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Warawdekar et al. CTCs from patients with metastatic breast cancer

FACSDiva software v6.1.3 (BD Biosciences). the study. For quantification of a gene transcript, a
TM
TaqMan quantitative PCR assay was done for the
RNA isolation and cDNA synthesis gene transcript for every dilution in duplicate and an
A Trizol/RNeasy hybrid RNA extraction protocol external calibration curve was obtained by plotting the
was used for isolation of total RNA under RNase- concentration as copy number vs. the corresponding
free conditions. The aqueous phase obtained was threshold cycle. Each of these calibration curves
[25]
passed through a gDNA eliminator column to remove were repeated at least thrice.
genomic DNA contamination. Total RNA isolated
from each sample was suspended in RNAse-free Quantitative PCR
water. Concentration and purity were determined on TaqMan assays were done for cytokeratin-19 (CK19),
Nanodrop ND-1000 and stored at -80 °C until further epithelial cell adhesion molecule (EpCAM), Mucin1
use. cDNA synthesis was carried out with high capacity (MUC1), Her2-neu, and Glyceraldehyde-3-phosphate
reverse transcriptase kit (Applied Biosystems) dehydrogenase (GAPDH). Reactions for all the above
according to manufacturer’s instructions. genes were performed with 10 µL volume with cDNA
corresponding to 20 ng. All the samples were analyzed
Preparation of qRT-PCR calibrators in duplicates and the average value of the two was
QRT-PCR calibrators were prepared adapting the used as the quantitative value. A non-template control,
Aerts et al. [22] method with selection of the average also in duplicate, was used for all genes.
cell number obtained in the positive fraction post-
immunomagnetic enrichment from patient samples. Statistical analysis
Serial dilutions corresponding to five log steps of cell For all groups studied, values were represented as
number by diluting RNAzol lysates of MCF-7 in the mean ± SD. A simple linear regression model was used
4
WBC cell line (Jurkat) with ratios of 1:1 to 1:10 of to assess the fit for the recovery of expected cells.
CK19+ and EpCAM+ cells per CK19- and EpCAM-
cells were made. Dilution of lysates was performed RESULTS
to avoid the need for permanent maintenance of cell
cultures for preparation of cell-cell dilutions. RNA Flow cytometry for CTCs analysis
extraction and cDNA synthesis were performed as A tricolor setup was configured to detect the CD45
described. Each sample was measured in triplicate. PerCP signal in 670 nm long pass, EpCAM-APC
signal in 660 nm bandpass, and Cytokeratin-FITC
We also prepared qRT-PCR calibrators by adapting signal in 530 nm band pass filter. Events that fell within
the Strati et al. method as a qRT-PCR requires the region P2, i.e. EpCAM, and CK dual-positive were
[23]
analysis of samples across time frames. Individual counted as meeting criteria for human tumor cells
PCR amplicons corresponding to gene-targets CK- [Figure 1A and B]. Thresholds for specific EpCAM
19, EpCAM, MUC-1, and Her2Neu that could serve APC and CK FITC signals were determined using the
as quantification calibrators were generated. For this sample stained with isotype control antibodies. The
purpose, total RNA was extracted from MCF-7 and same gating strategy was then applied for detecting
BT474 cells, DNase-treated, and quantified. cDNA was EpCAM+CK+CD45- cells in the clinical sample
synthesized from 1 µg of DNase-treated RNA using a stained with the specific antibodies.
high capacity cDNA kit according to manufacturer’s
instructions. PCR for each target gene was carried Specificity (ability to differentiate between
out with TaqMan primers on a Bio-Rad Peltier Thermal epithelial tumour cellsand WBC designate)
cycler to ensure production of calibrators from the EpCAM-APC (clone HEA-125) and Pan-Cytokeratin-
same amplicon that would be synthesized during the FITC (clone CK3-6H5) Abs were found to be specific
qRT-PCR of clinical samples. PCR amplicons were for human mammary cancer cells, with no non-specific
separated on a 3% Agarose gel, excised under UV, binding to Jurkat cells [Figure 1C-E]. Similarly, human
purified using QIAquickGel Extraction Kit (Qiagen), CD45 PerCP antibody (clone 30F11.1) was found to be
and quantified on Nanodrop. Copy number was highly specific for leukocytes and did not stain human
calculated from the concentrations using Avogadro mammary tumour cells [Figure 1F-H]. To determine
constant and molecular weight of each amplicon applicability of the method to various human mammary
number of bases of the PCR product multiplied by the cancer cell lines, the ability to detect MCF-7, T-47D,
mean molecular weight of a pair of nucleic acids, which ZR-75-1, BT-474, and MDA-MB-468 spiked in Jurkat
is 660. Serial dilutions of these stock amplicons were cells was tested. In all cases, tumor cells exhibited
made in TE buffer (1 × 10 copies to 1 × 10 copies) similar staining to EpCAM and CK antibodies and
0
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and used as quantification calibrators throughout could be clearly differentiated from Jurkat cells.
26 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ February 23, 2017