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Tumor Discovery CTC characterization for EGFR mutations

in the clinics has been hampered by challenges related 2.4. Cell culture
to reproducibility, low throughput, and intricate work A lung adenocarcinoma cell line (PC-9) obtained from
45
mechanisms. This study aimed to describe the design and the European Collection of Authenticated Cell Cultures
43
functionality of an immunomagnetic microfluidic device (ECACC, www.phe-culturecollections.org.uk) was cultured
that isolates epithelial cell adhesion molecule (EpCAM)- in RPMI-1640 media (Lonza, UK) containing 10% (v/v) fetal
positive CTCs from the blood of patients with NSCLC and bovine serum (Labtech.com, USA), with a final concentration
to evaluate exon 18 – 21 mutations of the EGFR gene with of 50 µg/mL penicillin and 250 µg/mL streptomycin (Lonza,
the aim of providing a rapid and robust way of obtaining UK). Cells were maintained in a humidified incubator at
clinically relevant information that can aid in treatment 37°C with an atmosphere of 5% CO (Galaxy 170 S, New
decisions. Brunswick Scientific, Stevenage, UK). Cells were used for
2
2. Materials and methods experiments when they were 80% – 90% confluent.
2.1. Materials 2.5. Spiking experiments
HPC Laser LS3060 60W CO laser cutter (HPC Laser, PC-9 cell lines were harvested from culture media, and
2
Halifax, UK); polymethylmethacrylate (PMMA) sheet a cell viability count was undertaken. Cells were always
(Vink Plastics, Manchester, UK); PC-9 cell lines (obtained at least 85% viable when used. PC-9 cells were added
from European Collection of Authenticated Cell Cultures); to 12 mL of RPMI-1640 media (Lonza, UK) at the
6
4
5
neodymium iron boron (NdFeB) magnets (Integrated following concentrations: 1 × 10 , 2 × 10 , 4 × 10 , and 8
3
Magnetics, California, USA); RPMI 1640 media (Lonza, × 10 cells/mL. Thereafter, the cells were isolated using the
Slough, UK); Dynal anti-EpCAM magnetic beads (Thermo microfluidic device. Cells isolated were thereafter subjected
Fischer Scientific, Loughborough, UK); 4,6-diamidino- to PCR to validate the device’s ability to process CTCs for
2-phenylindole (Vector Laboratories, Newark, USA); downstream analysis and detection of mutations. A range
fluorescein-conjugated pan-cytokeratin monoclonal of cell concentrations were used for spiking experiments
antibody (San Diego, USA); rhodamine-conjugated mouse to validate the utility of the device in isolating EpCAM-
anti-human CD45 antibodies (BD Biosciences, Fremont, positive cell lines. Some of these concentrations reflected
California USA); and PCR mix and primers (Stab Vida concentrations of EpCAM-positive CTCs reported in the
Genetics, Laboratory, Lisbon, Portugal). blood of NSCLC patients (1 – 80,000 cells/mL). 46,47

2.2. Manufacture of the PMMA chip 2.6. Patient recruitment/sample processing for CTC
analysis
The dimensions of the PMMA chip were first drawn using an
AutoCAD DXF file and then transferred to the laser cutter Fifty-nine patients aged between 47 and 81 years diagnosed
software to cut the design from a 3-mm PMMA sheet. Two with NSCLC and admitted to the Castle Hill Hospital
pieces of PMMA measuring 98 × 98 mm were cut using an were recruited for the study after ethical approval had
HPC Laser LS3060 60W CO laser cutter. Afterward, the been received from the North East-Newcastle and North
2
pieces were bonded together using ethanolic treatment and Tyneside Local Research Ethics Committee (REC13/
then underwent ultraviolet irradiation for 25 s as described NE/0242). Written informed consent was obtained from
previously. A single PMMA sheet measuring 57 × 66 mm all participants (Appendix A1). Patients’ demographic/
44
was also fabricated to form the lid. clinicopathological data were obtained from their medical
records by Professor Michael Lind and presented in a
2.3. Fabrication of the electromagnetic arm of the pseudo-anonymized format.
device A total of 13.5 mL of whole blood was collected into
The magnetic arm is made of a C-shaped polycarbonate three 3.2% trisodium vacutainer sample bottles (BD, USA).
piece (measuring 10 cm in height and 4 mm in thickness) Each sample bottle contained 4.5 mL of blood. Within
to which the NdFeB magnets (Integrated Magnetics, USA), 15 min of sample collection, the blood was transported
measuring 20 × 5 × 4 mm, are attached at the top and on ice to the laboratory where processing was done in a
bottom arms (Figure 1). The movement of the magnetic class II biological safety cabinet (ESCO Scientific). Blood
arm is controlled by a high-voltage power supply that was pooled in a sterile 50-mL Falcon tube, and 2 mL of
moves the arm in the x, y, and z axes. A magnetic actuator phosphate-buffered saline was added to reduce blood
program ensures precise movement of the arm. The viscosity. The tube was mixed thoroughly by inverting the
magnetic device and controller unit were manufactured by tube three times and then immediately loaded into the
Micro-Lab Devices Leeds, UK. chip (Section 2.7).

Volume 3 Issue 4 (2024) 3 doi: 10.36922/td.3987