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Warawdekar et al. CTCs from patients with metastatic breast cancer

and suitable approach. in expression of the selected genes across clinical
samples, estimate copies/mL in blood samples and
There are several technologies which have been enable direct comparison of samples across time
developed and are available for CTC enrichment points of collection. Thus, besides characterization, a
and detection as cited in literature. [12,13] The quantitative measure of gene expression translating
current standard method that has been widely to numbers of CTCs can be obtained.
used in large multicenter clinical trials world-wide
and which continues to be preferred is the FDA- This methodology was substantiated with blood
approved quantitative, semi-automated, Cell Search samples obtained from patients and healthy
system, [14,15] which assesses 7.5 mL of blood. It is normal individuals. Blood from 22 patients
based on the epithelial cell adhesion molecule with metastatic breast cancer and 20 healthy
(EpCAM/CD326) conjugated immunomagnetic individuals was separated on a Ficoll gradient to
enrichment followed by microscopic imaging obtain the peripheral blood mononuclear cells
using positive immunostaining of cytokeratin (CK), (PBMCs) fraction, positively enriched for EpCAM,
negative immunostaining of leucocyte common quantitatedby flow cytometry and validated with
antigen CD45, and DNA staining with DAPI. QRT-PCR for the presence of CTCs.

The literature shows that almost 60% of studies on METHODS
detection and enumeration of CTCs have preferred
methods using reverse transcription polymerase Materials
chain reaction (RT-PCR) because of its sensitivity DMEM, RPMI 1640, Fetal Calf Serum (FCS), Trizol,
and low cost as compared to using the Cell Search High capacity cDNA kit, TaqMan assays for CK-19,
technology. One such RT-PCR kit-based technology EpCAM, MUC1, Her2neuand GAPDH, 2 X TMM, and
is the Adnagen assay. [16,17] This entails enrichment on PBS were procured from Invitrogen Life Technologies
the basis of epithelial and tumour-specific antigens,
isolation of RNA, conversion to cDNA and subsequent (Carlsbad, CA, USA). RNeasy plus micro kit was
amplification of tumour-associated genes and a control from Qiagen, GmBh, D-40724, Hilden, Germany.
gene for normalization. Amplified cDNA samples are FcR blocking reagent, CD326 (EpCAM) Microbeads,
then analyzed on a chip system (Bioanalyzer) and MACS BSA Stock Solution, rinsing buffer, and
simultaneously compared with positive controls of washing buffer were purchased from Miltenyi Biotec
each gene provided in the kit. GmBH, Germany. Ficoll-Paque PLUS was procured
from GE Healthcare, Bio-Sciences AB, Uppsala,
Strategies using cell-imaging, quantitative RT-PCR Sweden. EDTA vacutainer tubes were from Greiner
[18]
(QRT-PCR) and flow cytometric detection [19-21] for Bio-One, Austria. All other chemicals were obtained
enumeration and characterization of CTC shave also locally and were of analytical grade.
been employed.
Antibodies
The purpose of this study was to devise a feasible, CD326 (EpCAM) antibody conjugated to APC (clone
relatively low-cost methodology which would HEA-125), pan-Cytokeratin antibody conjugated to
enumerate CTCs as well as validate their assessment. FITC (clone CK3-6H5), CD45 antibody conjugated to
The protocol for enumeration was developed using PerCP (clone 30F11.1), mouse IgG1 isotype control
tumor cells from cell lines of breast cancer subtypes antibodies (clone-IS5-21F5) conjugated to FITC,
spiked in a lymphoid cell line and its quantitation by flow APC, and PerCP were purchased from Miltenyi Biotec
cytometry. Cells of human mammary cancer subtypes GmBH, Bergisch-Gladbach, Germany.
were spiked into a Jurkat cell line which served as
the lymphocyte designate in numbers from 10 to Buffers
500 per 10 Jurkat cells. This mixed population was Separation buffer composition: phosphate-buffered
5
then probed for CD45, EpCAM, and pancytokeratin saline (PBS; pH 7.2), 5% bovine serum albumin
acquired on a flowcytometer and quantitated. Similar (BSA), 2 mmol/L EDTA prepared by diluting MACS
spiking experiments were done for QRT-PCR and BSA Stock Solution with auto MACS™ rinsing solution
expression of CK-19, MUC-1, EpCAM, and GAPDH. (1:20); FACS buffer contains 0.02% NaN , 1% FCS
3
The methods described by others [22,23] have been in PBS (pH 7.4); Saponin buffer for permeabilization
adapted for quantitation of CK 19, EpCAM, MUC-1, contained 0.1% saponin in FACS buffer.
and Her2neu. Data pertaining to these methods have
been generated and any one of these methods can Cell lines
be used. Our method will overcome the differences Human mammary carcinoma cell lines: MCF-7,
24 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ February 23, 2017