Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.

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The enumeration and capture of circulating tumor cells (CTCs) are potentially of great clinical value as they offer a non-invasive means to access tumor materials to diagnose disease and monitor treatment efficacy. Conventional immunoenrichment of CTCs may fail to capture cells with low surface antigen expression. Micropore filtration presents a compelling label-free alternative that enriches CTCs using their biophysical rather than biochemical characteristics. However, this strategy is prone to clogging of the filter microstructure, which dramatically reduces the selectivity after processing large numbers of cells. Here, we use the resettable cell trap (RCT) mechanism to separate cells based on their size and deformability using an adjustable aperture that can be periodically cleared to prevent clogging. After separation, the output sample is stained and analyzed using multi-spectral analysis, which provides a more sensitive and unambiguous method to identify CTC biomarkers than traditional immunofluorescence. We tested the RCT device using blood samples obtained from 22 patients with metastatic castrate-resistant prostate cancer while comparing the results with the established CellSearch® system. The RCT mechanism was able to capture ≥5 CTCs in 18/22 (82%) patients with a mean count of 257 in 7.5 ml of whole blood, while the CellSearch system found ≥5 CTCs in 9/22 (41%) patients with a mean count of 25. The ~10× improvement in the CTC capture rate provides significantly more materials for subsequent analysis of these cells such as immunofluorescence, propagation by tissue culture, and genetic profiling.

The extraordinary deformability of red blood cells gives them the ability to repeatedly transit through the microvasculature of the human body. The loss of this capability is part of the pathology of a wide range of diseases including malaria, hemoglobinopathies, and micronutrient deficiencies. We report on a technique for multiplexed measurements of the pressure required to deform individual red blood cell through micrometer-scale constrictions. This measurement is performed by first infusing single red blood cells into a parallel array of ~1.7 μm funnel-shaped constrictions. Next, a saw-tooth pressure waveform is applied across the constrictions to squeeze each cell through its constriction. The threshold deformation pressure is then determined by relating the pressure-time data with the video of the deformation process. Our key innovation is a self-compensating fluidic network that ensures identical pressures are applied to each cell regardless of its position, as well as the presence of cells in neighboring constrictions. These characteristics ensure the consistency of the measurement process and robustness against blockages of the constrictions by rigid cells and debris. We evaluate this technique using in vitro cultures of RBCs infected with P. falciparum, the parasite that causes malaria, to demonstrate the ability to profile the deformability signature of a heterogeneous sample.