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Detection of circulating tumor cells in patients with breast cancer using the conditionally reprogrammed cell culture method and reverse transcription-PCR of hTERT and MAGE A1-6

The present study aimed to verify the efficacy of the conditionally reprogrammed cell (CRC) culture method for the detection of circulating tumor cells (CTCs) in breast cancer. CTCs were isolated from the peripheral blood of patients with breast cancer, and culture of the collected CTCs was performed according to the conditional reprogramming protocol. Total RNA was extracted from cultured CTCs, and the hTERT and MAGE A1-6 genes were amplified using reverse transcription-PCR (RT-PCR). In addition, RNA extraction from another blood sample was performed and the expression of the two genes was analyzed by RT-PCR only. Following CRC culture, grown CTCs were observed in 7 samples (23.3%). The CTC detection rates by RT-PCR for the hTERT and MAGE A1-6 genes in CTCs grown using the CRC culture method were 26.7 and 10.0%, respectively. The positive expression rates for the hTERT and MAGE genes in CTCs assessed by RT-PCR only were 44.1 and 23.5%, respectively. When combining the positive expression rates of RT-PCR only and CRC culture for the hTERT and MAGE A1-6 genes, CTC detection rates increased to 53.3 and 23.3%, respectively. Additionally, when combining the positive expression rates of the two genes by either method, the CTC detection rate was the highest value observed. In conclusion, the present study revealed the potential of CRC culture in the detection of CTCs in breast cancer. Furthermore, a combination of CRC culture and RT-PCR for the hTERT and MAGE A1-6 genes is useful in enhancing the detection rate of CTCs in the blood.


Introduction
Liquid biopsy is often used as a technique for detecting biomarkers in body fluids, including blood, urine, saliva, pleural effusions, ascites and cerebrospinal fluid (1). Human body fluids contain biologic materials such as cell-free DNA and RNA, proteins, cells and vesicles (1). These materials can be representative of the tissues from which they originated, and thus liquid biopsy has the potential to be used instead of tissue-based biopsy in clinical practice and for research purposes. In addition, liquid biopsy allows clinicians to perform biopsies repeatedly and non-invasively.
Circulating tumor cells (CTCs) are tumor cells derived from primary tumors and/or metastatic lesions, and circulate freely in peripheral blood (2). CTCs have characteristics specific to their tumor of origin (2) and can migrate to the circulatory system causing metastasis (3). Recently, many studies have described the potential applications of CTCs in the diagnosis, evaluation of treatment response, and targeted therapy of cancer (4)(5)(6)(7)(8).
Multiple technologies have been developed to detect and isolate CTCs, but quality detection of CTCs remains a central problem in the study of CTCs. Most detection methods are based on the detection of epithelial cell adhesion molecule (EpCAM), cytokeratin, and lack of the leukocyte common antigen CD45 (3). Furthermore, CTCs can be sorted based on physical characteristics such as size, density, deformability, and electrical charge (1). The CellSearch ® system is an automated EpCAM-based system and is the only technology approved by the US Food and Drug Administration in a detection of CTCs for the clinical use (3). Although the CellSearch ® system is considered the standard method for the detection of CTCs, several issues still exist regarding this system. Not all CTCs express EpCAM (2), and CTCs are heterogeneous among cancer type and even within individuals (3). Moreover, CTCs isolated by the CellSearch ® system are fixed and unavailable for functional analysis (9). Thus, there is a need to develop more specific and efficient methods to distinguish viable CTCs from other cells.
The melanoma antigen encoding gene (MAGE) family is known to encode tumor-specific antigenic peptides (10) and is dysregulated in many cancers (11), and the human telomerase reverse transcriptase (hTERT) gene is also known to be expressed in most cancer cells (12). In our previous study, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of the MAGE A1-6 and the hTERT genes to detect CTCs in blood, and our results showed good sensitivity and specificity (13).
It has been suggested that ex vivo culture and characterization of CTCs may be utilized in the study of cancer metastasis and patient-derived tumor models (7,9), and in recent years, several studies have shown the results of CTCs culture in different culture conditions (7,(14)(15)(16)(17). Liu et al (18) showed that small numbers of cells can be cultured effectively by generating conditionally reprogrammed cell (CRC) (18). Conditional reprogramming (CR) is a cell culture technique culturing patient-derived cells with feeder cells and a Rho kinase (ROCK) inhibitor (18). Recently, Zheng et al (19) investigated CRC culture method that is efficient for the culture of CTCs in vitro and showed that the cultured cells preserve their original phenotype. Detection of CTCs using CRC cultures appears to be a very efficient alternative method, but a significant amount of research is still required.
In this study, we aimed to verify the efficacy of a CRC culture method for the detection of CTCs. Further, to enhance the detection rate of CTCs, we utilized a joint method combining CRC culture with RT-PCR of the MAGE A1-6 and the hTERT genes in blood.

Materials and methods
Patients and specimens. A total of 34 patients with breast cancer who had undergone surgery at Daegu Catholic University Hospital (Daegu, Korea) were included. To detect CTCs and analyze RNA levels of target genes, two 6-ml samples of peripheral blood were drawn into ethylenediaminetetraacetic acid (EDTA) coated tubes from each patient before surgery. Specimens were stored at 4˚C until further analysis. All data were recorded prospectively, and the clinicopathologic characteristics of the patients were evaluated from medical records. Cancer staging was assessed according to the 7th edition of the American Joint Committee on Cancer staging manual for breast cancer. Written informed consent was obtained from all patients according to the protocol approved by the Institutional Review Board of the School of Medicine, Catholic University of Daegu.
CTCs isolation. To detect CTCs, we collected peripheral blood mononuclear cells (PBMC) after red blood cells (RBCs) lysis from the blood. CTCs were isolated by negative immunomagnetic separation technology (20) using the CD45 antibody capture system as previously described (13). Briefly, the RBCs were lysed with RBC lysis buffer (Roche Diagnostics GmbH). Following RBC lysis, the cells were resuspended in 80 µl of CD45 binding buffer and reacted with 20 µl of microbeads conjugated to monoclonal antihuman CD45 antibodies (cat. no. 130 045 801; Miltenyi Biotec GmbH) for 15 min at 4˚C. The CD45 + cells were captured using a magnetic separator, and the CD45 cells were eluted and collected.
RNA extraction. Total RNA of PBMCs was extracted from one blood sample using the RNeasy Mini kit (Qiagen AB), or TRIzol™ reagent (cat. no. 15596026; Invitrogen (Carlsbad); Thermo Fisher Scientific, Inc.) and TRIzol Plus kits (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction. Briefly, the samples were lysed and homogenized in 1 ml of TRIzol™ reagent, and then incubated for 5 min at room temperature. Chloroform (0.2 ml) was added to the samples in TRIzol™ reagent, and incubated for 2-3 min at room temperature. After centrifugation for 5 min at 12,000 x g at 4˚C, RNA in the samples was precipitated by adding isopropanol. The samples were washed with 1 ml of 75% ethanol and the supernatant was discarded after the centrifugation. RNA was then dissolved with RNase free water and 0.5% SDS solution. RNA yield was determined by measuring absorbance at 260 and 280 nm.

Reverse transcription polymerase chain reaction (RT PCR).
RNA extracted from PBMC was then amplified for the hTERT and the MAGE A1-6 genes following the method described in previous studies (13,21). Briefly, extracted RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega Corporation) according to the manufacturer's protocol. The hTERT and the MAGE A1-6 genes were amplified using the LightCycler FastStart DNA Master System (Roche Diagnostics GmbH) according to the manufacturer's instruction, and detection was performed using the LightCycler 2.0 (Roche Diagnostics GmbH). The GAPDH gene was used as a housekeeping gene for normalization. Each experiment was independently repeated at least three times.
We identified cells grown in the culture plates and the cells were defined as CTCs grown by CRC culture, because the lifespan of CTCs is short and most of CTCs cannot survive for a long time after isolation, and would only be present if they were able to grow in culture condition. Following culture of CTCs by CRC culture methods, culture plates were treated with 1ml of TRIzol™ reagent and total RNA was extracted as above. Then, the hTERT and the MAGE A1-6 genes were amplified and analyzed using RNA extracted from cultured CTCs.
Statistical analysis. Statistical analysis was performed using SPSS software (version 15.0; SPSS, Inc.). CTCs were defined as cells isolated by negative immunomagnetic separation with positive expression of the MAGE A1-6 or the hTERT gene. The detection rate was defined as the percentage of samples with positive CTCs among all samples included in the analysis. The gene expression rates were defined as the percentage of samples with positive expression of the MAGE A1-6 or the hTERT gene among all samples included in the analysis. We collected two sets of blood to test for CTC detection. For the first set of blood, we used RT-PCR of the MAGE A1-6 and hTERT genes and for the second set of blood we performed CRC culture and analyzed RT-PCR of the MAGE and hTERT genes in the CRC-cultured CTCs. We defined detection of CTCs as positive expression of the genes by either RT-PCR only or the CRC culture method. The Pearson's Chi-square test was used to compare the detection rate of CTCs using RT-PCR of the MAGE A1-6 and hTERT genes, and the CRC culture method. The association between CTC detection methods and clinicopathologic characteristics was analyzed using Student's t-test for continuous parameters and the Pearson's Chi-square test for categorical data. Analysis of variance (ANOVA; parametric test) or non-parametric Kruskal-Wallis test was performed to compare continuous data of multiple groups. Multiple comparisons tests were performed when there were statistically significant differences between groups. A Bonferroni correction was applied as the post hoc test. All tests were two tailed. P<0.05 was considered to indicate a statistically significant difference.

Results
Clinicopathologic characteristics of the patients. The mean age of the patients was 57.2±12.0 years (mean ± standard deviation; range, 39-82). Among 34 patients, 30 patients had invasive ductal carcinoma, while the others were one each of mucinous carcinoma, metaplastic carcinoma, malignant phyllodes tumor and ductal carcinoma in situ. Most of patients with breast cancer were in an early stage, while 1 patient had metastatic breast cancer in the spine. Table I shows patients' clinicopathologic characteristics.
CRC culture of CTCs. CRC culture was carried out on 30 samples except 4 samples lacking sufficient quantity. Following CRC culture, grown CTCs were observed in 7 out of 30 samples (23.3%). Among the samples with growth of CTCs after CRC culture, 4 samples showed positive expression for the hTERT and the MAGE A1-6 genes. The minimum number of cells in the culture plates after CRC culture was 6 cells. Fig. 1 shows the representative CTCs cultured using CRC culture method and the cultured cells were confirmed as cancer cells.   (Fig. 3). Specifically, in 30 specimens subjected to CRC culture, 5 showed positive expression of the hTERT or MAGE A1-6 gene, although the expression of both genes in the same patients were negative by RT-PCR only.
Association of CTC detection rate with clinicopathologic characteristics by methods. When comparing CTC detection rates between RT-PCR only and CRC culture according to clinicopathologic characteristics of the patients, the positive expression of the MAGE A1-6 gene by RT-PCR only was significantly associated with higher breast cancer stage and molecular subtype of breast cancer (P= 0.006 and P= 0.039, respectively) (Table II). Also, when combining two methods of RT-PCR only and CRC culture, the positive expression rates of the hTERT and MAGE A1-6 genes were significantly higher in stage III or IV breast cancer than in stage I or II breast cancer (P=0.032 and P=0.032, respectively) (Tables II and III). There was no association between the combined positive expression of both genes and clinicopathologic characteristics (data not shown).

Discussion
Recent advances in research on CTCs have led to the development of multiple technologies to detect and isolate CTCs. In particular, ex vivo culture of CTCs using CRC culture methods is thought to be useful for the detection of CTCs and use in CTC-based studies. However, there are few studies on CRC culture in CTC detection. In this study, we aimed to verify the efficacy of CRC culture in CTC detection and as a result, we showed the growth of CTCs after cell culture using CRC culture in breast cancer for the first time. Also, consistent with the results of previous studies (18,19), we found that cultured   CTCs preserved the characteristics of the primary cancer cells. Furthermore, our study showed that the detection rate of CTCs was enhanced using a combination of CRC culture methods and RT-PCR of hTERT and MAGE A1-6 genes in the blood. CRCs have the capacity to grow indefinitely without genetic manipulation (18,22) and CRC culture methods have been proven to easily establish patient-derived CRC cultures from both normal and cancer tissue (22). In this regard, enhancing the growth of CTCs using CRC culture is helpful in improving the release of viable CTCs and effective in increasing the efficiency of CTC-based studies. In a previous study (19), the authors used a spiking model from metastatic lung cancer Table II. Association between gene expression levels of MAGE A1-6 of circulating tumor cells and clinicopathological characteristics according to the detection methods in breast cancer. Since the first human cancer cell line was established from cervical carcinoma, various cancer cell lines have been generated (14). However, ex vivo culture of CTCs has been challenging because of the limited number of viable CTCs. With recent advances of cell culture techniques that optimize culture conditions (14,18), several studies have shown the results of ex vivo culture of CTCs in various culture conditions (7,15,17). Table III. Association between the gene expression levels of hTERT of circulating tumor cells and clinicopathologic characteristics according to the detection methods in breast cancer. Zhang et al (15) showed the results of CTC culture from lung cancer using a three-dimensional (3D) co-culture environment using a mix of collagen, matrigel and cancer associated fibroblasts derived from a primary pancreatic tumor as culture medium. Cayrefourcq et al (17) established of colon cancer CTC line using stem cell culture medium mixed with DMEM, insulin, EGF, fibroblast growth factor, fetal calf serum and other substances at first, before switching to another culture medium later. In breast cancer, Yu et al (7) reported that long-term oligoclonal CTC cultures were established from CTCs isolated from metastatic breast cancer patients. They cultured CTCs in serum-free media supplemented with EGF and basic fibroblast growth factor under hypoxic conditions (4% O 2 ). Interestingly, the authors tried to test several culture conditions including CRC culture methods, but the results were unsuccessful. On the other hand, unlike previous results (7), our study showed the growth of CTCs using CRC culture in breast cancer. Although the previous study (7) did not explain why the CRC culture methods were unsuccessful in CTC culture, there are several reasons that may explain these conflicting results. First, the technique for isolating CTCs was different from ours and previous studies which used microfluidic technology. Second, we isolated CTCs from patients with primary cancer before surgery, while in the previous study CTCs were isolated from metastatic breast cancer patients who were either off therapy or progressing on treatment. Third, we cultured CTCs using CRC culture method for 4 weeks. In CRC culture methods, epithelial cell colonies are readily visible after 2 days, and cultures usually reach confluence in 5 days (22). For CRC culture of CTCs, culture of CTCs should be started within few hours and once epithelial cell colonies are formed, cultured cells can continuously grow to yield more cells. In previous study, the results showed that 'Fast-growing' tissues such as prostate, lung, cervix, skin and salivary gland tissues can yield up to 2 million cells after 6-7 days (22). Tissues with an intermediate growth rate including breast and kidney tissues yield 1-2 million cells after 2 weeks. 'Slow-growing' tissues such as colon, pancreas, ovary and thyroid tissues yield up to 10,000 cells after 4 weeks. Our CTCs were derived from breast tissue and it was suitable to culture CTCs for at least 2 weeks, so we cultured for 4 weeks to enhance more cell growth. Further clarification requires future studies comparing the various culture conditions. In addition to the detection of CTCs using CRC culture, we combined RT-PCR of the hTERT and MAGE A1-6 genes to enhance CTC detection rates in breast cancer. The hTERT and MAGE A1-6 genes are known to be specific for cancer. If the positive expression rate of the direct RT-PCR for hTERT and MAGE A1-6 gene is sufficient for the detection of CTCs, CRC culture, which takes 4 weeks, is unnecessary. In previous studies using RT-PCR of the hTERT and MAGE A1-6 genes (13,23), both genes were specifically expressed in CTCs of breast cancer patients. The positive expression levels reported for the hTERT and MAGE A1-6 genes in breast cancer varied from 19.6 to 63.6 and from 13.0 to 63.6%, respectively (13,(23)(24)(25)(26). Both methods of RT-PCR of hTERT and MAGE A1-6 genes and CRC culture have advantages and disadvantages (Table IV). We hypothesized that if CTC detection using CRC culture is possible, a combination of CRC culture and RT-PCR only for the hTERT and MAGE A1-6 genes in the blood would enhance the detection rate of CTCs. In our results, the detection rate of CTCs using RT-PCR was lower after CRC culture than without which may be due to possible alteration of these genes in cultured CTCs. However, when combining the two methods, CTC detection rates were increased to 53.3 and 23.3% for hTERT and MAGE A1-6 gene, respectively, compared to 26.7 and 10.0% using CRC culture. To the best of our knowledge, this is the first study to show results for the combination of CRC culture and RT-PCR for the hTERT and MAGE A1-6 genes to detect CTCs in breast cancer. Our results suggest that the combination of methods used in this study may be beneficial if the positive expression rate of these genes is not sufficient for the detection of CTCs in breast cancer.
Although CRC culture of CTCs in breast cancer was shown in this study, the growth rates of CTCs using CRC culture were lower than expected. This may be due to a low number of isolated CTCs, because most of the patients in this study were in an early stage of breast cancer. The detection rate of CTCs in breast cancer is known to be higher in metastatic disease than in early breast cancer (28), and the presence of CTCs has been reported to be associated with disease progression. Our results confirmed these findings, as CTC detection rates were significantly associated with breast cancer stage. Therefore, in early breast cancer, especially stage I, the number of isolated CTCs may be small. In addition, the cell culture conditions can affect the growth of CTCs using CRC culture. In a study to determine optimal growth conditions for CTCs, four different culture environments were tested, and the results showed that a 3D co-culture environment using a mix of collagen, matrigel and cancer associated fibroblasts exhibited the highest level of cell expansion, compared to two-dimensional (2D) or mono culture environments (15). In our study, we used plates containing medium for CRC culture, which is a 2D co-culture environment. Even if CRC culture enables CTCs to grow indefinitely, this 2D environment may have limited the growth of CTCs. To enhance the growth of CTCs in breast cancer, we are planning to test a 3D co-culture environment using matrigel and CRC culture.
Our study has several limitations. First, healthy donor blood was not included so we could not analyze the specificity of the method. We designed this study based on the results of previous studies showing that the MAGE A1-6 and hTERT genes are specifically expressed in cancer cells (29)(30)(31)(32)(33)(34). To show more reliable results, a negative control is required. Second, we did not enumerate CTCs after isolation to determine the number of CTCs isolated. Instead of enumeration, we detected CTCs using RT-PCR of the MAGE A1-6 and hTERT genes and it was difficult to determine the average number of isolated CTCs and the percentage of viable isolated CTCs using this method. Therefore, it remains uncertain how many CTCs were culturable after isolation. Further study is required to confirm the proportion of viable CTCs that are considered culturable after isolation. Third, we could not prove that the colonies originated from CTCs. Because we didn't enumerate CTCs after isolation and detected CTCs only using RT-PCR of the MAGE A1-6 and hTERT genes, it was difficult to investigate CTCs microscopically. Instead, we tried to compare the CTC colonies to the microscopic findings of cancer cells in breast cancer tissue. There were some similarities between the CTC colonies and cancer cells in breast cancer tissue, but the findings were not identical. Previous studies described that there is broad morphological and immunophenotypical variation within CTCs derived from the same tumor of origin and detecting CTCs microscopically is still challenged (35)(36)(37).
Further investigation is required to prove that the colonies were derived from CTCs. Fourth, we did not evaluate the EpCAM as a positive control for detection of CTCs. Although the detection rate of EpCAM may not be sufficient for detection of CTCs, an old positive control is needed to reliably compare the results. Finally, the blood volume collected in this study was lower than that used in other studies. To increase the sensitivity of our assay methods, it may be necessary to collect more blood. Also, the total number of samples was small, and further studies with a larger sample size and appropriate control group are needed to show more reliable results.
In conclusion, our study shows a potential of CRC culture in the detection of CTCs in breast cancer. We also showed that a combination of CRC culture and RT-PCR for the hTERT and MAGE A1-6 genes is useful in enhancing the detection rate of CTCs in the blood. If we can increase the efficiency of CRC culture to expand CTCs by improving culture conditions, CRC culture could be used for CTC detection in a clinical setting, and CTC lines could be established from breast cancer patients using CRC culture method. To properly apply CRC culture to the utilization of CTCs in breast cancer in clinical applications, further studies with larger numbers of samples and multiple culture conditions are needed.
Hospital. Written informed consent was obtained from all patients according to the protocol.

Patient consent for publication
Written informed consent was obtained from all patients according to the protocol.