Mesenchymal stem cells enhance the metastasis of 3D-cultured hepatocellular carcinoma cells
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Accumulating evidences have demonstrated that mesenchymal stem cells (MSC) could be recruited to the tumor microenvironment. Umbilical cord mesenchymal stem cells (UCMSC) were attractive vehicles for delivering therapeutic agents against cancer. Nevertheless, the safety of UCMSC in the treatment of tumors including hepatocellular carcinoma (HCC) was still undetermined.
In this study, an in vitro co-culture system was established to evaluate the effect of UCMSC on the cell growth, cancer stem cell (CSC) characteristics, drug resistance, metastasis of 3D-cultured HCC cells, and the underlying mechanism was also investigated.
It was found that after co-cultured with UCMSC, the metastatic ability of 3D-cultured HCC cells was significantly enhanced as indicated by up-regulation of matrix metalloproteinase (MMP), epithelial-mesenchymal transition (EMT)-related genes, and migration ability. However, cell growth, drug resistance and CSC-related gene expression of HCC cells were not affected by UCMSC. Moreover, EMT was reversed, MMP-2 expression was down-regulated, and migration ability of HCC cell was significantly inhibited when TGF-β receptor inhibitor SB431542 was added into the co-culture system.
Therefore, these data indicated that UCMSC could significantly enhance the tumor cell metastasis, which was due to the EMT of HCC cells induced by TGF-β.
KeywordsThree-dimensional cell culture Umbilical cord mensenchymal stem cells Hepatocellular carcinoma Metastasis TGF-β
Mesenchymal stem cells (MSC) are typically characterized by their ability to differentiate into a variety of mesenchymal cells. In recent years, MSCs have aroused a lot of interests due to their ability to give rise to bone, cartilage, fat, and muscle cells, which could be extensively used in regenerative medicine . MSC reside in many adult organs or tissues, such as bone marrow (BM), adipose, fetal liver, lung, and umbilical cord (UC). UCMSC were attractive seed cells due to the least invasive source and their characteristics similar to those of BMMSC . In addition, they have unique properties compared with other stem cells, such as high proliferation rate and hypoimmunogenicity .
There was growing evidence that MSC could be recruited to the injured sites in many pathological conditions, such as inflammation, tissue repair and tumor [4, 5, 6]. The migrating ability to tumor makes them useful as anti-tumor gene or drug carriers. The recent suggestion that MSC can be recruited by tumors has triggered a series of studies that aimed at examining their potential role in cancer progression. However, the effect of MSC on the tumor progression can be pro- [7, 8, 9] as well as anti-tumorigenic [10, 11] due to the different source of MSC and the tumor models used .
Besides, the role UCMSC played in tumor progression was also controversial. A few studies suggested UCMSC could inhibit tumor growth [12, 13, 14]. Ayuzawa et al. found UCMSC attenuated breast cancer growth by attenuation of Erk-1/2 and PI3K/AKT signaling pathway . Ohta et al. showed FST over-expressing human UCMSC significantly reduced the growth of breast cancer cells . The results of Chao et al. showed that when co-cultured with UCMSC, breast cancer cell number decreased significantly, which was caused by the tumorigenesis suppressing ability of UCMSC. They found that UCMSC induced the apoptosis of breast cancer cells by direct cell contact or by cell-in-cell phenomenon after internalization . Nevertheless, UCMSC have been also reported to promote esophageal carcinoma cancer growth and metastasis both in vivo and in vitro . The results concerning the effect of UCMSC on tumor growth were still mixed, and most of the in vitro studies were carried out under two-dimensional (2D) culture conditions.
Currently, HCC was the third most deadly and fifth most common cancer worldwide . A few studies showed that BMMSC could inhibit cell division of HCC cells and potentiate their death [17, 18, 19]. Still there were some studies found that BMMSC in the inflammatory microenvironment of HCC promoted the development of chemoresistance and metastasis of HCC cells [20, 21]. The paradoxical effect of BMMSC in HCC progression was currently poorly understood, as the in vitro investigation was mostly performed in 2D culture system. In those studies, HCC cells were directly co-cultured with MSC, or treated with conditioned medium of MSC as indirect co-culture, both of which failed to mimic the interaction between HCC cells and MSCs in HCC microenvironment in vivo. In addition, as promising vehicles for delivering therapeutic agents, the safety of UCMSC in HCC treatment remains to be determined.
In our previous study, we established a three-dimensional (3D) culture system with alginate gel (ALG) beads. In this 3D culture system, adhesion (intergrin β1, ICAM 1), and ECM-related (typeIand type IV collagen) gene expression in HCC cells were up-regulated compared with 2D culture and close to those in liver cancer tissue, which represented a in vivo-like HCC cell culture model .
So in this study, HCC cells were cultured in ALG beads, and then co-cultured with UCMSC. The aim of this study was to evaluate the effect of UCMSC on the growth, CSC characteristics, drug resistance and metastasis of human HCC cells and investigate the underlying mechanisms to figure out the role that UCMSC play in HCC progression.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. SB-431542 was added to cells at a concentration of 20 μM. The sodium alginate (MW: 500 kDa, G/M ratio was 33:67) was purified by removing protein and endotoxin according to the protocol used in our laboratory.
Cell culture and encapsulation
HCC cell line, HCCLM3, was kindly provided by the Liver Cancer Institute, Zhongshan Hospital, Fudan University. HCCLM3 cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (high glucose DMEM, Invitrogen, San Diego, CA) supplemented with 10 % fetal bovine serum (FBS) (HyClone, Logan, UT). Cells were encapsulated within ALG beads and cultured according to the previous study . Cells were harvested from ALG beads by treatment with 55 mM sodium citrate. Images were taken using an inverted phase contrast microscope (Eclipse, Nikon, Tokyo, Japan) every other day.
Human UCMSC cells and the culture medium were kindly provided by Zhongyuan Union Stem Cell Bioengineering Corporation (Tianjin, China). Primary UCMSC were isolated according to the standard operating procedure of UC blood bank. Medium was changed every 3 days and cell passage was performed when 90 % confluence was reached.
Co-culture of UCMSC and 3D-cultured HCCLM3
After 15-day culture in ALG beads, 3D cultured HCCLM3 cells (8 × 104/cm2) were co-cultured with UCMSC (1.6 × 104/cm2) for 5 days with or without SB431542 (20 μM) in flasks.
Cell counting kit-8 (CCK8) (Dojindo Laboratories, Kumamoto, Japan) assay was use to detect cell proliferation according to the manufacturer’s instructions. The absorbance was recorded using a microplate reader (Well Scan MK3, Labsystems Dragon, Finland).
ALG beads with HCCLM3 cells were collected and incubated with live/dead staining working solution composed of 2 μM calcein AM and 4 μM ethidium homodimer-1 (ED-1) at 37 °C for 2 h. After washing with normal saline, HCCLM3 cells were observed using a confocal laser scanning microscopy (CLSM) (SP2, Leica, Heidelberger, Germany).
Quantitative real time RT-PCR
Real-time PCR was carried out with the SYBR Premix Ex Taq™ (Perfect Real Time) (Takara) method. Total RNA was isolated using RNAiso Plus (TaKaRa, Shiga, Japan) according to the manufacturer’s instruction. Reverse transcription was performed with the PrimeScript™ RT reagent kit (TaKaRa). PCR amplification and fluorescence detection were performed using the Mx3000P Real-Time Cycler (Agilent Technologies, Santa Clara, CA, USA). Primers (listed in Additional file 1: Table S1) were designed by Takara Biotechnology (Dalian) Co., Ltd. (Dalian, China). Each sample was tested in triplicate, and β-actin was used as an internal control. The results obtained from three independent experiments were presented as the calculated comparative expression ratios of target sample to 2D cell by using CT method (2-∆∆CT).
Drug resistance test
HCCLM3 cells cultured under different conditions were treated with cisplatin (5 μg/ml) for 48 h. The viability of surviving cells was measured by CCK8 assay.
Enzymatic activity of MMP2 and MMP9 was tested by gelatin zymography . After incubation for 24 h, conditioned medium was collected and equal amounts of protein from each sample was loaded. MMP2 and MMP9 were differentiated according to their molecular weight, 72 kDa and 92 kDa, respectively.
In vitro invasion assay
The invasiveness of HCCLM3 cells cultured under different conditions was evaluated by Matrigel invasion assay according to the previous study . The transwell chambers (8 μm pore size) (Corning, Tewksbury, MA, USA) were coated with matrigel (BD, San Jose, CA, USA) according to the manufacturer’s instructions. HCCLM3 cells (105 cells per insert) were seeded on the top of matrigel with serum-free DMEM. The lower chamber was filled with DMEM/10 % FBS. After incubated at 37 °C for 48 h, HCCLM3 cells migrated through the membrane were stained with crystal violet and counted under microscopic observation. The data were shown as the means ± SD of three independent assays.
All experiments were performed three times independently as individual experiments. Data were expressed as means ± SD. Student’s t-test was used to determine the statistical significance between two groups. One-way ANOVA was used to specify differences between groups when more than two experimental groups were evaluated. Differences were considered to be significant for p < 0.05.
Effect of UCMSC on the proliferation of 3D-cultured HCCLM3 cells
Effect of UCMSC on CSC characteristics of 3D-cultured HCCLM3 cells
Effect of UCMSC on chemoresistance and metastatic properties of 3D-cultured HCCLM3 cells
Expression profile of EMT-related genes in co-cultured HCCLM3 cells
Decreased metastatic ability of 3D-cultured HCCLM3 cells by inhibition of TGF-β in co-culture system
For the less invasive source and lack of ethical concerns, UCMSC might be easily used as anti-tumor reagent delivery vehicle . Nevertheless, the effect of UCMSC on tumor progression is still controversial, so the safety of UCMSC for cytotherapy was still undetermined. Almost all the in vitro studies on investigating the role of UCMSC in tumor development were performed under 2D cell culture conditions. As is well known, 3D-cultured tumor cells might more resemble the in vivo tumor tissue. So in this study, a co-culture system was used to evaluate the effect of UCMSC on the proliferation, CSC characteristics, drug resistance and metastatic ability of 3D-cultured HCCLM3 cells. And we found that HCC cell metastasis was significantly enhanced by UCMSC through TGF-β.
Jing et al. pretreated 2D-cultured BMMSC with IFNγ and TNFα to mimic the inflammation condition in tumor, and they found that the conditioned medium of MSC could promote the metastasis of HCC cell line SMMC-7721 and HeP-3B though the elevated expression of TGF-β, which induced the EMT of HCC cells . In this study, our results supported their findings. However, Li et al. reported BMMSC inhibited the metastasis of HCC cell line MHCC97-H both in vitro and in vivo . They found that the conditioned medium of 2D-cultured BMMSC enhanced in vitro proliferation, but suppressed the invasive ability of HCC cells through down-regulating TGF-β expression of HCC cells. These controversial findings might be attributed to the different cell lines and different co-culture methods. Cell lines originated from different stages of tumor development had distinct migration ability and might lead to the distinct reaction to the soluble factors in MSC conditioned medium. In addition, MSC cultured under normal and inflammation condition might secret different kinds of factors due to the different microenvironment where they resided. Therefore, further research should be performed to clarify the mechanism of BMMSC or UCMSC on tumor cell metastasis in different culture conditions.
ALG beads used in this work provided a 3D environment for HCCLM3 cells. After 15 days of culture, HCC cells formed tumor spheroids, which created a more in vivo-like tumor microenvironment. So we assumed that the secreted soluble factors of UCMSC would be more similar to that in vivo. Moreover, in this study, it was found that after co-cultured with UCMSC, TGF-β gene expression in HCCLM3 cells was almost the same as the control group (data not shown), which suggested that UCMSC didn’t affect the TGF-β expression of HCC cells. In addition, there was no direct contact between HCCLM3 cells and UCMSC in this study, so the secreted TGF-β in co-culture system would be the main culprit for the elevated metastatic ability of HCC cells.
Our results indicated that UCMSC would favor the metastasis of HCC cells, however, in vivo study should be performed to confirm it. Nevertheless, it should be cautious when using UCMSC as therapeutic vehicles, at least under hepatocellular carcinoma condition.
In this study, a co-culture system was established to investigate the effect of UCMSC on 3D-cultured HCC cells. It was found that UCMSC could significantly enhance the metastasis of HCC cells by the induction of EMT which was regulated by TGF-β.
2D, two-dimensional; 3D, three-dimensional; ALG, alginate; BMMSC, bone marrow mesenchymal stem cell; CSC, cancer stem cell; EMT, epithelial to mesenchymal transition; HCC, hepatocellular carcinoma cell; MMP, matrix metalloproteinases; MSC, mesenchymal stem cell; UCMSC, umbilical cord mesenchymal stem cell
The authors would like to acknowledge the generous giving of HCCLM3 from Professor Jia Fan (Fudan University). And we would like to acknowledge Zhongyuan Union Stem Cell Bioengineering Corporation (Tianjin, China) for kindly providing Human UCMSC cells and the culture medium. This work was supported by a grant from Important National Science & Technology Specific Projects of China (2012ZX10002011-013), National Natural Science Foundation of China (No.31271055, No.31470944).
The design of the study was supported by Important National Science & Technology Specific Projects of China (2012ZX10002011-013), and data collection and analysis were supported by National Natural Science Foundation of China (No.31271055, No.31470944).
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
CL performed most of the experiments and wrote the main manuscript text. YL helped review the manuscript and analyze some data of this paper. X-XX helped prepare Fig. 5. XG helped culture 2D HCC cells. G-WS designed this paper, analyzed the data of this paper, and reviewed the manuscript. X-JM helped review the manuscript. All authors have read and approved the manuscript.
The authors declare that they have no competing interests.
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