Human Mesenchymal Stem Cells Derived from the Placenta and Chorion Suppress the Proliferation while Enhancing the Migration of Human Breast Cancer Cells

Background Breast cancer is the most frequently diagnosed malignancy among women, resulting from abnormal proliferation of mammary epithelial cells. The highly vascularized nature of breast tissue leads to a high incidence of breast cancer metastases, resulting in a poor survival rate. Previous studies suggest that human mesenchymal stem cells (hMSCs) play essential roles in the growth, metastasis, and drug responses of many cancers, including breast cancer. However, hMSCs from different sources may release different combinations of cytokines that affect breast cancer differently. Methods In this study, we have isolated hMSCs from the placenta (PL-hMSCs) and the chorion (CH-hMSCs) and determined how these hMSCs affect the proliferation, migration, invasion, and gene expression of two human breast cancer cells, MCF-7 and MDA-MB-231, as well as the possible mechanisms underlying those effects. Results The results showed that the soluble factors derived from PL-hMSCs and CH-hMSCs inhibited the proliferation of MCF-7 and MDA-MB-231 cells but increased the migration of MDA-MB-231 cells. The study of gene expression showed that PL-hMSCs and CH-hMSCs downregulated the expression levels of the protooncogene CyclinD1 while upregulating the expression levels of tumor suppressor genes, P16 and P21 in MCF-7 and MDA-MB-231 cells. Furthermore, hMSCs from both sources also increased the expression levels of MYC, SNAI1, and TWIST, which promote the epithelial-mesenchymal transition and migration of breast cancer cells in both cell lines. The functional study suggests that the suppressive effect of CH-hMSCs and PL-hMSCs on MCF-7 and MDA-MB231 cell proliferation was mediated, at least in part, through IFN-γ. Conclusions Our study suggests that CH-hMSCs and PL-hMSCs inhibited breast cancer cell proliferation by negatively regulating CYCLIND1 expression and upregulating the expression of the P16 and P21 genes. In contrast, hMSCs from both sources enhanced breast cancer cell migration, possibly by increasing the expression of MYC, SNAI1, and TWIST genes in those cells.


Background
The uncontrolled proliferation of mammary epithelial cells causes breast cancer, which is the most prevalent malignant tumor in women [1]. Breast cancer metastasis is common due to the high density of blood and lymphatic vessels in breast tissue, resulting in a low survival rate in patients. Although surgical procedures and systemic treatments, such as hormone therapy, chemoradiation, and immunotherapy, have improved significantly, the overall survival rate of patients with advanced-stage breast cancer remains poor due to resistance to treatment and relapse [2,3].
Several studies have found that human mesenchymal stem cells (hMSCs) play a crucial role in cancer growth, metastasis, and the response to chemotherapeutic drugs. hMSCs are multipotent stem/progenitor cells found in several tissues, including bone marrow, adipose tissue, umbilical cord, placenta, and chorion [4][5][6][7]. hMSCs have been recognized as prospective cell sources for several clinical applications due to their ability to release many beneficial bioactive compounds that reduce inflammation, increase cell viability, and enhance neovascularization [8][9][10]. Additionally, some sources of hMSC, such as bone marrow and adipose tissues, can be harvested from patients and used for autologous transplantation, thus circumventing the need for immunosuppression [10,11].
In animal models, hMSCs have also been shown to migrate from the bloodstream to cancerous areas [12][13][14], where they form cancer-associated fibroblasts (CAFs) that promote cancer growth. CAFs derived from hMSCs released multiple soluble factors that induce tumor neovascularization, increase cancer survival, and enhance metastasis [15][16][17][18][19][20][21][22][23]. Furthermore, hMSCs are believed to promote tumor growth by suppressing immune surveillance against tumor cells [24]. Despite these data, some studies have shown that hMSCs can also limit the growth and spread of several cancers [25][26][27][28]. These discrepancies could be caused by differences in the types of hMSCs and cancer used in these investigations.
Although bone marrow-derived hMSCs (BM-hMSCs) have been widely used in most research and therapeutic applications, their harvesting requires an invasive process and their numbers decrease with age [29,30]. As a result, hMSCs derived from gestational tissues, such as the placenta and chorion, have been deemed more appropriate for therapeutic applications since they are easily available in high quantities using a noninvasive procedure. However, the bioactive molecules produced by hMSCs originating from gestational tissue and their impact on breast cancer cells are still poorly characterized. Therefore, this study is aimed at investigating the effects of placenta-derived hMSCs (PL-hMSCs) and chorionderived hMSCs (CH-hMSCs) on proliferation, migration, invasion, and gene expression profiles of two different human breast cancer cells, estrogen receptor-expressing MCF-7 cells and estrogen receptor-negative MDA-MB231 cells.

Methods
2.1. Subjects. This study was approved by the Ethics Committee for Human Research of the Faculty of Medicine of Thammasat University (project number: MTU-EC-DS-2-001/62). The study was carried out according to the ICH-GCP. After labor, healthy women donated their placentas and chorions. All donors signed an informed consent form.
2.2. Isolation and Culture of hMSCs. As described in our earlier study [31], the placenta and the chorionic membrane were manually separated, minced into small pieces, and incubated for 30 minutes at 37°C in 0.25% (w/v) trypsin-EDTA (Invitrogen Corporation, USA). The digested tissues were then washed twice with PBS, resuspended in DMEM +10% (v/v) Fetal Bovine Serum (FBS) (Lonza, USA), and cultured in 25 cm 2 flasks (Corning, USA) at 37°C. The medium was changed every three days, and the cells were passaged when their density reached 80% confluence. An inverted microscope (Nikon ECLIPSE Ts2R, Japan) was used to examine and photograph the morphological characteristics of hMSCs. . Human breast cancer cells, MCF-7 and MDA-MB231, were purchased from  ATCC. MCF-7 is an estrogen receptor-expressing human  breast cancer cell line derived from the pleural effusion of a  female patient with metastatic (stage IV) breast cancer. MDA-MB231 is a triple negative (ER -/PR -/EGFR -) human breast cancer cell line derived from the pleural effusion of a female patient with metastatic (stage IV) breast cancer. Both cells were cultured in DMEM (Invitrogen Corporation, USA) supplemented with 10% (v/v) FBS (Lonza, USA) at 37°C. The medium was changed every three days, and the cells were passaged when their density reached 80% confluence.
2.5. Osteogenic and Adipogenic Differentiation of hMSCs. As described in our previous study [31], 5 × 10 4 hMSCs were cultured in NH OsteoDiff® Medium (Miltenyi Biotec, Germany) for osteogenic differentiation. The cells were cultured for 28 days with media replacement every three days. At the end of the culture, the cells were fixed with 4% paraformaldehyde, stained with a 40 mM alizarin red S solution (Sigma-Aldrich, USA) for 20 minutes at room temperature, and viewed under an inverted microscope (Nikon ECLIPSE Ts2R, Japan). For adipogenic differentiation, 5 × 10 4 hMSCs were cultured in NH AdipoDiff® medium (Miltenyi Biotec, Germany) for 28 days with media replacement every three days. At the end of the culture, the cells were fixed with vaporized 37% formalin for 10 minutes at room temperature, stained with 0.5% (w/v) oil red O (Sigma-Aldrich, USA) in 6% (v/v) isopropanol for 20 minutes at room temperature and observed by light microscopy.
2.6. Preparation of hMSC-Conditioned Medium. As described in our previous study [31], 7 × 10 5 hMSCs were initially expanded in DMEM +10% FBS until their density reached 90% confluence. The medium was then removed, the cells were rinsed twice with 10 ml sterile PBS, fresh 15 ml DMEM +10% FBS medium was added, and the cells were incubated for another 24 hours. The conditioned medium (CM) was collected after incubation and centrifuged at 1,000 g for 10 minutes at 4°C before being filtered through a 0.45 μm syringe filter (Corning, USA). Finally, the filtered CM was lyophilized and diluted to the appropriate concentrations. 2.11. Statistical Analysis. The mean and standard error of the mean (SEM) was used to present the data. The significance of the differences between the observed data was determined using unpaired student's t-test. Statistical significance was defined as a P value of less than 0.05.

Characteristics of hMSCs Derived from the Placenta and
Chorion. The placenta-derived hMSCs (PL-hMSCs) and chorion-derived hMSCs (CH-hMSCs) exhibited typical characteristics of hMSCs according to the minimal criteria suggested by the international society for cell therapy [32]. The hMSCs of both sources showed a fibroblast-like morphology (Figure 1(a)), expressed typical surface markers of hMSCs (positive for CD73, CD90, and CD105 and negative for hematopoietic markers, CD34, and CD45; Figure 1(b)), and could differentiate to adipocytes and osteocytes as determined by oil red O staining and alizarin red S staining, respectively (Figures 1(c) and 1(d)).   Stem Cells International

The Expression Levels of Cytokine Genes in hMSCs Derived
from the Placenta and Chorion. The expression levels of 5 cytokine genes commonly involved in cancer growth [33][34][35], including Dickkopf WNT signaling pathway inhibitor 1 (DKK1), transforming growth factor-β (TGF-β), interferon-β (IFN-β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in CH-hMSCs and PL-hMSCs were determined by quantitative real-time PCR. The results showed that the CH-hMSCs and PL-hMSCs derived from most donors expressed high levels of DKK1, TGF-β, and IFN-γ ( Figure 2). Although CH-hMSCs and PL-hMSCs also expressed IFN-β and TNF-α genes, their expression levels were lower than those of DKK1, TGF-β, and IFN-γ ( Figure 2). ). Due to the very low migratory and invasive capacity of MCF-7 cells in our study, which is also described elsewhere [36][37][38], we only use MDA-MB231 cells for our migration and invasion assays.  The results showed that the addition of CHIR99021 or SB431542 did not decrease the ability of CH-hMSCs and PL-hMSCs to suppress the proliferation of MCF-7 ( Figure 8) and MDA-MB231 cells (Figure 9). The results demonstrate that CH-hMSCs and PL-hMSCs did not suppress the proliferation of MCF-7 and MDA-MB231 cells through the action of DKK1 and TGF-β. Unlike CHIR99021 and SB431542, XMG1.2 significantly reduced the ability of CH-hMSCs and PL-hMSCs to suppress MCF-7 and MDA-MB231 cell proliferation (Figures 8 and 9). These findings suggest that IFN-γ is one of the factors involved in the suppressive effect of CH-hMSCs and PL-hMSCs on MCF-7 and MDA-MB231 cell proliferation.

Discussion
hMSCs have long been regarded as one of the most promising sources for cell therapy. The capacity of hMSCs to release a wide range of bioactive chemicals that alter various processes, including immune response, cell growth, and neovascularization, is crucial to their therapeutic potential [39]. hMSCs also play a role in cancer progression by releasing angiogenic factors, such as IGF-1, SDF-1, and PDGF, which promote tumor neovascularization, enhance tumor engraftment, and suppress immune responses to cancer cells [16,17,19,20,22,24,[40][41][42]. However, several other studies found that hMSC prevented the growth and spread of several malignant neoplasms in animal models, including colon   [25][26][27][28]. The variations could be attributed to differences in the sources of hMSCs and types of cancer cells used in the studies. The hMSCs derived from the placenta and chorion (PL-hMSCs and CH-hMSCs) have been considered to be more appropriate sources of hMSCs for therapeutic applications than the commonly used BM-hMSCs, since they may be easily obtained in high quantities using a noninvasive technique. However, the types of bioactive chemicals pro-duced by hMSCs originating from these gestational tissues and their impact on the properties of breast cancer, the most prevalent cancer in women, have yet to be determined.
Our study found that bioactive compounds released from PL-hMSCs and CH-hMSCs inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Both sources of hMSC suppressed breast cancer cell proliferation in the same way, and there was no difference between hMSCs generated from different donors. According to our

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Stem Cells International study of gene expression, soluble factors derived from CH-hMSCs and PL-hMSCs inhibited the cell proliferation of MCF-7 and MDA-MB-231 by negatively regulating the expression level of the protumorigenic gene Cyclin-D1 and upregulated the tumor suppressor gene P16 and the cyclindependent kinase inhibitor genes P21 in these cells. All of these genes have been shown to play a critical role in the onset and progression of breast cancer. The inactivation of p16 INK4a , a CDK inhibitor that inactivates CDK4/6 and prevents cell cycle progression at the G1/S checkpoint, resulting in the loss of cell cycle control, while dysregulation of the CDK4/6-cyclin D1 complex is a crucial stage in the genesis of breast cancer [43][44][45][46]. In contrast to their effect on breast cancer cell proliferation, the soluble factors derived from CH-hMSCs and PL-hMSCs enhanced MDA-MB231 cell migration in a dosedependent manner. Consistent with this observation, our gene expression study showed that the soluble factors derived from CH-hMSCs and PL-hMSCs upregulated the expression levels of MYC, SNAI1, and TWIST that promote epithelial to mesen-chymal transition and migration of breast cancer cells [47][48][49][50][51]. There are variances in the ability of PL-hMSCs and CH-hMSCs obtained from different donors to enhance MDA-MB231 migration, which could be due to differences in the amount of released cytokines. Interestingly, although both sources of hMSCs increased the migration of MDA-MB231 cells, they did not affect the invasion of MDA-MB231 cells. These findings suggested that the cytokines that cause MDA-MB231 cells to migrate may be different from those that cause them to invade.
According to the study of CH-hMSC and PL-hMSC gene expression, we hypothesized that DKK1, TGF-β, and IFN-γ might be responsible for the inhibitory effects of hMSCs on the proliferation of MCF-7 and MDA-MB231 cells. Our functional study using CHIR99021, SB431542, and XMG1.2 suggests that CH-hMSCs and PL-hMSCs suppressed MCF-7 and MDA-MB231 cell proliferation, at least in part, through IFN-γ rather than DKK1 and TGF-β. This result contrasts with a previous study showing that BM-hMSCs promote breast cancer cell proliferation, migration, and invasion under   Data are presented as mean ± SEM of four experiments. MCF-7 cultured in DMEM medium +10% FBS supplemented with XMG1.2 (control+IFNGi) served as controls. * P < 0:05 between the compared groups. CH-CMs were prepared from 3 CH-hMSCs (CH9, CH15, and CH16), and PL-hMSC-CMs were prepared from 3 PL-hMSCs (PL11, PL14, and PL17).  14 Stem Cells International hypoxic conditions by releasing TGF-β1 [52]. The discrepancies are most likely due to the different types of hMSCs used in each study. Due to the heterogeneous nature of hMSCs at the time of isolation and subsequent expansion, all sources of hMSCs, including BM-hMSCs, likely consist of several subpopulations that might release different combinations of cytokines and therefore exert different effects on breast cancer cells. Therefore, hMSCs derived from different tissues or those derived from the same tissue from different donors could exhibit distinct characteristics and biological properties, making the outcome of therapy difficult to predict. Consequently, we believe that all hMSCs, regardless of the sources, should be characterized in terms of their cytokine expression before being used for various therapeutic purposes. The knowledge gained from this study could help identify soluble factors derived from hMSCs that could be used to inhibit breast cancer cell growth, so hMSCs derived from many tissue sources or donors could be screened for those therapeutically beneficial factors and, therefore, the suitable MSCs could be selected for treatment.

Conclusion
Our study suggests that CH-hMSCs and PL-hMSCs inhibited breast cancer cell proliferation by negatively regulating CyclinD1 expression and upregulating the expression of the P16 and P21 genes. The suppressive effect could be mediated, at least in part, by IFN-γ released from the hMSCs. On the contrary, hMSCs from both sources enhanced breast cancer cell migration, possibly by increasing the expression of the MYC, SNAI1, and TWIST genes in these cells. We believe that the findings of this study could be used to design more effective treatments for patients with advanced-stage breast cancer by modulating the interaction between breast cancer cells and hMSCs.