Abstract
The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 out of 8 samples from patients diagnosed with metastatic prostate cancer (mean ± SEM = 21 ± 2.957 CTCs/mL, median = 21 CTCs/mL), demonstrating the potential clinical utility of this device.
Highlights
The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments
The holder is connected to a fully automated flow control unit (Fig. 2b), which consists of a diaphragm compressor as pressure source, an electronic proportional valve that controls the pressure applied to the sample and reagents reservoirs, an electronic rotary valve to select from which reservoir liquid is expelled, and a flow sensor that provides the necessary feedback to precisely compute the pressure needed in the selected reservoir to maintain the desired flow rate
CTCs have demonstrated their potential as a blood-based biomarker that can be used on a broad range of cancer-related clinical applications
Summary
The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. The first report describing the existence of CTCs dates from 18694, the heterogeneity and the www.nature.com/scientificreports extremely low concentration of these cells in regard to the cellular components of blood, about 1–10 CTCs per 109 blood cells, makes their capture extremely challenging[5,6] It was not until the recent development of technologies with the required sensitivity and reproducibility, that the possibility to perform CTC-based clinical assays started to become a reality. An effective alternative to these technologies are microfiltration devices, which rely on the differences in size and deformability between blood cells and CTCs in order to capture them These platforms have consistently proven their effectiveness at isolating a greater number of CTCs in samples from patients with different types of cancer, even capturing CTC subtypes that no longer express EpCAM antigens, when compared with approaches based on capture antibodies[28,29,30]
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