High-throughput isolation and characterization of heterogeneous viable circulating tumor cell using tapered-slit filters with paraformaldehyde treatment

Abstract

Circulating tumor cells (CTCs) have received a great amount of attention, as they are known to promote metastatic spread of cancer. Despite a wide variety of techniques developed to utilize CTCs as a diagnostic and prognostic tumor biomarker, clinical trials based on CTC analysis have shown inconsistent benefits due to difficulties in isolating these heterogeneous rare cells with high sensitivity, as well as maintaining their viability. First, we developed a tapered slit filter (TSF) for high throughput isolation of viable heterogeneous CTCs. The numerical analysis and in vitro cell capture assay revealed that the optimal TSF prototype was capable to reduce the stress applied on cells by 10% compared to straight-walled slit filters (SSFs), which in turns enabled high throughput (25 mL/h), viable (87-94%), and highly sensitive (83-100%) capture of cancer cells, regardless of their epithelial/mesenchymal features. As a result, TSF outperformed conventional CTC isolation devices, as an EpCAM-targeting approach demonstrated low capture efficiency for mesenchymal-like SW620 cells (~10%), while sharp edges of the SSFs significantly reduced the cell viability (~70%). Second, we developed a viable CTC isolation method based on paraformaldehyde (PFA) treatment for sensitive capture of mesenchymal cells. In vitro cell line experiments revealed that both the epithelial-like and mesenchymal-like cancer cells improved the Young's modulus by more than 25 and 42% at 0.1% PFA concentrations, while preserving viability (90%) and size (99.2%). Consequently, PFA treatment enabled high throughput (50 mL/h) and very sensitive capture (100%) of mesenchymal cancer cells, with negligible reduction in specificity or cell viability, when using one of the filter-based CTC isolation devices. The clinical study using blood samples obtained from colorectal cancer patients further demonstrated that TSF and PFA treatment were capable to isolate heterogeneous CTC subtypes, which expressed epithelial, mesenchymal, or hybrid of epithelial/mesenchymal markers. The results presented in this study support that the new device has potential to provide in-depth information on pathological status of the patients.