Introduction: The critical role of circulating tumor cells (CTCs) in distant cancer metastasis is well established. However, their metastatic propensity when in the form of cell clusters far exceeds that in single cell form. This underscores the pressing need for further investigation into the biology of CTC clusters. For the study of either patient blood or cultured cell clusters spiked into in healthy blood, the initial and crucial step in investigating CTC clusters is their isolation from other cells. Additionally, the exceedingly low proliferation rate of CTCs renders their expansion in vitro impractical. Consequently, accessing primary CTCs becomes paramount for gaining insight into their formation and survival in vivo. Microfluidics, as a technology for handling fluid and cells at a microscale level, can be an invaluable tool for isolating CTC clusters, which are extremely rare in whole blood. The non-Newtonian nature of blood rheology, its diverse cell composition, and the rarity of CTC clusters collectively pose challenges for their isolation.
Materials and
Methods: The microfluidic device used in this study contains many triangular prism obstacles in a staggered configuration with specific gaps between them to trap CTC clusters while allowing other cells in blood to pass through unimpeded [1], as shown in Figure 1. Therefore, the CTC clusters are expected to be trapped between obstacles. The 5-inch Silicon wafer, and SU8-2075 were used for soft lithography, and a device thickness of 100 μm. Polydimethylsiloxane (PDMS) elastomer base was crosslinked by its curing agent in a 1:10 ratio, degassed using a desiccator and poured onto a mold and kept in the oven for 90 min in 65°C to be cured, and then a microscopy glass slide was bonded to the PDMS using oxygen plasma. The internal surface of the device was treated with 10% BSA (Bovine Serum Albumin) and incubated overnight. For processing blood samples, whole blood was either used in unprocessed form or diluted by a 1:10 ratio with PBS to reduce risk of clot formation. Injection was carried out in two steps. The first step involved a flow rate of 2.5 ml/hr for the sample, and the second step involved the same flow rate for the buffer to deplete anything but CTC clusters. Thereafter, a reverse flow was applied with 250 ml/hr flow rate to extract CTC clusters. All steps were carried out at 4°C. Furthermore, to mimic CTC clusters, LNCaP (human prostate adenocarcinoma) cells cultured using superhydrophobic microwell arrays for 3D cell culture, developed in the King Lab, were utilized [2].
Results, Conclusions, and Discussions:
Results: Utilizing a surface treatment using overnight incubation in a 10% BSA solution, and maintaining the device at 4 °C, effectively prevented both cell adhesion to PDMS and the formation of blood clots. This device demonstrates remarkable efficacy in the isolation of CTC clusters from a variety of sample sources, including unprocessed patient blood samples, diluted patient blood samples, and LNCaP clusters (Figure 1). While undiluted patient blood samples carry an increased risk of clot formation, their structural integrity is compromised by shear stress and dynamic flow, leading to eventual disintegration. Conversely, CTC clusters were found to be resistant to shear stress and remained in cluster form.
Discussion and
Conclusions: We have fabricated a microfluidic device to isolate CTC clusters from whole blood patient samples or diluted blood, as well as LNCaP clusters with appropriate surface treatment. The ability to isolate CTC clusters within a closed microfluidic system enables testing of drugs for cluster disintegration and further investigations into the biology of how such metastatic cells respond to fluid forces present in the circulation.
