Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines, CMT-U229 and MCF-7, and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44+/CD24low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4, E-cadherin, N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44+/CD24low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (P<0.05). Immunofluorescence staining showed increased E-cadherin expression (P<0.05) and decreased expression of OCT4, N-cadherin and vimentin (P<0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover, treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (P<0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs, suggesting its potential anti-metastatic role in canine and human breast cancer cell lines.
Citation: Gonçalves NdN, Colombo J, Lopes JR, Gelaleti GB, Moschetta MG, Sonehara NM, et al. (2016) Effect of Melatonin in Epithelial Mesenchymal Transition Markers and Invasive Properties of Breast Cancer Stem Cells of Canine and Human Cell Lines. PLoS ONE 11(3): e0150407. https://doi.org/10.1371/journal.pone.0150407
Editor: Fernando Schmitt, University of Toronto, CANADA
Received: July 14, 2015; Accepted: February 12, 2016; Published: March 2, 2016
Copyright: © 2016 Gonçalves et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (www.fapesp.br; NNG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Breast cancer is the most prevalent cancer in women worldwide representing 23% of all cases of cancer . Mammary tumors are also common in female dogs, representing approximately 52% of all neoplasms that affects this animal population [2, 3]. In both species, breast cancer has a high rate of mortality and morbidity mainly due to the tumoral recurrence and metastasis .
Cancer stem cells (CSCs) are responsible for tumor initiation, recurrence, metastasis and resistance to therapy in several tumor types, including breast cancer [5–7]. These cells, also called tumor-initiating cells, constitute a distinct fraction in the tumor mass, and they have the capacity of self-renewal and pluripotency, reproducing the heterogeneity of the original tumor from which they are derived [6, 8]. These features also characterize embryonic stem cells, therefore suggesting common molecules might exist between CSCs and embryonic stem cells, such as, octamer-binding transcription factor 4 (OCT4), a essential regulator for the self-renewal and pluripotency .
The subpopulation of breast CSCs is characterized by phenotype CD44+/CD24low/- [9, 10], by their tumor-initiation capability in immune-compromised mice  and by an in vitro ability to form mammospheres [12, 13]. The undifferentiated cells derived from epithelium are the only ones capable to survive in suspension and to form mammospheres, the other cell types die by anoikis .
The epithelial mesenchymal transition (EMT) is a mechanism to generate cancer stem cells endowed with an invasive and metastatic phenotype [14, 15]. EMT occurs in the embryogenesis process, during the organ and tissue formation as well as in trauma restoration and organ fibrosis and carcinogenesis [16, 17]. This process is mediated by the activity of growth and transcription factors, resulting in loss of the epithelial cells’ typical intercellular junction structure, acquisition of mesenchymal morphology, loss of apical-basal cell polarity and motility and invasion ability . Studies have also demonstrated that EMT is involved in cell plasticity, process by which non-stem cells acquire stem cell characteristics .
The major EMT molecular marking include loss of the epithelial marker E-cadherin, and overexpression of mesenchymal markers as N-cadherin and vimentin . E-cadherin, a member of the cadherin superfamily, is a structural component of adherent junctions, fundamental to the polarity and adhesion of epithelial cells [18, 20, 21]. N-cadherin, another member of the cadherin family responsible for the integrity of adherent junctions, is usually expressed in mesenchymal cells [22, 23]. Vimentin, is a main component of the intermediate filament family of proteins and it is expressed in the mesenchymal cells .
Currently there has been growing interest in identifying new therapeutic agents that may interact with molecular markers present in cancer stem cells, formed in the EMT process. Thus, these new agents could interfere in the metastatic process, which is the main cause of mortality among cancers, including breast cancer .
Melatonin (N-acetyl-5-methoxytryptamine), a naturally hormone produced and secreted by pineal gland, has been proven effective in tumor inhibition, in both in vitro and in vivo studies [25–27]. This hormone has oncostatic activity through a variety of mechanisms including antiproliferative actions, modulation of oncogenes expression, antioxidant and antiangiogenic effects .
According to Lopes et al. , melatonin inhibits cell viability and proliferation and induces apoptosis in canine breast cancer cells, especially ER-positive with high expression of MT1 receptor. Studies also suggest that melatonin has anti-invasive and anti-metastatic action, which involves multiple cellular models including EMT [30–32]. According to Mao et al. , melatonin has inhibited EMT in MCF-7 cells because it induces the degradation of β-catenin, an E-cadherin repressor, via activation of kinase protein GSK3β. Nowadays some studies have shown inhibitory effect of melatonin in cancer stem cells. Thus, previous studies demonstrated that the treatment with melatonin was able to decrease the cell proliferation and induced the cell death by apoptosis and autophagy of colorectal and glioma CSCs [34, 35].
However, the specific action of melatonin in cancer stem cells, which result from EMT, has been underexplored [30, 36]. Therefore, the objectives of this study were to evaluate the effects of melatonin treatment on modulation of molecular markers: OCT4, E-cadherin, N-cadherin and vimentin, as well as, in the cell viability and invasiveness of the cell mammospheres.
Materials and Methods
The canine mammary cancer cell line CMT-U229, previously cultivated  was kindly provided by Dr. Eva Hellmén. The histological type this canine cell lineage it is a benign mixed mammary tumor according to WHO (World Health Organization). The human breast cancer cell line MCF-7 was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).
The CMT-U229 and MCF-7cells were grown in a humidified incubator at 5.0% CO2 at 37°C until they were 80–90% confluent in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Cultilab, Campinas, SP, Brazil) and HAM-F12 (Cultilab, Campinas, SP, Brazil), respectively, supplemented with 10% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), penicillin (100 IU/mL) and streptomycin (100 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA).
Mammospheres culture assay
Mammosphere culture was performed as previously described . Cells were grown in the MammoCult medium (Stem Cell Technologies, Vancouver, Canada) supplemented with MammoCult Proliferation Supplements (Stem Cell Technologies, Vancouver, Canada) and plated in ultra low attachment individual wells of 8.96 cm2 at a density of 10,000 viable cells/mL and grown for 7 days. The mammosphere formation rate was calculated as previously described  using the following equation: (number of mammospheres per well/number of cells seeded per well) × 100%.
Flow cytometry analysis
By using a Guava easyCyte flow cytometer (Millipore), the expression of stem cell markers in a breast cancer panel was distinctly evaluated in cells from mammospheres. The antibodies used were phycoerythrin (PE)-conjugated anti-CD24 (BDbiosciences) and fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BDbiosciences). Staining was done according to the instructions of the manufacturer and analysed in flow cytometer. For the analysis, 80,000 cells were detected and quantified by percentage (%).
MTT cell viability assay
The cell viability potential of cells treated or not with 1 mM melatonin (Sigma-Aldrich, St. Louis, MO, USA) was measured by MTT assay (3(4,5- dimetiliazol-2-il)-2,5difeniltetrazolium bromide), which is based on the ability of live cells to convert tetrazolium salt into purple formazan.
To MTT assay, the mammospheres were disaggregated by the action of the enzyme trypsin and the cells were plated in a 96 well plate. Thus, the MTT assay was performed with adherent cells provided from mammospheres.
Regarding treatment, melatonin was diluted with 50% of PBS (Phosphate Buffered Saline) solution and 50% of ethanol and the cells received treatment in the absence of light, because the hormone is photosensitive. Melatonin has been shown to act decreasing the cell viability of breast cancer cell lines [40, 41]. It should be emphasized here that the concentration of 1 mM melatonin used for the treatment of the cells was defined according to the literature. This is the pharmacological concentration used in several studies about the effects of melatonin in neoplastic cells [42–47].
Briefly, individual well of 0.31 cm2 was inoculated with 100 μL of supplemented medium containing 5 x 104 cells. After 24 h, 10 μL MTT stock solution (5 mg/mL, Sigma Chemical Co) was added to each well, and the plates were further incubated for 4 h at 37°C. To solubilize the MTT formazan crystals, the cells were incubated with SDS-HCl (10 mM) (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 4 h. The absorbance at a wavelength of 570 nm was measured by the ELISA reader (Thermo Plate, Waltham, MA,USA). For the analysis, the cell viability (%) was calculated for all groups compared to control samples. All experimental samples were done in triplicate.
Cells from mammospheres, attached to 8-well chamber slides (Sarstedt, Newton, NC, USA) and incubated or not with 1 mM melatonin at 37°C for 24 h, were fixed immediately in 4% paraformaldehyde and permeabilized 0.4% Triton X-100 for 20 minutes. Cells were blocked with 10% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA) and then incubated with the antibodies E-cadherin (1:400 Abcam), N-cadherin (1:400 Abcam), Vimentin (1:100 Sigma Aldrich, St. Louis, MO, USA) and OCT-4 (1:1000 Abcam) at 4°C overnight, followed by incubation with secondary Alexa Fluor 488 anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) per 1 hour at room temperature. Nuclear staining was performed by 4',6-diamidino-2-phenylindole (DAPI, Life Technologies, Eugene, OR, USA) and mounted with Prolong Gold (Life Technologies, Eugene, OR, USA). Images were captured on a confocal microscopy (ZEISS, modelo LSM 710, software ZEN 2010, Thornwood, NY, USA). The spheroids were measured at 405 nm for nuclei staining and 488 nm for cytoplasmic staining.
Immunofluorescence staining evaluation
Two different photomicrographs were taken at 40X magnification under bright field and the intensity of the staining was quantified by Image J Software (NIH, Bethesda, MD, USA). Each photograph was divided into four quadrants and 20 spots (small circular ROI) were randomly selected in each photomicrography. A negative control section of the corresponding staining was used to measure background activity in S1 Fig. The slides were observed in a microscope Nikon Eclipse E200. The values were obtained in arbitrary units (a.u.) and showed the mean optical density (M.O.D.) for each sample. The pinhole size was 90 μm.
The invasion assay was performed with the adherent cells provided from mammospheres.
The invasiveness of CMT-U229 and MCF-7 cells treated or not with melatonin was assessed using transwell chambers with 8 μM pore size membrane (Corning Matrigel Invasion Chamber; Bedford, MA, USA). In the upper compartment of the chamber, approximately 2.5 x 104 cells/insert were added into the culture medium without serum, while to the other compartment were added 750 μL of culture medium with the chemoattractant (0.5% and 10% FBS to negative and positive control’s, respectively) and 10% FBS was associated with 1 mM melatonin.
The cells were incubated at 37°C in 5% of CO2 for 24 h. After incubation, the transwell membranes were washed and impermeabilized. The invading cells were stained with hematoxylin and the nonmigrating cells were removed from the upper surface of the transwell membrane by a cotton swab. The counting was made with an inverted optic microscope by putting the insert over a plate containing glycerol at 50%.
Cell invasion (%) of each group was calculated from the average values of cells that migrated and invaded the matrigel membrane in duplicate. The positive control group was taken as 100%. The result was calculated from the difference compared to the positive control group.
The cell lines were separated in two groups, according to the treatments performed (control and melatonin). First, the results were submitted to analysis of normal distribution using Column statistics and the Gaussian distribution test. The average of densitometric analysis, cell viability and the percentage of total cells with migratory and invasive capacity were subjected to Student's t test. All values were expressed as the average ± Standard Deviation (SD). The value of P<0.05 was considered statistically significant. All analyses were performed using GraphPad PRISM5 software (GraphPad Software, Inc., La Jolla, CA).
CMT-U229 and MCF-7 cell lines mammospheres
The mammospheres were generated from the canine mammary cancer cell line CMT-U229 and human breast cancer cell line MCF-7 in MammoCultTM medium (StemCell Technologies) and efficiently formed compact mammospheres (Fig 1). CMT-U229 and MCF-7 cells were continuously capable of forming mammospheres through repeated subcultures in MammoCultTM medium (StemCell Technologies). The mammosphere formation rate was 0.5.
Relationship of CMT-U229 and MCF-7 mammospheres and cancer stem cell population
To confirm the phenotype of breast cancer stem cells (CD44 + / CD24- / low) flow cytometry was performed in mammospheres. For reaction control monolayer of CMTU-229 and MCF-7 cells were used. Our results confirmed the phenotype of breast cancer stem cells. The results indicated that the mammary stem cells population for CMTU-229 cell line was consisted of 26.89% of cell positive for the anti-CD44 antibody and 6.75% for the anti-CD24 antibody (Fig 2C; P = 0.0001). Similarly, for the MCF-7 mammospheres 16.29% were positive for CD44 and 12.3% for CD24 (Fig 2D; P = 0.0001). However, cells grown in monolayer culture showed the non-stem cell phenotype, in CMTU-229 cell line 13.60% CD44+ and 27.59% CD24- (Fig 2A; P = 0.0404). The same phenotype was observed for MCF-7 strain, from 0.47% CD44 + and CD24- 82.69% (Fig 2B; P = 0.0001).
Fig 2. Graphical representation demonstrating the flow cytometry percentage for CD44/CD24 cancer stem cell phenotype in cells.
Flow citometry for cells grown in mololayer for control group of (A) CMTU-229 cell line and (B) MCF-7 cell line. Flow citometry for mammospheres for both cell lines (C) CMTU-229 and (D) MCF-7. *P<0.05 Statistical significance compared to CD44 group was determine by Bonferroni.
Melatonin reduces cell viability of CMT-U229 and MCF-7 mammospheres
Thereby, it was performed an MTT assay to estimate the number of viable cells after the treatment with 1 mM melatonin for 24 h. The viability of cells from mammospheres, in both cells lines, was significantly decreased after melatonin treatment when compared to control groups (P = 0.0147, P = 0.0286, respectively; Fig 3).
Fig 3. Effect of melatonin on viability of cells from mammospheres.
(A) CMT-U229 and (B) MCF-7 cells were treated with 1 mM of melatonin for 24 h and cell viability was measured by MTT assay. The white column corresponds to control group. Each column represents the mean ± standard error of triplicate experiments. *P<0.05 Statistical significance compared to control group was determine by Student´s t- test.
Melatonin induces differential proteins expression in EMT mammospheres process
In order to examine the effects of melatonin on breast cancer stem cells from canine and human cell lines, the protein expression of the cancer stem cell marker OCT4, the epithelial marker E- cadherin, the mesenchymal markers N-cadherin and vimentin, were measured in cells from mammospheres.
In CMT-U229 cells treated with 1 mM of melatonin, OCT4 protein expression was significantly decreased compared to the control group (P = 0.0426; Fig 4A and 4E) and E-cadherin protein expression increased after melatonin treatment compared to the control group (P = 0.0002; Fig 4B and 4E). On the other hand, N-cadherin and vimentin protein expression were significantly decreased in the melatonin treated cells compared to the control groups (P = 0.0002; Fig 4C and 4E and P = 0.0411; Fig 4D and 4E, respectively).
Fig 4. Immunofluorescence.
Detection of (A) OCT4, (B) E-cadherin, (C) N-cadherin and (D) vimentin in CMT-U229 cells mammospheres after melatonin treatment compared to control groups. E. Statistical analysis of OCT4, E-cadherin, N-cadherin and vimentin proteins expression. Data are shown as mean ± standard deviation. The magnification was 40 X. *P<0.05 Statistical significance compared to control group was determine by Student´s t- test.
For MCF-7 cells, OCT4 protein expression was also decreased in melatonin treated cells compared to the control group (P = 0.0001; Fig 5A and 5E) and E-cadherin protein expression increased after melatonin treatment compared to the control group (P = 0.0001; Fig 5B and 5E). For N-cadherin and vimentin proteins, low expression occurred in the treated cells compared to the control groups (P = 0.0430; Fig 5C and 5E and P = 0.0001; Fig 5D and 5E, respectively).
Fig 5. Immunofluorescence.
Detection of (A) OCT4, (B) E-cadherin, (C) N-cadherin and (D) Vimentin in MCF-7 cells after melatonin treatment compared to control groups. E. Statistical analysis of OCT4, E-cadherin, N-cadherin and Vimentin protein expression. Data are shown as mean ± standard deviation. The magnification was 40 X. *P < 0.05 Statistical significance compared to control group was determine by Student´s t- test.
Melatonin decreased migration and invasion of CMT-U229 and MCF-7 mammospheres
To confirm the above-mentioned effect of melatonin on canine and human mammospheres, CMT-U229 and MCF-7 cells were treated with 1 mM of melatonin for 24 h and it was verified reduction in migration and invasion in both cell lines (40.4% for CMT-U229 and 56.3% for MCF-7), when compared to the positive control (P = 0.0017, P = 0.0377, respectively; Fig 6A–6D).
Fig 6. Migration and invasion rate of CMT-U229 and MCF-7 mammospheres after melatonin treatment.
(A) CMT-U229 and (C) MCF-7 cell lines after 24 h of treatment with 1 mM melatonin compared with positive control groups. (B) CMT-U229 and (D) MCF-7 cell lines correlation between positive and negative control groups. Data are shown as average ± Standard Deviation. *P < 0.05 Statistical significance compared to control group was determine by Student´s t- test.
The main purpose of this study was to evaluate the effects of melatonin treatment in breast CSC of canine and human cell lines and the results demonstrated that melatonin has similar activity in the canine CMT-U229 and human MCF-7 cell lines. This fact reinforces the concept that breast tumors in women and female dogs have similar biological characteristics and dogs tumors can be used to better understand the pathogenesis of this disease and valid comparative models for the study of breast tumors [48, 49].
The presence of stem cells in canine [19, 37, 50–54] and human [19,52] breast cancer cell lines have been demonstrated in several studies. CSC exhibit surface markers, whereby the subpopulation of stem cells can be separated from non-stem cells . According to Dontu et al.  stemness' of tumor cells is measured in vitro by its ability to form mammospheres. As can be seen in this study, canine and human breast cancer efficiently formed mammospheres, consisting of CD44+/CD24low/- cells, which confirms the cancer stem cell phenotype. The selection of CSCs from both lineages was performed in serum-free culture medium permissive for growing of stem cells.
Breast cancer cells with CD44+/CD24low/- surface phenotype have tumor initiating properties with pluripotency characteristics and invasive capacity. The transition from an epithelial phenotype (CD44-/CD24+) to a mesenchymal phenotype (CD44+/CD24low/-) enables the cell to move from the primary tumor to metastatic site . Ponti et al.  found that breast cancer cell line that grown as spheroids also had CD44+/CD24low/- phenotype and expressed the transcription factor OCT4. OCT4 is a member of the POU family, expressed in embryonic stem cells, germ cells and human stem cells and it is responsible for maintaining an undifferentiated state in the cells . This transcription factor is an essential regulator for self-renewal and pluripotency of embryonic stem cells. Since CSCs share features with embryonic stem cells, it is suggested that transcription factors are expressed commonly in both cells .
The expression of OCT4 has an important role in carcinogenesis and provides a possible mechanism by which cancer cells acquire or maintain the therapy resistance phenotype . Linn et al.  related overexpression of OCT4 with drug resistance in prostate cancer cell line. Furthermore, overexpression of this gene has also been associated with metastasis and poor prognosis in several types of cancer, including colorectal , lung  and glioma .
In our study, it was showed the expression of OCT4 protein in canine and human breast cancer cell lines, and this expression was decreased after 1 mM of melatonin treatment. Pang et al.  and Ferletta et al.  also noted OCT4 expression in mammospheres of canine mammary tumor cell lines, REM134 and CMT-U229 avl2, respectively. However, few studies have evaluated the action of melatonin in molecular markers of stem cells [21, 63, 64] working with embryonic stem cells ES-E14TG observed a transient reduction in the expression of OCT4 after treatment with melatonin. The molecular mechanism by which melatonin inhibits the expression of OCT4 is not yet understood.
The CSC have an invasive and metastatic phenotype and can be generated by epithelial mesenchymal transition mechanism (EMT) . Previous studies showed that EMT activation of human neoplastic mammary epithelial cells is associated with enrichment of cells with stem-like properties [55, 62]. Thus, due to the relationship between EMT and cancer stem cell, we verified the presence of EMT molecular markers in CMT-U229 and MCF-7 stem cells. Also, we analyzed the action of melatonin treatment on these EMT markers. Our results showed that the CSCs from both cell lines, CMT-U229 and MCF-7, presented decrease in E-cadherin expression and increase in N-cadherin and vimentin expression. Furthermore, after melatonin treatment, there was an increase of E-cadherin expression and a decrease of N-cadherin and vimentin expression. Some studies have demonstrated low expression of E-cadherin associated with metastasis, lymph nodes involvement and poor prognostic in breast [22, 65] and liver cancer . On the other hand, overexpression of N-cadherin and vimentin is correlated with poor prognosis in several types of cancer, including colorectal , bladder  and hepatocellular carcinoma .
However, few studies have evaluated the action of melatonin on EMT markers. According to our results, Cos et al.  found high expression of E-cadherin in MCF-7 cells treated with melatonin. Ma et al.  also verified an increase of E-cadherin and survival, in mice with mammary tumor treated with melatonin. According to Mao et al. , melatonin has inhibited EMT in MCF-7 cells because it induces the degradation of β-catenin, an E-cadherin repressor, via activation of kinase protein GSK3β. The β-catenin can translocate to the nucleus and complex with the transcription T-cell factor/lymphocyte enhancer factor (TCF/LEF) to induce the transcription of Wnt target genes, including the zinc finger protein Snail, a transcriptional repressor of E-cadherin .
Cos et al.  found that there was no vimentin expression in both treated and untreated MCF-7 cells after melatonin treatment, probably due to the fact that vimentin is a specific marker of mesenchymal cells. In our study it was observed vimentin expression in both cell lines and its expression decreased after treatment with melatonin. According to Gilles et al.  and Mao et al. , a signaling model of Wnt/β-catenin also induces the expression of vimentin. Therefore, melatonin may decrease vimentin expression by GSK3β activation and consequential β-catenin degradation. Besides that, melatonin action on N-cadherin expression has been little explored in the literature, but the Wnt/β-catenin signaling model also induces the expression of N-cadherin , therefore, melatonin can suppress N-cadherin expression by a similar mechanism to that described above for vimentin.
It was also observed that melatonin decreased the viability of stem cells in both cell lines. Currently, some studies have shown inhibitory effect of melatonin on CSC [34, 35]. Kannen et al.  by using colorectal CSCs demonstrated that melatonin reduces cell proliferation and promotes apoptosis of cancer stem cells. Similar results were found by Martin et al. , which showed that melatonin induces cell death by autophagy and simultaneously increased the effect of chemotherapy in glioma stem cells.
However, the exact mechanism through which melatonin inhibits cell growth in both in vitro and in vivo models is not fully understood, although some mechanisms have been proposed. These include induction of apoptosis, changes in the lipidic metabolism and an increase in the activity of natural killer cells, as well as stimulation of cytokines production, such as interleukins IL-2, IL-6, IL-12 and IFN .
Some studies also indicate that melatonin is able to reduce migration and invasion in some cancer types, such as glioblastoma , hepatocarcinoma , lung  and breast cancer [68, 77, 78]. According to Cos et al. , melatonin treatment reduces the invasiveness of MCF-7 cells, causing a decrease in cell attachment and cell motility, probably by interacting with the estrogen-mediated mechanisms of MCF-7 cells. Ortíz-López et al.  verified that melatonin inhibits the migration process and cell invasion in MCF-7 cells via ROCK-1 protein by modulating dynamic cytoskeleton. In turn, Mao et al.  found that melatonin exerts an inhibitory effect in breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of metalloproteinase 2 and 9 expression and activity. According to that, we also found that 1 mM of melatonin reduced the invasiveness of stem cells in both cell lines, CMT-U229 and MCF-7, and this fact may be associated with the anti-metastatic properties of melatonin described above. This result is of great interest since the invasiveness is one of the main characteristics of tumor stem cells.
In conclusion, our results show that the breast cancer stem cells, in both a canine and a human cell line, are responsive to melatonin treatment, reducing the viability and the invasiveness cellular capacity, as well as, the expression of stem cell and EMT markers. Few studies have investigated the melatonin effects on breast cancer stem cells, making this study the first one to simultaneously evaluate canine and human cell lines. In addition, due to the breast CSCs being highly resistant to chemotherapy, drugs that act successfully on this subpopulation can represent an effective therapeutic option for the breast cancer patient.
We thank Laboratory of Molecular Research in Cancer–LIMC from Faculdade de Medicina de São José do Rio Preto (FAMERP) for providing structure to carry out this project.
Conceived and designed the experiments: DAPCZ NNG. Performed the experiments: NNG JC JRL GBG MGM NMS CFZ. Analyzed the data: NNG JC GBG NMS CFZ SMO. Contributed reagents/materials/analysis tools: DAPCZ EH. Wrote the paper: NNG JC JRL GBG MGM NMS EH DAPCZ.
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Department of Obstetrics and Gynecology, University of Duesseldorf, Moorenstraße 5, 40225 Duesseldorf, Germany
Received 12 January 2014; Revised 25 March 2014; Accepted 26 March 2014; Published 8 May 2014
Copyright © 2014 Natalia Krawczyk et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Evaluation and characterization of circulating tumor cells (CTCs) have become a major focus of translational cancer research. Presence of CTCs predicts worse clinical outcome in early and metastatic breast cancer. Whether all cells from the primary tumor have potential to disseminate and form subsequent metastasis remains unclear. As part of the metastatic cascade, tumor cells lose their cell-to-cell adhesion and undergo epithelial-mesenchymal transition (EMT) in order to enter blood circulation. During EMT epithelial antigens are downregulated; thus, such tumor cells might elude classical epithelial marker-based detection. Several researchers postulated that some CTCs express stem cell-like phenotype; this might lead to chemoresistance and enhanced metastatic potential of such cells. In the present review, we discuss current data on EMT and stem cell markers in CTCs of breast cancer and their clinical significance.
Presence of disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in peripheral blood of primary breast cancer patients was shown to be associated with impaired clinical outcome [1, 2]. Moreover, the persistence of CTCs/DTCs after completion of adjuvant treatment also represents a negative prognostic factor [3–5]. These cells are therefore assumed to be a surrogate marker of minimal residual disease and precursors of distant metastasis. Despite the prognostic relevance of tumor cell dissemination, detection of tumor cells in blood or bone marrow is not necessarily followed by relapse of disease. While most of these cells are already apoptotic or dead and others will successfully be eliminated by shear forces of the bloodstream, only a small group of CTCs possesses the ability to extravasate and migrate through the endothelial cell layer [6–10]. Merely a fraction of those is able to survive at secondary sites and cause tumor growth “metastatic inefficiency” [11, 12]. Although factors determining the fate of CTCs still remain to be elucidated, one presently discussed theory considers epithelial-mesenchymal transition (EMT) to be a crucial step in tumor cell dissemination.
EMT is a phenomenon hypothesized to contribute to cancer progression and metastasis . In this process epithelial cells of the primary tumor undergo a series of phenotypic changes, such as reduction of cell-cell adhesion, increment in cell mobility and invasiveness, loss of epithelial markers, and acquisition of mesenchymal phenotype . Moreover, it has been demonstrated that the process of EMT can generate cells with stem cell-like properties . Cancer cells with stem cell-like, self-renewal capabilities (cancer stem cells: CSCs) are currently regarded to be the source of metastatic tumor spread . Since CTCs have been shown to express mesenchymal and stem cell markers, it has been recently postulated that EMT plays a key role in the process of tumor cell dissemination [17–20]. In consequence, tumor cells undergoing EMT may migrate into peripheral blood as CTCs. Due to their mesenchymal stemness features, these cells might be able to reach distant sites of the body and initiate metastases. In the following review we will discuss current data on the EMT and stem cell markers in CTCs of breast cancer and their clinical relevance.
2. Tumor Cell Dissemination and Its Role in the Metastatic Cascade
Distant metastasis represents the major cause of morbidity and mortality in breast cancer patients [21, 22]. Tumor cell dissemination is a phenomenon that occurs in the very early stage of carcinogenesis and is thought to be a potential source of metastatic disease . Disseminated tumor cells in bone marrow can be detected in up to 30–40% of primary breast cancer patients at the time of diagnosis and are strongly associated with impaired prognosis . Depending on the sensitivity of the assay used and stage of disease, the detection rates of CTCs in peripheral blood range from 10 up to 80%; prognostic relevance of CTCs has been recently confirmed by several clinical trials both in the adjuvant and in the metastatic setting. However, data on CTC prevalence and their clinical significance, especially in early breast cancer, are to date incoherent [24–37]. Hematogenous spread of tumor cells into blood circulation of patients with solid malignancies has been a known phenomenon for a long time [35, 38, 39]. While numerous tumor cells daily reach peripheral blood, only a small fraction of these cells has the ability to survive and to arrive at secondary homing sites “metastatic inefficiency” [11, 12]. Moreover, their seeding at the secondary sites is not a random process. As suggested by Paget in the “seed and soil” hypothesis from 1889 and confirmed by several studies, the interactions between circulating tumor cells “seeds” and the microenvironment of their potential homing sites “soil” play a crucial role in the formation of metastasis [38, 40–42]. These findings are in accord with clinical data; a pooled analysis of nine studies involving 4703 primary breast cancer patients demonstrated that more than half of patients with disseminated tumor cells in bone marrow at the time of diagnosis do not develop metastatic disease . CTCs seem to represent a highly heterogeneous cell population with regard to their morphology, molecular characteristics, implantation efficiency after dissemination and their metastatic potential [43–45].
Epithelial-mesenchymal transition is a process well known from embryogenesis. In order to reach their final destination, embryonic epithelial cells acquire functional and phenotypic properties of migratory, invasive mesenchymal cells and thus become detached from the surface of the embryo [46, 47]. Interestingly, epithelial-mesenchymal transition represents a reversible mechanism; once the target localization has been reached, these cells undergo a reverse process of mesenchymal epithelial transition (MET) and recover their epithelial character to proliferate and form differentiated tissues . This phenomenon, essential for embryonic development, has been recognized to represent a crucial step in tumor progression and metastasis .
The process of EMT involves the loss of cell-to-cell adhesions, loss of apicobasal cell polarity, and increment of migratory and invasive features of mesenchymal cells . EMT can therefore compromise the mechanical integrity of the tissue . EMT, once induced in tumor cells, may allow them to escape from primary tumor, migrate through the blood unaffected by therapeutic agents, and reach the site of future metastasis. Furthermore, it has been postulated that MET also represents the part of metastatic formation and that tumor cells regain their epithelial properties at their secondary homing sites [50, 51]. This hypothesis is in accord with the observation that metastatic lesions generally share epithelial features of the primary tumor (e.g., E-cadherin expression) [52, 53].
EMT process can be induced by extracellular factors like transforming growth factor β (TGFβ), Wnt, Notch, epidermal growth factor (EGF), hypoxia, and others . Numerous transcription factors inducing EMT, like SNAIL, TWIST, SLUG, ZEB1, ZEB2, and FoxC2, have been evaluated . Loss of E-cadherin, overexpression of N-cadherin, and cytoskeletal alterations (e.g., expression of vimentin) hallmark this process causing phenotypical and structural changes that lead to acquisition of motility and invasiveness of cells that have undergone EMT. Several studies have shown a correlation between EMT process and high aggressiveness of breast cancer. EMT markers seem to be associated with basal-like breast cancer phenotype and, therefore, with high invasiveness and metastatic potential [55, 56]. Table 1 summarizes markers used for detection and characterization of CTCs showing epithelial as well as mesenchymal phenotypes.
Table 1: CTC detection and characterization markers.
4. Detection of Tumor Cell Dissemination
The challenge in identifying and detecting CTCs is based on their rare number as well as the lack of a universal breast cancer marker. The majority of methods currently used are based on the detection of epithelial markers. The main disadvantage lies in the fact that cells undergoing EMT or with a mesenchymal phenotype might thus be missed. Only a few markers useful in the isolation of CTCs with a mesenchymal phenotype have been evaluated (Table 1). In the past ten years the number of assays to detect and characterize CTCs has increased steadily. All techniques have in common the fact that, due to the low frequency of the isolated tumor cells, they have to be extremely sensitive. In several cases the first step is the enrichment of tumor cells . The choice of enrichment and characterization steps depending on the markers analyzed (especially EpCAM) is crucial to allow as well as to limit the detection of cells undergoing EMT or not. A short perception of enrichment and detection methods in regard to EMT and stem cell markers, some of them commercially available, will be given in the following. These methods are summarized in Table 2.
Table 2: Detection and characterization methods of CTCs.
One way to enrich disseminated tumor cells is density gradient centrifugation. Mononuclear cells are isolated using Ficoll and are subsequently spun on glass slides. Visualization of the tumor cells beside the leukocytes is effected by means of immunocytochemistry. Due to the lack of a general marker, tumor cells are characterized as epithelial cells which are positive, among others, for EpCAM or cytokeratins . Theodoropoulos et al. could identify CTCs with a putative stem cell-like phenotype in the blood of metastatic breast cancer patients using either cytokeratin, CD44, and CD24 or cytokeratin, ALDH1, and CD24 after density gradient centrifugation .
Another way to enrich CTCs is to label the cells with specific antibodies which are conjugated with magnetic particles. There are several tests commercially available which are based on the immunomagnetic enrichment of epithelial markers, especially EpCAM [24, 60], therefore limiting the possibilities to detect mesenchymal tumor cells which have undergone EMT. They differ in the subsequent characterization of the CTCs: commonly used techniques are the antibody-based detection of specific markers on the protein level and also on the RNA level using RT-PCR.
The semiautomatic CellSearch system (Janssen Diagnostics, Raritan, NJ, USA) which has been approved by the FDA is based on an immunomagnetic enrichment of epithelial cells using EpCAM-specific antibodies coated with magnetic beads. CTCs are quantified and further characterized by immunofluorescence detecting cytokeratins 8, 18, and 19 and CD45 to exclude leucocytes as well as staining of the nuclei (DAPI) [24, 61]. Additional staining of CD44 could be shown by Lowes et al. . Using the CellSearch Profile Kit which consists only of the immunomagnetic enrichment step of EpCAM+ cells without further characterization allows the individual subsequent characterization of the CTC, using among others ALDH1 [63, 64].
Additional assays are commercially available to detect CTCs based on the analysis of the expression levels of epithelial or tumor-specific genes, where applicable with a preceding enrichment step. In case of the AdnaTest Breast Cancer (AdnaGen GmbH, Langenhagen, Germany) this enrichment step is performed using magnetic beads which are coated with EpCAM- and MUC1-specific antibodies. Subsequent RT-PCR allows the quantitative analysis of the expression levels of MUC1, GA733-2, and HER2 [60, 65, 66]. The additional characterization of the CTCs is effected by means of detection of the EMT and stem cell markers TWIST, Akt2, PI3K, and ALDH1, respectively [17, 20].
There are several approaches to enrich CTCs using special chips combining microfluidics and immobilization of CTCs by binding of specific antibodies (e.g., CTC-chip, Herringbone Chip) [67, 68]. The latter chip was used by Yu et al. to establish an RNA in situ hybridization assay to detect and quantify CTCs with either an epithelial or mesenchymal phenotype or with a phenotype in between (partial EMT). The expression levels of seven pooled epithelial transcripts (EpCAM; cytokeratins 5, 7, 8, 18, and 19 and cadherin 1) and three pooled mesenchymal transcripts (SERPINE1/PAI1, cadherin 2, and fibronectin 1) were analyzed to characterize CTCs which were detected by binding at least one of the following antibodies on a herringbone chip: EpCAM, HER2, or EGFR .
Another technique to enrich CTC which is solely based on the size of the cells is filtration. Several systems are available, for example, the ISET filter using pores with a diameter of 8 μm . The same pore size was used in another study combining Whatman Nuclepore track-etched membranes and immunofluorescent staining of cytokeratins 8, 18, and 19 as well as CD45 to exclude leucocytes .
Flow cytometry is another technique which allows an individual characterization of rare cells like CTCs. Using flow cytometry, Giordano et al. could detect a subpopulation of cancer stem cells expressing either ALDH1, CD44, and low amounts of CD24 or ALDH1 and CD133 .
Although the majority of assays use EpCAM as detection marker, different markers are currently used to detect and enrich CTC (Table 2). Due to the fact that CTCs change their phenotype during EMT and MET, false negative results can be obtained depending on which detection marker was used. EpCAM-based assays involve the risk that CTC showing a mesenchymal phenotype might be missed.
5. Can EMT Be Detected in CTCs?
To date, several methods have been developed to detect isolated tumor cells in peripheral blood and bone marrow of breast cancer patients. Since there is no breast cancer specific marker to identify these cells, most detection assays rely on their epithelial characteristics [72, 73]. Based on the assumption that the acquisition of a mesenchymal phenotype by a small fraction of tumor cells disseminated from primary tumor represents a crucial step in the metastatic cascade allowing these cells to migrate to their secondary homing sites and build metastasis, it is possible that EMT markers can be detected among the CTCs of breast cancer patients . This hypothesis has been recently confirmed by various studies in both metastatic and early breast cancer [18–20, 20, 74–78]. Mego et al. demonstrated that EMT markers positive CTCs can be detected in up to 26% of metastatic breast cancer patients. Moreover, a high expression of EMT markers predicted shorter progression free survival in these patients . Aktas et al. showed in their trial on 39 metastatic breast cancer patients that EMT markers, such as TWIST1, Akt2, and PI3Kα, can be codetected in up to 62% of CTC positive blood samples; EMT markers were more likely to be found in patients resistant to therapy, suggesting increased invasiveness of tumor cells undergoing this process. Interestingly, cells undergoing EMT have also been detected in the blood of 7% of patients negative for CTCs . Similar findings in primary breast cancer were presented by Kasimir-Bauer et al.; EMT markers could be detected in 72% of CTC positive and 18% of CTC negative patients, respectively . Raimondi et al. demonstrated the expression of EMT markers (e.g., vimentin, fibronectin) in up to 38% of breast cancer patients tested by the standard definition as CTC negative . These findings suggest that, in addition to CTCs expressing epithelial antigens, a fraction of CTCs with exclusively mesenchymal phenotype could exist and thus remain undetectable for assays based on epithelial character of these cells. However, due to the methodology, morphological features of the cells were not evaluated in these trials and false positive results cannot be excluded [17, 20]. In this regard, CTCs coexpressing mesenchymal and epithelial markers have been visualized in three other studies in breast cancer patients confirming that both kinds of markers can be expressed in the same cell [69, 75, 76]. Additionally, in the analysis by Armstrong et al. vimentin-positive CTCs were detected in peripheral blood of metastatic breast cancer patients while paired metastases from the same patients were shown to be negative for this marker . This suggests a reversibility of the EMT process once tumor cells reach their destination resembling the phenomenon of epithelial plasticity known from embryonic development . Available literature on EMT in CTCs of breast cancer patients is summarized in Table 3.
Table 3: EMT markers in CTC of breast cancer patients.
6. Are CTCs Cancer Stem Cells?
One recently discussed hypothesis indicates that tumor progression and metastatic spread can be traced to a small fraction of tumor cells with stem cell-like characteristics [81, 82]. These cancer stem cells have been identified in breast cancer tissue and were shown to be associated with tumors of aggressive behavior . Assuming that CSCs are responsible for tumor cell dissemination and further metastasis, it seems likely that putative stem cell-like features should be found among tumor cells disseminated from primary tumor. This hypothesis has been confirmed by several researchers [17–20, 77–79]. As reported by Balic et al., most disseminated tumor cells in bone marrow of breast cancer patients presented with / phenotype . Moreover, it has been shown that DTCs with / phenotype are associated with increased prevalence of metastases and with tumors characterized by aggressive biology [80, 84].
According to recent data both stem cell and EMT markers are frequently coexpressed in CTCs of breast cancer patients [18, 77]. These findings support the theory that EMT generates a cell population with stem cell-like features, a phenomenon that has been confirmed by numerous experimental trials [15, 85]. CTCs presenting stem cell-like characteristics have been found in both primary and metastatic breast cancer. In a recent study by Kasimir-Bauer et al. on 502 primary breast cancer patients 46% of CTC positive and 5% of CTC negative blood samples were positive for ALDH1, a common stem cell marker . Similar findings have been shown by Aktas et al. in the metastatic situation. Moreover, a presence of stem cell-like CTCs in peripheral blood of breast cancer patients was shown to be associated with therapy resistance; stem cell markers or EMT factors or both were detected in 74% (25/34) of nonresponders and in 10% (2/21) of patients who responded to systemic treatment . In the trial by Raimondi et al. an overexpression of stem cell markers in CTCs was correlated with advanced stage of disease . Cancer stem cells are currently believed to be the cause of therapy resistance and treatment failure in breast cancer . Data on stem cells markers in CTC of breast cancer patients are summarized in Table 4.
Table 4: Stem cell markers in CTC of breast cancer patients.
7. Therapeutic Consequences
To date, systemic therapies target either highly proliferative tumor cells (cytotoxic therapy) or cells with a specific phenotype (e.g., HER2-targeted treatment). However, such therapies are not able to identify cells that act as a source for subsequent metastasis in a selective manner. Tumor cells with putative stem cell-like expression profile are assumed to enter the blood circulation early in the course of disease and might elude therapy precisely because of their stem cell character [20, 75]. Hypothetically, specific elimination of these cells could prevent the colonization of secondary homing sites and metastasis formation. Thus, the potential existence of a stem cell-like cancer cell might lead to a paradigm shift in oncologic treatment.
Detection and characterization of CTCs have become an important focus of oncologic research; several clinical trials have been initiated during the last decade that evaluate not only CTCs within accessory translational projects, but also ones that focus exclusively on CTCs and stratify therapy according to CTC levels . Most of these trials (e.g., SWOG0500, CirCe01, TREAT CTC, and DETECT III and IV) are based on immunocytochemical detection of CTCs using the FDA-approved CellSearch system (Veridex, Warren, NJ, USA), a semiautomated antibody-based quantitative technique . Since CTCs are enriched by immunomagnetic beads linked with anti-EpCAM antibodies and detected using antibodies against epithelial antigens, loss of epithelial markers during EMT could make these cells “invisible” to the assay and possibly influence treatment decisions [78, 89]. Gorges et al. reported that use of EpCAM-based enrichment techniques may lead to failure in CTC detection; in an animal based model EpCAM-based AdnaTest failed to detect CTCs despite clinically apparent metastasis. However, CTCs could be detected by PCR without the enrichment step . Recently, antibody-based therapies against tumor cells expressing epithelial markers have been introduced in the treatment of cancer of epithelial origin. Catumaxomab, a trifunctional antibody directed against EpCAM, is a potent therapeutic agent for malignant ascites in EpCAM-positive advanced cancer (e.g., ovarian cancer) [90, 91]. Since EMT involves at least temporary downregulation of EpCAM expression, it might influence the efficacy of EpCAM-directed therapy on tumor cells undergoing EMT.
Therefore, signaling pathways involved in EMT and responsible for the formation of CSCs represent potential targets for future treatment regimens, and drugs inhibiting these pathways are being tested in preclinical and clinical trials . In this regard everolimus (RAD001), an oral inhibitor of PI3K/Akt/mTOR pathway, was shown to inhibit cancer stem cells in vitro and in vivo and demonstrated potential effectivity in treatment of breast cancer cells resistant to standard therapy possibly through this mechanism[93–95]. These data are in accordance with clinical results; in a phase II study RAD001 was shown to restore sensitivity to tamoxifen in metastatic breast cancer patients with endocrine resistance improving the clinical benefit rate at six months in these patients . A phase III BOLERO-2 trial demonstrated a 6-month improvement in progression-free survival in patients with resistance to nonsteroidal aromatase inhibitor treated with everolimus in combination with exemestane versus exemestane alone . Everolimus is currently being evaluated for its potential to overcome trastuzumab resistance as well. A phase III BOLERO-1 trial compares trastuzumab and paclitaxel with and without everolimus, while the phase III BOLERO-3 trial compares trastuzumab and vinorelbine with and without everolimus.
Hedgehog, Notch, and Wnt represent further signaling pathways involved in formation of breast cancer stem cells [98–100]. Since the expression of Notch ligands has been demonstrated to be significantly elevated in triple negative breast cancer, Notch has become a promising target in breast cancer treatment . In this context blocking of Notch by γ-secretase inhibitors (GSIs) has been the most extensively used approach. GSIs were shown to induce apoptosis and decrease proliferation in breast cancer cell lines and to eliminate breast cancer stem cells in vitro [102, 103]. GSIs like MK-0752 or RO4929097 have been tested in phase I and II clinical trials in primary and metastatic breast cancer providing early clinical evidence of effectiveness for these agents in breast cancer therapy [104, 105]. A phase I study analyzes RO4929097 in combination with Hedgehog pathway antagonist vismodegib in metastatic breast cancer patients . Vismodegib, established in the therapy of advanced basal cell carcinoma, was also shown to inhibit tumor cell growth in tamoxifen resistant breast cancer in vivo and in vitro . Furthermore, PKF118-310 an inhibitor of Wnt signaling pathway was recently reported to eradicate breast cancer stem cells in a mouse model overexpressing HER2, thus also representing a potential drug candidate for the treatment of breast cancer .
An additional agent that was demonstrated to be effective against breast cancer stem cells is all transretinoic acid (ATRA). In a recent experimental approach, ATRA was able to eliminate breast cancer cells that gained CSC properties, suggesting its effectiveness in cancer resistant to conventional oncologic therapies . However, ATRA has to date performed poorly in clinical trials; in a pilot phase II study 17 metastatic or recurrent breast cancer patients were treated with ATRA in combination with paclitaxel showing time to progression and survival rates similar to those reported for paclitaxel alone .
Another promising drug candidate in this context is salinomycin, which was shown to inhibit tumor growth in mice by eradicating breast cancer stem cells . Recent preclinical trials demonstrated that salinomycin is particularly effective against cancer growth in combination with conventional chemotherapeutics, supporting the postulation that targeting different cell populations is essential in cancer therapy .
Multiple studies have shown that single tumor cells undergo transdifferentiation which enables intravasation; this important step of metastatic cascade is termed epithelial-mesenchymal transition. Through EMT, circulating tumor cells downregulate epithelial antigens and cell-to-cell adhesion and thus enhance their motility and invasive potential. Cells that undergo EMT seem to gain stem cell-like properties; such cells represent a small fraction of tumor cells capable of self-renewal and highly resistant to cytotoxic treatment. Since the majority of CTC detection systems are based on the presence of epithelial markers, tumor cells that have undergone EMT might elude classical detection methods, which may lead to false-negative results.
|CSC:||Cancer stem cell|
|CTC:||Circulating tumor cell|
|DTC:||Disseminated tumor cell|
|EGF:||Epidermal growth factor|
|EGFR:||Epidermal growth factor receptor|
|EpCAM:||Epithelial cell adhesion molecule|
|GA733-2:||Gastrointestinal tumor-associated antigen|
|HER2:||Human epidermal growth receptor 2|
|MET:||Mesenchymal epithelial transition|
|mTOR:||Mammalian target of rapamycin|
|PCR:||Polymerase chain reaction|
|TGFβ:||Transforming growth factor beta.|
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.