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Warawdekar et al. CTCs from patients with metastatic breast cancer

T-47D, ZR75-1, BT-474, and MDA-MB-468 and the quantitative RT-PCR.
lymphoid cell line, Jurkat, were used for cell spiking
and standardization of the flow cytometry assay, Cell spiking experiments
and qPCR. MCF-7, BT-474, and MDA MB-468 were Human mammary cancer cell lines representing the
maintained in DMEM containing 10% FCS; T-47-D and molecular subtypes were grown as adherent cultures
ZR75-1 in RPMI-1640 with L-glutamine (2 mmol/L) and harvested using sterile trypsin-EDTA solution at
and 10% FCS; Jurkat in DMEM containing 10% 80% confluency. The Harvested cells were collected,
FCS and antibiotic mixture containing gentamycin, washed, and re-suspended in a fixed volume of culture
streptomycin and anti-fungal, Forcan. Cell line medium. An average of 3 cell counts and viability were
ZR75-1 was purchased from the National Centre for determined by the trypan blue dye exclusion method.
Cell Science, Pune, India. All other cell lines were This cell suspension was used for serial dilution in cell
available in the institute or laboratory and procured spiking experiments and cells were spiked into the
for ongoing studies. Jurkat cell line, which served as the WBC designate
of the PBMC fraction. Jurkat cell number was selected
Patients and controls on the basis of the average cell count obtained in the
A total of 22 breast cancer patients with metastatic positive fraction following immunomagnetic enrichment
disease were included in this study. In addition, blood of 20 mL of peripheral blood, which is approximately
samples from 20 healthy volunteers were accrued. 1 × 10 cells counted with a hemocytometer. Human
5
5
After collection, blood samples were immediately mammary tumor cells were spiked into 1 × 10 Jurkat
processed for isolation and detection of CTCs. cells at levels ranging from 0.5% to 0.001%. These
cell suspensions were labeled with antibodies as
Blood collection and PBMCs isolation described below, acquired on the flowcytometer and
Peripheral blood (25 mL) was drawn by phlebotomy the percentage expected to the percentage recovered
in EDTA vacutainer tubes. The initial 5 mL was routed was plotted as shown below. For each sub-type,
[24]
for other routine tests to avoid contamination with cells experiments were repeated at least thrice.
from the skin and blood vessels. Tumor cells were
isolated along with the PBMCs on a Ficoll Hypaque Antibody labeling and acquisition on flow
gradient. Blood diluted with PBS was layered over cytometer
Ficoll Hypaque and centrifuged at 400 g for 30 min at Serially diluted samples of tumor cells spiked into Jurkat
25 °C. The interphase was collected, washed twice, cells were surface-stained with monoclonal antibodies
with separation buffer, spun at 400 g for 15 min at that target epithelial cell antigens EpCAM (CD326) and
4 °C to obtain a cell pellet, which was suspended in CD45 or the corresponding isotype control antibody by
separation buffer and the total yield and viability was incubating the cells in 50 μL of FACS buffer for 30 min in
assessed. the dark at room temperature. Cells were then washed
with FACS buffer and fixed with 1% paraformaldehyde
Immunomagnetic enrichment and CTCs for 15 min at 4 °C before permeabilization for
isolation intracellular staining. To permeabilize cells, the pellet
CTCs were enriched from the total cell pellet. was resuspended in 0.1% saponin buffer. Cells were
Depending on the total cell count, initially 50-100 µL of subsequently stained with anti-pan CK antibody or the
FcR blocking reagent (Miltenyi Biotec) was added to relevant isotype control and incubated for 45 min in the
block Fc receptors and eliminate non-specific binding. dark at room temperature. After staining, cells were
Subsequently, 50-100 µL of micro beads conjugated washed with saponin buffer, resuspended in 300 μL
to monoclonal antibody for EpCAM (Miltenyi Biotec) of FACS buffer, and immediately acquired on a FACS
was added and this mixture was incubated for 30 min Aria flowcytometer (Becton Dickinson, USA), which
TM
at 4 °C. Subsequently, cells were washed and re- is equipped with a 488 nm blue laser (for excitation
suspended in 700 µL of separation buffer. This labeled of FITC and PerCP) and a 633 nm red laser (for
cell suspension was then acquired on autoMACS Pro excitation of APC) and the following filters: FITC: 530
®
Separator (Miltenyi Biotec) with a double-positive nm band pass; PerCP: 670 nm long pass; APC: 660
selection. The positive fraction constituted cells that nm band pass. Setup and automatic compensation
expressed EpCAM and thus was an enriched fraction were performed using cells stained for each marker.
of EpCAM-expressing cells. The negative fraction PMT voltages used for recording fluorescence signals
constituted lymphocytes, cells that do not express were as follows: FITC = 429, PerCP = 599, APC =
EpCAM and flow through the column. The positive 396. The absolute number of spiked tumor cells was
and negative fractions were divided into two aliquots: estimated by acquiring a minimum of 10,000 events
one for multi-parameter flow cytometry, the other for in the analyzed sample. Data were analyzed with BD
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ February 23, 2017 25