**2.6. RT‐PCR**

istics of the patients are shown in **Table 1**, including mean age, TNM phase, histopathology, lymph node status, metastasis and so on. All these patients were incipient and were treated by systemic therapy, including 12 patients cured by cytokine‐induced killer cells therapy (CIK).

Carcinoma cell line SKBR‐3 (breast) was used to estimate the sensitivity and specificity of the

Antibodies, which were used for multiparameter flow cytometry, were as follows: anti‐CD45‐ PerCP, anti‐CD44‐APC (allophycocyanin, clone G44‐26, catalog number 559942) and anti‐ CD24‐FITC (fluorescein isothiocyanate, clone ML5, catalog number 555427) were from Becton Dickison Crop., USA. Ep‐CAM (epithelial‐cell adhesion molecule) and CK‐8, CK‐18, CK‐19

Every patient with breast cancer drew 20 ml blood for CTCs detection and three healthy volunteers. The blood of healthy volunteers was treated as negative control. About 5 ml blood was discarded to avoid contamination with skin cells as previously described [26]. We separated mononucleocytes from 15 ml blood by Ficoll‐Paque (Haoyang Biological Production Limited Company, Tianjin, China) for 20 min with 1800 × *g* at 25°C. One half of the mononu‐ clear cells was resuspended in phosphate‐buffered saline (PBS) for multiparameter flow cytometry on the account of at least 2–3 × 106 cells for each sample and the other half was kept

Mononucleocytes were enriched and washed twice with PBS and then labelled antibodies that

SKBR‐3 breast cancer cells were used to evaluate the sensitivity of the flow cytometry (tumor cells recovery). Tumor cells were resuspended and counted. 1, 10, 50, 500 cells were spiked, respectively, into 7.5 ml of blood from healthy person. And then, it was processed as described as earlier and control the preparation for the same volume of the cell suspension and cells were counted to accurately estimate the number of cells spiked into the blood. The average number of Ep‐CAM and CK‐8, CK‐18, CK‐19 positive cells on FACS Caliber™ was used to calculate

), kept in dark at 4°C for 30 min. We added 20 μl monoclonal antibodies for each sample. Cell pellets were resuspended in 250 μl PBS and enumerated by FACS Caliber™ (Becton

, CK‐8, CK‐18, CK‐

flow cytometry, which was maintained in RPMI 1640 plus 10% fetal calf serum.

(CK‐8, CK‐18, CK‐19 phycoerythrin staining) were from Abcam, USA.

in Trizol reagent (Invitrogen, UK) at -70°C until RNA extraction for RT‐PCR.

target white cell antigens and epithelial cell antigens (CD45‐, Ep‐CAM<sup>+</sup>

And all the patients were drawn blood for the detection of CTCs.

**2.2. Cell line**

198 Tumor Metastasis

**2.3. Antibodies**

**2.4. Preparation of samples**

**2.5. Flow cytometry**

the cells recovery.

Dickison Crop., USA) at last.

19<sup>+</sup>

We extract RNA with 1 ml trizol from the mononucleocytes, which were separated from 7.5 ml blood and then kept at -70°C. Add 0.2 ml chloroform and then centrifuge the sample at 12,000 × *g* for 15 min at 4°C. The intact RNA, which was contained in the supernatants, was removed into a new tube. RNA was dissolved in 10 μl RNase‐free water after precipitating RNA with isopropyl alcohol and washing with 75% ethanol. Then, RNA was transcribed to cDNA by a reverse transcriptase in a total 10 μl RT reaction solution, which was contained of 2 μl 5x Reverse Transcriptase Buffer, 1 μl dNTP (10 mM each), 0.25 μl RNase inhibitor (10 U), 1 μl oligo (dT)15 primer (25 pmol), 5.25 μl RNase‐free water containing RNA (>0.5 μg), 0.5 μl avian myeloblastosis virus (AMV) reverse transcriptase (5U) (TaKaRa, China). The resulting cDNA was subjected to PCR amplification. PCR was composed of 2 μl cDNA, 10 μl Mix, 2 μl primer of EpCAM, 6 μl H2O in a total volume of 20 μl. The primer of EpCAM was as follows: 5'‐GGACCTGACAGTAAATGGGGAAC‐3'; 5'‐CTCTTCTTTCTGGAAATAACCAGCAC‐3' [18]. GAPDH mRNA primer detail was as follows: 5'‐TGCACCACCAACTGCTTAGC‐3'; 5'‐ GGAGGCAGGGATGATGTTCT‐3' which was designed by Primer 5.0. The reaction condition was 95°C for 2 min to activate Taq DNA polymerase and it finally elongated 72°C for 7 min with 35 cycles. We detected the PCR products by ethidium bromide staining on a 1% agarose gel.

#### **2.7. Statistical analysis**

Correlation, regression analysis and a Mann‐Whitney rank sum test were performed on titration experiments. Chi‐square test was used to compare across CTC groups. Overall survival was performed to describe the condition of CTCs and prognosis and was estimated by the Kaplan‐Meier. Comparison of groups used the log‐rank test. All statistical tests were two‐sided, and *P* values < 0.05 were considered statistically significant. Analyses above were performed by using the SPSS 13.0.
