre-enrichment of the tumor cell fraction is necessary to ensure specific and sensitive cell capture and prevent clogging of the microfluidic device as well as excess cell background. The Parsortix™ system (ANGLE plc) is a size-based, label-free, pre-enrichment platform that has been intensively tested at the UKE. First proof-of-principle studies on patient blood samples demonstrated the feasibility of our new system in a clinical setting.
Taken together, the improved version of this chip is now ready for clinical testing. Moreover, we have submitted a patent application to protect the IP and made promising contacts to industry partners for future commercialization of the chip platform. The successful development of CTC profiling technologies for therapeutic targets and resistance mechanisms as well as the establishment of cell culture conditions for CTCs has furthermore enabled us now to implement short-term CTC cultures for future drug testing purposes.
