Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology.

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells

Nanotechnology applied to translational oncology: Developing tools for liquid biopsy

Liquid biopsy represents a powerful tool to support precision medicine, allowing the study of the subset of circulating components that derived from cancer tissue. Among all these circulating materials, the Circulating tumour cells (CTCs) represent one of the most promising biomarkers. However, the evaluation of CTCs has not been incorporated yet into current clinical guidelines for treatment decision. This might be due to CTCs are infrequent, appearing at an estimated level of one against the background of millions of surrounding normal peripheral mononuclear blood cells (PBMCs). The objective of this thesis project is to develop innovative nanoparticles that can address two of the critical points that make challenging the use of CTCs in translational studies of breast cancer: ex vivo culture and isolation. Nanoemulsions composed by a combination of lipids with potential to improve cell viability were formulated. The use of proliferative nanoemulsions (NEs) was successfully translated to ex vivo CTC cultures from metastatic breast cancer patients to expand these cells for their characterization. The analysis of these cells in culture not only showed that the precursor cells had mesenchymal and stem features but also it was determined that the capability of CTCs to grow ex vivo using the established protocol is a predictive factor in metastatic breast cancer. Finally, the NEs were functionalized with peptides (Pept-NEs) to endow them with specific recognition capabilities and it was confirmed that Pept-NEs can be immobilized on surfaces for their use as a potential isolation system