Ninety percent of cancer related deaths are not caused by the primary tumor but by metastases damaging distant and vital organs. Metastasis is initiated by dissemination of tumor cells from the primary tumor and spread of circulating tumor cells (CTCs) through the blood stream. However, clinical decision-making in oncology is usually based on the analysis of a single tissue biopsy of the primary tumor and/or metastases, if obtainable. As metastases predominantly evolve many years after primary tumor resection, they are described to harbor unique genomic alterations. In addition, intra-patient heterogeneity of metastases makes this tissue-based approach even more complicated, as it increases the likelihood of missing the most aggressive tumor clones in need of eradication by therapy within the lesion accessible by the needle biopsy. Furthermore, tissue biopsy 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. Thus, the capture and analysis of CTCs as “liquid biopsy” will become a valuable diagnostic tool, once a reliable, cost-effective method is established. Patients will benefit by better diagnostics, follow up observation with less invasive multiple sequential blood analyses and better individualized therapies. 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.
In our last ERC-PoC funding period, we established the optimal microfluidic structure and streptavidin pattern design for our novel CTCapture chip device, leading to peak recoveries of 96%, assessed by spiking experiments with cell culture cell line cells. In addition, the size-based, label-independent pre-enrichment technology Parsortix™ (ANGLE, plc) was found to be best suited in combination with our platform. Building on these results, three main areas of focus were pursued during CAPTURE-CTC_2.0 each relevant for the future validation and commercialization of the CTC chip. First, the evaluation of downstream single cell analysis following capture of tumor cells on the CTCapture chip. Second, the establishment of an antibody cocktail targeting multiple capture antigens, and third the application of our platform to clinical cancer patient samples.
In cell culture experiments with an EpCAM-based capture antibody, we were able to show that 90% of tumor cells captured on our CTCapture Chip remain viable at time point of capture. This represents an important prerequisite for subsequent cell culture. In addition, DNA of single tumor cell line cells (of breast cancer and melanoma origin) isolated on our chip after capture and staining, could successfully be amplified by whole genome amplification (WGA). Amplified DNA products of highest quality (4/4 bands in quality control) were reached for 50 % of the analyzed single cells for each of the two cell lines. Successful WGA is a pivotal step in downstream molecular analysis allowing for further in-depth genomic analysis, such as detection of clinically relevant mutations and other genomic aberrations. We therefore extended our work and evaluated whether mutation detection on a single cell level was possible from melanoma cell line cells, following capture and staining on the CTCapture-chip. In a first proof-of-principle experiment, we were able to detect a described BRAF V600E mutation in single picked tumor cells, using the AGENA iPlex HS melanoma mutation panel, thereby demonstrating compatibility of our platform with downstream mutational analysis.
Furthermore, we performed extensive evaluations of various potential capturing antibodies targeting epithelial, mesenchymal, prognostic and therapeutically relevant molecular targets (e.g. E-Cadherin, N-Cadherin, CEA, EGFR and ERBB2), making use of the enormous versatility of the CTCapture-chip. Through in depth testing of the specificity, sensitivity and capturing performance of the potential antibodies via immunocytochemichal stainings and simulation experiments using magnetic cell separation (MACS) technology (Miltenyi Biotec), we established an antibody cocktail targeting various therapeutically relevant markers in combination with EpCAM. In parallel, a separate melanoma antibody-capture panel was developed for analysis of CTCs in melanoma as the most aggressive skin cancer. To further demonstrate utility for cancer diagnostics, the identified capturing antibody cocktails were applied to blood samples of metastatic breast cancer (mBCa) and metastatic melanoma patients. The encouraging proof-of-principle results obtained with our optimized CTCapture-chip demonstrated feasibility of future testing in clinical studies. The IP of the CTCapture-chip has been protected by our patent application (WO 2016/128125A1 and US20180024134A1) and we made promising contacts to industrial partners for future commercialization of the chip platform.
