Background
Mesenchymal stem cells (MSCs) were first identified by Friedenstein and were described as an adherent, fibroblast-like population in the in vitro culture of bone marrow, which were also found to be able to differentiate into bone in vivo [
1] Subsequently, the concept expanded, it proved that MSCs are not only bone marrow resident cells but are also found in many other tissues of the body including adipose, umbilical cord, fetal liver, muscle and lung [
2]-[
4]. MSCs possess an innate ability for self-renewal and can differentiate into multiple cell types, such as osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons [
5]. Accumulating studies of the past few years support their use for treating both genetic and acquired human diseases associated to loss of specialized tissues [
6],[
7]. In addition, MSCs have received intensive attention in the field of tumors. Tumor tissue contains abundant growth factors, cytokines and matrix-remodeling proteins, explaining why tumors are likened to wounds that never heal [
8]. It has been reported that MSCs migrate to a variety of tumors, this migratory ability points to MSCs as attractive candidates for delivery vehicles of antitumor agents [
9],[
10]. However, several co-injection experiments in animal studies revealed that MSCs promote tumor growth and metastasis [
11],[
12] which would present a serious obstacle to using MSCs as delivery vehicles for anti-cancer therapy. But prior studies on the biology and therapeutic application of human MSCs in human malignancies have reported mixed results. MSCs injected intravenously in a mouse model of Kaposi's sarcoma were shown to home to sites of tumorigenesis and potently inhibit tumor growth [
13]. MSCs have also been shown to have anti-angiogenic effect both in vitro and in mouse models of melanoma [
14]. The inconsistent results are clear indicators that the effect of MSCs on tumor cells is poorly understood and need further investigation.
Mesenchymal stem cells used in the experiment are mostly acquired from adult BM. Wharton's jelly (WJ) of the umbilical cord exhibits the characteristics of stromal cells and is a novel source of mesenchymal stem cells [
15]. Mesenchymal stem cells that are derived from WJ of human umbilical cord (hUCMSCs) have been shown to evidence characteristics similar to those of bone marrow stromal cells (BMSCs). Compared to BMMSCs, UCMSCs have many advantages to use in cell-based therapy because of their relatively large ex vivo expansion capacity, low risk of viral infection, lack of donor morbidity, and less pronounced immunogenicity [
16]-[
18]. So, it offers an attractive alternative to BMSCs for cell-based therapy. However, the MSCs used in the foundation researches and clinical experiments are mostly acquired from adult BM. Though similarly, there were evidence showed that hUCMSCs have unique properties compared to BMMSCs [
19]. However, there is little data on the relationship between hUCMSCs and tumors.
To explore the role of hUCMSCs on tumors, we studied the effects of human hUCMSCs on the esophageal carcinoma (EC) because it occurs with high prevalence in many areas of the world especially in China [
20],[
21]. We investigated the influence of hUCMSCs on EC growth in vivo. We also investigate in vitro co-culture of two different types of EC cell lines with hUCMSCs to explore the mechanism that how hUCMSCs affected tumor growth.
Discussion
The aim of the current study was to study the interaction between hUCMSCs and esophageal carcinoma. hUCMSCs were transplanted into BALB/c nude mice in an effort to observe the outgrowth of the tumor. The results indicated that all of the groups into which mixed cells were injected evidenced larger tumor size than the groups injected solely with Eca109 cells, thereby indicating that hUCMSCs could favor esophageal tumor formation. Furthermore, we also found that in the mice with pre-established esophageal carcinoma, i.v. injection of hUCMSCs also promoted the tumor growth.
Multiple mechanisms may be responsible for the hUCMSCs induced increase of tumorigenesis and tumor growths. hUCMSCs have been shown to have immunmodulatory action in vivo and in vitro [
22],[
23]. In this experiment, we used nude mice for xenotransplantation. Therefore, increased allogenic tolerance via co-injection with hUCMSCs cannot provide a reasonable explanation for this phenomenon.
In this study, we offer two possible explanations for the enhanced tumor growth in response to hUCMSCs. Firstly, hUCMSCs directly stimulate the growth of esophageal cancer cells. The findings of this study, namely that the mixed culture or cultured by transwell system with hUCMSCs increased the proliferation of esophageal cancer cells in vitro, indicated that hUCMSCs induce the increased proliferation of transplanted tumor cells. It also appears that the promoted effects of hUCMSCs on esophageal carcinoma cells may be both cell-contact dependent as well as mediated via diffusible factors secreted by the hUCMSCs. It was difficult to distinguish tumor cells from hUCMSCs directly cocultured with them, so we only detected the molecular changes in the tumor cells induced by the hUCMSCs-transwell co-culture. The in vitro molecular data showed that the increase in the proliferation of tumor cells were associated with the up-regulation of Bcl-2 and survivin expressions.
In vivo, we observed that tumors formed by Eca109 cells admixed with hUCMSCs increased in blood vessel formation in gross analysis, compared with tumors formed by Eca109 alone. Angiogenesis is critical for tumor growth so that the blood vessel in the tumor environment could provide sufficient nutrients and oxygen to the cells, which are essential for the growth and survival of tumor cells [
24]. It is known that MSCs could produce various growth factors that stimulate angiogenesis, [
25] so it is possible that enhanced angiogenesis may account for the esophageal tumor growth-promoting effects by hUCMSCs.
In addition, we noticed that in the 20 axillary lymph nodes of 5 mice received mixed-cell 2 lymph nodes metastases were observed, but lymphatic metastasis was not detected in the other two groups of mice. Our results from in vitro assays showed that hUCMSCs promoted migration and invasion ability of esophageal carcinoma cells in vitro. In our in vitro molecular experiment, MMP-2 and MMP-9 were found to be up regulated in EC cells co cultured with hUCMSCs. Since MMP-2 and MMP-9 promotes cell migration and invasion, [
26],[
27] this may be possible mechanism in which hUCMSCs promote esophageal carcinoma cells invasion and thus may be a possible explanation for hUCMSCs promoted the lymph node metastasis of esophageal cancer.
In a paper published in 2010, Li's results revealed that hMSCs inhibited the proliferation and invasion of Eca-109 cells in vitro [
28]. It' seems that their result is quite different from ours. But the MSCs they investigated were derived from bone marrow. The different sources of MSCs for assessment may be one of the factors accounting for the variability results of pro-tumorigenic or anti-tumorigenic effects. In agreement with this, results of Akimoto's study demonstrated that umbilical cord blood-derived mesenchymal stem cells inhibit, but adipose tissue-derived mesenchymal stem cells promote glioblastoma multiforme proliferation [
29]. So, differences must be considered when choosing a stem cell source for safety in clinical application.
Methods
Cell culture
The human esophageal cancer cell lines Eca109 and TE-1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), where they were characterized by mycoplasma detection, DNA-Fingerprinting, isozyme detection and cell vitality detection. The cell lines were maintained in culture as an adherent monolayer in RPMI-1640 (GIBCO) medium supplemented with 10% FBS. All cells were incubated at 37°C in 5% CO2 humidified cell culture incubator.
hUCMSCs preparation
Human umbilical cord samples were collected from 8 healthy donors. Written informed consent was obtained from the pregnant women before labor. The umbilical cord samples were collected in sterile boxes that contained PBS solution. The collected human umbilical cord tissues were washed three times with Ca2+ and Mg2 + -free PBS. They were mechanically cut by scissors in a midline direction and the vessels of the umbilical artery, vein and outlining membrane were dissociated from the WJ. The jelly was then extensively cut into pieces smaller than 0.5 cm3. The explants then were cultured in DMEM/F12 (1:1) containing 10% fetal calf serum (FCS). They were left undisturbed for five to seven days to allow for migration of the cells from the explants. The cellular morphology became homogenously spindle-shaped in cultures after 4-8 passages.
HUCMSCs identification
The ability of MSCs to differentiate into osteoblasts and adipocytes was confirmed prior to use. Osteogenic differentiation was evaluated by calcium deposition staining using the Alizarin Red staining. The induction of adipogenic differentiation was apparent by intracellular accumulation of lipid-rich vacuoles that stained with Oil Red O. The specific surface molecules of HUCMSCs were characterized by flow cytometric analysis. The cells were stained with the following antibodies: CD14-FITC, CD19-ECD, CD29-FITC, CD34-PE, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC (BD Pharmingen, USA). Thereafter, the cells were analyzed using a Becton Dickinson flow cytometer (Becton Dickinson, San Jose, CA).
Animal experiments
36 female BALB/c nude mice (4-week-old) were purchased from the Beijing HFK Bioscience CO., LTD. Animals used in this study were maintained in accordance with the Policy of Animal Care and Use Committee of Bethune International Peace Hospital. Animals were housed in micro-isolator cages under sterile conditions and observed for at least 1 week to ensure proper health before study initiation. Animals were injected with 5 × 10
6 Eca109 cells alone or mixed with an equal number of hUCMSCs, subcutaneously into the Armpit region. Treatment with hUCMSCs was also conducted in animals with pre-established tumors by i.v. tail vein injection at a dose of 5 × 10
6 per mouse. In some experiments, mice were given additional doses of hUCMSCs by tail vein injection. Tumor growth and progression were monitored by every four days measurements of tumors with calipers. The volume of tumor was calculated by using the following equation as reported previously [
30]: volume = Length × Width
2/2.
Histopathology and immunhistochemistry
At the indicated time points, the animals were sacrificed. After which tumors were dissected out. The ipsilateral and contralateral axillary lymph nodes of the nude mice were also collected. Then the tumor tissues and the lymph nodes were fixed in 10% formaldehyde and processed using standard methods. The sections were stained using hematoxylin and eosin (H&E) in order to examine the histopathology.
Cell migration and invasion experiment
For the invasion assay, 24 well Transwell chambers (8.0 μm pore, Corning, USA) were coated with a 50 μl Matrigel (BD, Franklin Lakes). For migration assays, the ECMgel was not needed. Tumor cells were cultured for 24 h in the mixture of tumor cell culture medium and hUCMSCs-conditioned medium (1:1), and in control groups tumor cells were cultured for 24 h in the tumor cell culture medium supplemented with 50% DMEM/F12 (1:1) medium, and then tumor cells were collected and resuspended in the FBS-free RPMI 1640 medium at a concentration of 5 × 105 cell per ml by cell counting for three times. Then, the cell suspensions were put into the upper compartment of the transwell chambers (200 μl/well), and hUCMSCs (5000/well) was put into the lower compartment. After cultured for 24 h, the cells that did not penetrate the polycarbonate membrane at the bottom of the chamber were wiped off with cotton stickers. The membrane was removed and fixed with methanol and stained with Crystal Violet. Five vision fields were randomly selected under microscope and the number of cells that penetrated the membrane was counted. Each group consisted of three duplicates.
The migratory ability of hUCMSCs was also assayed by means of a 24-well microchamber plate with uncoated inserts (8.0 μm pore). Either 2.0 × 104 Eca109 or TE-1 cells in DMEM with 0.5% FBS or medium alone was plated into the lower chambers. After 4 hours of incubation at 37°C, upper chambers containing 2.0 × 104 hUCMSCs in DMEM were set into the lower chambers. Three wells were used for each experiment. After 16 hours of incubation, inserts were fixed with methanol and stained with Crystal Violet. The number of migrating cells was determined as described above.
Cells co-culture and measurement of cell proliferation
Esophageal cancer cells (Eca109 or TE-1 cells) were cultured either in directly contacting co-cultured or in indirectly co-cultured (transwell system) with hUCMSCs. Esophageal cancer cells and hUCMSCs directly contacting co-cultured was performed as follow: hUCMSCs were first seeded into 24 well plates (5 × 104/ml, 500ul/well). Following 24 hours of incubation, hUCMSCs were found tight attached to the bottom of the well. Before co-cultured with esophageal cancer cells, hUCMSCs were treated with mitomycin C (20 μg/ml) to prevent cell proliferation. Then esophageal cancer cells (5 × 104/ml, 500ul/well) were added to the 24 well plates and co-cultured with hUCMSCs at 37°C for 48 hours. Separately cultured cancer cells and mitomycin-treated hUCMSCs were used as control. Then cell proliferation was determined by Cell Counting Kit-8 (CCK-8, Dojindo, Japan), which allows sensitive colorimetric assays for the determination of the number of viable cells. Before CCK-8 assay, the co-cultured cells and control group cells were detached from the plates. Because the cancer cells can't easily separated from direct contact co-cultured hUCMSCs, the separately cultured hUCMSCs and cancer cells were put together as controls.
In transwell system, hUCMSCs were physically separated from esophageal cancer cells by a transwell membrane with 0.4-μm pore size (Corning, USA.). hUCMSCs were seeded on upper chamber of 24 well stranswell plate and Eca109 or TE-1 cells were seeded on the lower chamber of transwell plate. The cells were co-cultured for 48 hours. The proliferation of cancer cells was assessed by CCK-8 kit (Dojindo, Japan).
Every group had five wells. All the proliferation experiments were run in triplicate and the results expressed as mean ± SD.
Real-time RT-PCR
Quantitative PCR was performed using the SYBR Green realtime PCR method. Esophageal cancer cells and hUCMSCs were co-cultured by transwell system for 48 hours. Then, hUCMSCs were collected. Total RNA was isolated from hUCMSCs using an Rneasy Mini Kit (Qiagen, Valencia, CA, USA) and Trizol Reagent (Invitrogen). Quantitative RT-PCR was performed using an ABI 7000 PCR instrument (Applied Biosystems, Foster City, CA, USA). Each sample was tested in triplicate, and the samples obtained from three independent experiments were used for the analysis of relative gene expression using the 2−△△Ct method. The following primers were used for real-time PCR:
human β-actin, F 5′-cctcgcctttgccgatcc-3′, R 5′-ggatcttcatgaggtagtcagtc-3′;
human survivin, F 5′-agccctttcaaggaccac-3′, R 5′-gcactttcttcgcagtttcc-3′;
human Bcl-2, F 5′-tggccagggtcagagttaaa-3′, R 5′-tggcctctcttgcggagta-3′;
human Bax, F 5′-ttgcttcagggtttcatcca-3′,R 5′-agacactcgctcagcttcttg-3′;
human MMP-2, F 5′-tctcctgacattgaccttggc-3′, R 5′-caaggtgctggctgagtagatc-3′;
human MMP-9, F 5′-ttggcgacaagaagtgg-3′, R 5′-gccattcacgtcgtccttat-3′.
Western blotting
Esophageal cancer cells and hUCMSCs were co-cultured by transwell system for 48 hours. Then, hUCMSCs were collected. Equal amounts of whole cell lysates were resolved by SDS-PAGE and electrotransferred on a PVDF membrane. Primary antibody [anti-human survivin (1:2000; Cell Signaling, USA); anti-human Bcl-2, anti-human BAX and anti-human β-actin (1:1000; Dingguo, China), anti-human MMP-2, anti-human MMP-9 (1:1000; proteintech, USA)] incubation was carried out overnight at 4°C. The immunoreactive signals were detected with an enhanced chemiluminescence kit (Millipore). Quantitative analyses were performed using a Gel Doc 2000 scanner system and Quantity One image analysis software (Bio-Rad).
Statistical analysis
All statistical analyses were performed using SPSS 13.0 software package. Statistical significance was assessed by comparing mean values (means ± SD). The two-tailed student's t-test was used to test the probability of significant differences between samples. The significance level was set at P < 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XY carried out the cell culture, Real-time RT-PCR, participated in the animal experiments and drafted the manuscript. ZL carried out the immunoassays. YM carried out the cell migration and invasion experiment. SL and YG carried out the animal experiments. GJ performed the statistical analysis. GW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.