Mesenchymal stem cells (MSC) are strongly associated with tumor progression and have been used as novel cell-based agents to deliver anticancer drugs to tumors. However, controversies about the direct involvement of MSCs in tumor progression suggest that MSCs mediate tumor progression in a cancer type-dependent manner. In this report, we analyzed the functional interactions between human MSCs and lung adenocarcinoma (LAC) cells to determine the therapeutic potential of MSCs in lung cancer. We showed that MSCs effectively inhibited the migration, invasion, and cell-cycle progression of several LAC cell lines. MSCs also enhanced the mesenchymal–epithelial transition (MET) pathway, as evidenced by the reduction of several epithelial–mesenchymal transition-related markers in LAC cells cocultured with MSCs or in MSC-conditioned medium (MSC-CM). By cytokine array analysis, we determined that Oncostatin M (OSM), a differentiation-promoting cytokine, was elevated in the MSC-CM derived from primary MSC cultures. Furthermore, OSM treatment had the same effects as MSC-CM on LAC, whereas neutralizing antibodies to OSM reversed them. Notably, short hairpin RNAs against STAT1, an important downstream target of OSM, hindered the OSM-dependent induction of MET. In vivo xenograft tumor studies indicated that OSM inhibited tumor formation and metastasis of LAC cells, whereas neutralizing OSM in the MSC-CM hampered its inhibitory effects. In conclusion, this study showed that OSM is a paracrine mediator of MSC-dependent inhibition of tumorigenicity and activation of MET in LAC cells. These effects of OSM may serve as a basis for the development of new drugs and therapeutic interventions targeting cancer cells. Cancer Res; 72(22); 6051–64. ©2012 AACR.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide despite continuous efforts to find effective treatments (1). Lung adenocarcinoma (LAC) is the most common histologic type of lung cancer. The highly invasive and metastatic phenotypes of LAC are the major reasons for treatment failure and the poor prognosis associated with this disease. Stem cell-based therapy is an emerging strategy to treat various diseases, including cancer. Rat umbilical cord matrix stem cells have been shown to inhibit murine lung adenocarcinoma (LAC) growth (2). However, further work in human lung cancer models is still needed to clarify the therapeutic potential of this approach. Mesenchymal stem cells (MSC) have recently gained much attention for their potential applications in tissue engineering and disease therapy. Their tropism for sites of tissue damage and the tumor microenvironment has led to a great deal of interest in the functions of MSC in tumors and their ability to serve as delivery vehicles for therapeutic agents (3–5). For example, MSC-based delivery of the death receptor ligand TRAIL leads to the inhibition of tumor growth and the elimination of cancer metastasis in xenograft lung tumor models (6, 7) as well as a reduction in the number of putative lung cancer stem cells (8). However, the effects of MSCs themselves on cancer progression, as well as the crosstalk mechanisms between MSCs and cancer cells, remain controversial and unclear.

The epithelial–mesenchymal transition (EMT) is a critical process during embryonic development that is reengaged in adults during wound healing, tissue regeneration, and cancer progression (9). EMT is a key step in the induction of tumor metastasis and is associated with a poor clinical outcome in cancer patients (10). An aberrant upregulation of EMT transcription factors is associated with poor overall and metastasis-free survival in patients with non–small cell lung cancer (11, 12). Targeting the EMT pathway or inducing the mesenchymal–epithelial transition (MET), the inverse of EMT, has been reported to suppress lung cancer progression and metastasis (13–15) and may therefore represent a potential treatment strategy for LAC patients. Notably, the composition of the microenvironment, including oxygen tension, growth factors, and cytokines, was recently suggested to modulate the MET and EMT process in cancer cells (16–19). MSCs are an important source of many chemokines and cytokines that affect the tissue repair process (20). MSC-secreted cytokines have also been implicated in the MSC-mediated regulation of tumor cells. However, whether MSCs mediate LAC tumor progression through a paracrine mechanism and whether MSC-derived cytokines are involved in LAC tumorigenesis and MET remain open questions.

Oncostatin M (OSM) is a differentiation-promoting cytokine that has been reported to induce hepatocyte differentiation of human embryonic and putative liver cancer stem cells (21, 22). Recently, OSM was shown to promote MSC differentiation into osteoblasts (23). OSM is also reported to have antiproliferation effects against several types of cancers and is thus considered a potential therapeutic target (21, 24–28). However, the role of OSM in tumor progression and metastasis has yet to be evaluated. In this study, we aimed to investigate the role of MSCs and their mediators in the regulation of lung cancer progression. We first showed that both MSCs and MSC-conditioned medium (MSC-CM) inhibited in vivo tumor growth and the in vitro proliferation, migration, and invasion of LAC cells in a dose-dependent manner. Moreover, both MSCs and MSC-CM promoted the MET process in LAC cells, as evidenced by elevated pro-MET markers, inhibited pro-EMT regulators, and suppressed Nanog stemness factor. We then analyzed the cytokine composition of the MSC-CM and identified OSM as an important mediator of the MSC-dependent inhibition of LAC tumorigenicity. Treatment with recombinant OSM repressed LAC proliferation, migration, invasion, and promoted MET as well as epithelial-like phenotypic transformation. Conversely, neutralizing OSM in the MSC-CM hindered these effects on LAC cells. Most importantly, a xenograft tumor model revealed that OSM reduced the tumor size, suppressed the incidence of metastasis, and enhanced MET markers. In conclusion, this study identified OSM as a crucial paracrine mediator of the MSC-dependent inhibition of LAC tumorigenicity, in part by enhancing the MET pathway.

Cancer cell lines and culture conditions

A549 LAC cell line, H1299 large cell carcinoma cell line, A431 epidermal carcinoma cell line, and breast adenocarcinoma cell lines (including MCF-7 and MDA-MB-231) were obtained from the American Type Culture Collection before 2007 and tested positive for human origin. The CL1-5 LAC cell line was established previously (29). All cell lines were maintained in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS.

Collection of CM

MSCs were seeded at a density of either 5 × 105 (low) or 2 × 106 (high) cells/100-mm plate in complete RPMI-1640 or MesenPRO RS (Invitrogen) medium supplemented with pre-selected FBS, P/S, and l-glutamine. CM was collected after 24 hours of incubation with MSCs, filtered with a 0.2-μm pore size Millex filter unit (Millipore), and then stored at −80°C. Control medium was produced simultaneously after a 24-hour incubation without MSCs.

Animals and tumor cell transplantation

Luciferase-expressing CL1-5 (CL1-5-luc) cells were harvested, washed, resuspended in PBS, and mixed with an equal volume of Matrigel (BD Biosciences). CL1-5-luc cells, MSCs, or a mixed population of CL1-5-luc cells and MSCs (in a total volume of 100 μL) were injected subcutaneously into the right dorsolateral side of the flank region of 8-week-old male BALB/c nude mice (BioLasco Taiwan Co.). The tumors were measured with an IVIS Lumina II system (Caliper Life Science).

Migration and invasion assay

A FluoroBlok 24-Multiwell Insert System with an 8-μm pore size polyethylene terephthalate membrane (BD Falcon) was used to test cell mobility. Each well was filled with 700 μL medium, and cell suspensions were seeded into the insert chamber at a density of 2.5 × 104 cells in 300 μL medium. After 24 hours, the medium was removed, and the chamber was washed with 1× PBS and fixed in 100% methanol overnight. The reverse side of the membrane facing the lower chamber was stained with propidium iodide (Sigma-Aldrich) for 30 minutes, and the migratory cells were then visualized under an inverted microscope. Cell number was quantitated using ImageJ software. For the invasion assay, the membrane was coated with Matrigel (BD Biosciences) diluted with an equal volume of serum-free medium and incubated for at least 1 hour at 37°C before the cells were seeded.

Proliferation assay

Cells were seeded into 96-well cell culture plates at a density of 1 × 103 cells/well in 100 μL media and allowed to adhere overnight. The media was aspirated and replaced with MSC-CM or MesenPRO RS medium with or without recombinant OSM (Peprotech) as described. A WST-1 (Roche Applied Science) assay was carried out according to the manufacturer's protocol to assess the changes in relative cell density every 24 hours.

Statistical analysis

The results are reported as the mean ± SD. Statistical analyses were carried out using Student t test. A P value <0.05, as denoted with “*” in figures, was considered statistically significant.

Bone marrow-derived MSCs suppressed LAC tumor growth in vivo

Bone marrow-derived MSCs are known to specifically migrate to and engraft at tumor sites (30, 31). Although several reports in the past decade have investigated the physical and functional interactions between MSCs and different types of cancer cells (e.g., breast, liver, and colon), their relationships remain controversial. Moreover, the roles of MSCs in lung cancer progression and development are still unclear and need to be further investigated. To examine the effects of MSCs on lung cancer cell tumorigenicity, we first conducted xenotransplantation experiments in immunocompromised mice. Primary bone marrow-derived MSCs were isolated from clinical specimens taken from 3 healthy donors and analyzed by flow cytometry to confirm the expression of MSC markers and the absence of macrophage, endothelia, and hematopoietic cell markers (1 representative MSCs shown in Supplementary Fig. 1). The MSCs were then mixed with luciferase-labeled CL1-5 human LAC cells at a ratio of 1:5 (C+M (low)) or 1:10 (C+M (high)) before subcutaneous injection into the right flanks of male mice. Injections of either MSCs or CL1-5 cells alone were conducted simultaneously (Fig. 1A, top; n = 4), and tumor volumes were monitored for 7 weeks. As expected, the CL1-5-induced xenograft tumors significantly increased in size. However, when mixed with MSCs, regardless of the ratio, the growth of the CL1-5 xenograft tumors were dramatically inhibited (Fig. 1A). At day 49, the volume of the CL1-5/MSC tumors was approximately one tenth that of the CL1-5 tumors (Fig. 1A, bottom right). These data indicated an in vivo inhibitory effect of MSCs on LAC tumor growth.

Figure 1.

Bone marrow-derived MSCs inhibited LAC tumor growth in an animal model. A, immunocompromised mice were subjected to subcutaneous transplantation of 8 × 106 MSCs, 4 × 106 luciferase-labeled CL1-5 cells (CL1-5-luc), or mixed MSC:CL1-5 at the ratio of 1:5 (C+M-high, with 8 × 105 MSC) or 1:10 (C+M-low, with 4 × 105 MSC). The mean total photon flux, representing the tumor volume, was monitored weekly using an IVIS Lumina Live Imager. Representative tumor photographs of IVIS images taken on day 49 are shown (top). The tumor growth curves and the chart of tumor volumes on day 49 are presented (bottom, n = 4). B, Immunocompromised mice were subcutaneously transplanted with 4 × 106 CL1-5-luc cells and allowed to form tumors. Once the tumor locus could be visualized, 100 μL of control or MSC-derived conditioned medium (CM; supernatant from 2 × 106 cultured MSCs) was injected at the tumor locus every 7 days until day 55. The mean total photon flux was assessed on day 55 and presented as fold change in the graph (n = 3).

Figure 1.

Bone marrow-derived MSCs inhibited LAC tumor growth in an animal model. A, immunocompromised mice were subjected to subcutaneous transplantation of 8 × 106 MSCs, 4 × 106 luciferase-labeled CL1-5 cells (CL1-5-luc), or mixed MSC:CL1-5 at the ratio of 1:5 (C+M-high, with 8 × 105 MSC) or 1:10 (C+M-low, with 4 × 105 MSC). The mean total photon flux, representing the tumor volume, was monitored weekly using an IVIS Lumina Live Imager. Representative tumor photographs of IVIS images taken on day 49 are shown (top). The tumor growth curves and the chart of tumor volumes on day 49 are presented (bottom, n = 4). B, Immunocompromised mice were subcutaneously transplanted with 4 × 106 CL1-5-luc cells and allowed to form tumors. Once the tumor locus could be visualized, 100 μL of control or MSC-derived conditioned medium (CM; supernatant from 2 × 106 cultured MSCs) was injected at the tumor locus every 7 days until day 55. The mean total photon flux was assessed on day 55 and presented as fold change in the graph (n = 3).

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The inhibitory effect of MSCs on LAC cell-derived tumors could occur through direct cell–cell contacts or in a paracrine fashion without direct cell contact. To distinguish between these possibilities, we first subcutaneously transplanted luciferase-labeled CL1-5 cells in the right flank of immunocompromised mice. Two weeks after transplantation, when the tumor mass was visible using an IVIS Lumina Imaging system, control or CM that had been incubated for 24 hours without or with MSCs, respectively, was injected at the tumor locus. The injections were conducted on days 14, 21, 28, and 35, and the tumor volumes were assessed on day 55 by measuring changes in the mean total photon flux using the IVIS system. The CL1-5 tumors in the CM-treated mice were significantly smaller than those of the mice treated with control medium (Fig. 1B; n = 3). These data suggested that the inhibitory effect of MSCs on LAC tumor growth occurs through a paracrine pathway and may not require direct cell–cell contact.

MSC-CM inhibited the proliferation and cell-cycle progression of LAC cells

The MSC-mediated inhibition of tumor growth in the animal model indicated that MSCs may suppress cell proliferation or cell-cycle progression in LAC cells. We therefore carried out WST-1 proliferation assays for 2 LAC cell lines (CL1-5 and A549) incubated in control medium or 2 concentrations of MSC-CM. No significant differences in total cell number were observed between groups on the first day. However, after day 2, CL1-5 and A549 cell numbers were reduced by MSC-CM in a dose-dependent manner compared with the cells cultured in control medium (Fig. 2A, left). By day 4, treatment with high CM reduced the cell numbers by more than 50% compared with control cells (Fig. 2A, right). Flow cytometry analysis of the 2 cell lines revealed that treated cells were partially sequestered in G1 phase: MSC-CM increased the percentage of CL1-5 and A549 cells in G1 phase by 15% and 8%, respectively, compared with cells treated with control medium (Fig. 2B). This decreased rate of cell proliferation and restrained cell-cycle progression could explain the inhibition of tumor growth observed in the animal model.

Figure 2.

MSCs inhibited lung adenocarcinoma cell proliferation, cell-cycle progression, and wound-healing capacity. A, CL1-5 and A549 cells were subjected to a WST-1 proliferation assay in the presence of high density CM (supernatant from 2 × 106 MSCs), low density CM (supernatant from 5 × 105 MSCs), or control medium (medium incubated overnight without MSCs). The proliferation curves are shown on the left, and the fold changes in cell number on days 1 and 4 are shown on the right. B, CL1-5 and A549 cells were incubated in high density CM, low-density CM, or control medium for 24 hours and then subjected to flow cytometry cell-cycle analysis. The percentages of cells in each stage of the cell cycle are presented. C, CL1-5 and A549 cells were subjected to a wound-healing assay in the presence of control medium or CM. The numbers of cells within the gap at 0, 6, and 12 hours were calculated and shown in the graphs in the right panels.

Figure 2.

MSCs inhibited lung adenocarcinoma cell proliferation, cell-cycle progression, and wound-healing capacity. A, CL1-5 and A549 cells were subjected to a WST-1 proliferation assay in the presence of high density CM (supernatant from 2 × 106 MSCs), low density CM (supernatant from 5 × 105 MSCs), or control medium (medium incubated overnight without MSCs). The proliferation curves are shown on the left, and the fold changes in cell number on days 1 and 4 are shown on the right. B, CL1-5 and A549 cells were incubated in high density CM, low-density CM, or control medium for 24 hours and then subjected to flow cytometry cell-cycle analysis. The percentages of cells in each stage of the cell cycle are presented. C, CL1-5 and A549 cells were subjected to a wound-healing assay in the presence of control medium or CM. The numbers of cells within the gap at 0, 6, and 12 hours were calculated and shown in the graphs in the right panels.

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MSC-CM inhibited migration, invasion, and EMT in LAC cells

Migration and invasion are critical properties of the cancer cells that initiate metastasis. To clarify the involvement of MSCs in LAC migration, we carried out a wound-healing assay with both LAC cell lines. As shown in Fig. 2C, the numbers of migrating cells were decreased by MSC-CM in a dose-dependent manner compared with those treated with control medium. This effect was not due to a difference in proliferation rate, as we observed an approximately 3- to 5-fold difference in the number of migrating cells between control- and high CM-treated cells at 12 hours (Fig. 2C), whereas cell proliferation was not affected within the first 24 hours (Fig. 2A).

An indirect coculture assay was carried out to further assess cell mobility (Fig. 3A, left). Reduced migration of CL1-5 cells was observed when the bottom chamber was coated with MSCs or filled with MSC-CM compared with noncoated or control medium-filled controls, respectively (Fig. 3A, middle and right). The same experiment was also conducted with other 4 primary MSCs isolated from bone marrow or endometrium of 4 individuals (Supplementary Information). Each of the MSCs showed inhibitory effect on CL1-5 migration, indicating that the MSC-mediated inhibition of LAC cell migration is rather a general phenomenon than a unique case for a specific strain of MSC (Supplementary Fig. 2). Moreover, this phenomenon was not unique to CL1-5 cells; the migratory abilities of 2 other lung cancer cell lines (A549 and H1299) were also suppressed when cultured in MSC-CM (Fig. 3B, left). Apart from migration, invasion is another important property of malignant tumor cells. Using a Matrigel-coated Transwell culture chamber, we showed that the invasion of both CL1-5 and A549 cells was inhibited by MSC-CM (Fig. 3B, middle).

Figure 3.

MSC-CM inhibited cell migration, invasion, and induced the MET. A, diagram showing the indirect coculture of MSCs and LAC cells for the Transwell migration and invasion assays (left). CL1-5 cells were subjected to a 24-hour Transwell migration assay with or without either MSCs or MSC-CM. The migrating cells that moved through the 8-μm pores to the other side of the membrane were counted under a microscope (middle) and presented as a percentage relative to controls (right). B, another LAC cell line, A549, and the H1299 large cell carcinoma cell line, were also subjected to the migration assay with or without MSC-CM as indicated (left). CL1-5 and A549 cells were subjected to a Matrigel-coated Transwell migration assay with MSC-CM or control medium, and the results are presented as the percentage of migrating cells relative to the control (middle). Two breast cancer cell lines, MDA-MB-231 and MCF-7, an epidermal cancer cell line, A431, and the CL1-5 cell line were subjected to the migration assay in the presence or absence of MSC-CM (right). C, CL1-5 cells were cultured in either MSC-CM or control medium for 24 hours and then switched to ordinary RPMI maintenance medium for the indicated durations. Cells were subcultured when they reached 90% confluence. For the last 24 hours of RPMI incubation, cells were seeded in the Transwell and subjected to the migration assay. The procedure is summarized in the top left diagram. Blue arrow, control medium; red arrow, CM; black arrow, RPMI medium. The migrating cells were quantified and presented as the percentage of the control value in each set (bottom left). Micrographs of the migratory cells are presented (top right). D, CL1-5 cells with 24-hour treatment of CM or control medium were subjected to immunofluorescent staining and confocal microscopic observation.

Figure 3.

MSC-CM inhibited cell migration, invasion, and induced the MET. A, diagram showing the indirect coculture of MSCs and LAC cells for the Transwell migration and invasion assays (left). CL1-5 cells were subjected to a 24-hour Transwell migration assay with or without either MSCs or MSC-CM. The migrating cells that moved through the 8-μm pores to the other side of the membrane were counted under a microscope (middle) and presented as a percentage relative to controls (right). B, another LAC cell line, A549, and the H1299 large cell carcinoma cell line, were also subjected to the migration assay with or without MSC-CM as indicated (left). CL1-5 and A549 cells were subjected to a Matrigel-coated Transwell migration assay with MSC-CM or control medium, and the results are presented as the percentage of migrating cells relative to the control (middle). Two breast cancer cell lines, MDA-MB-231 and MCF-7, an epidermal cancer cell line, A431, and the CL1-5 cell line were subjected to the migration assay in the presence or absence of MSC-CM (right). C, CL1-5 cells were cultured in either MSC-CM or control medium for 24 hours and then switched to ordinary RPMI maintenance medium for the indicated durations. Cells were subcultured when they reached 90% confluence. For the last 24 hours of RPMI incubation, cells were seeded in the Transwell and subjected to the migration assay. The procedure is summarized in the top left diagram. Blue arrow, control medium; red arrow, CM; black arrow, RPMI medium. The migrating cells were quantified and presented as the percentage of the control value in each set (bottom left). Micrographs of the migratory cells are presented (top right). D, CL1-5 cells with 24-hour treatment of CM or control medium were subjected to immunofluorescent staining and confocal microscopic observation.

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Whether MSCs promote or inhibit cancer progression remains controversial. Karnoub and colleagues reported that MSCs within tumor stroma promote breast cancer metastasis (32), and similar phenomena have also been observed in colon cancer (33). On the other hand, accumulating reports show that MSCs inhibit the tumorigenesis of several types of cancers, such as hepatomas and lymphomas (34–37). To further validate our results, we carried out a migration assay using CL1-5 cells, the A431 epidermal carcinoma cell line, and the MCF-7 and MDA-MB-231 breast cancer cell lines, which have been reported to be stimulated, not inhibited, by MSCs in tumorigenicity assays (32, 38). As shown in Fig. 3B, only CL1-5 cells were inhibited by MSC-CM; migration of the other cell lines was slightly increased by this treatment. It should be noted that the ratio of MSCs to cancer cells used in this experiment was much less than that used in previous reports (32), which likely explains the decreased stimulatory effect of MSC-CM on the migration of breast and epidermal carcinoma cell lines compared with previous reports. However, even under this low ratio of MSCs:cancer cells, CL1-5 cell migration was still severely repressed (Fig. 3B, right).

Next, we investigated the reversibility of the inhibitory effect on migration. CL1-5 cells were cultured in control medium or MSC-CM for 24 hours, after which the medium was replaced with RPMI, the standard culture medium for CL1-5 cells, for the indicated durations (Fig. 3C, top left). Cells were subcultured when they reached 90% confluence and subjected to the migration assay for the last 24 hours of RPMI incubation. We found that a 24-hour rest in RPMI medium had little effect on cell mobility in the MSC-CM-treated cells. However, 72 hours of rest in RPMI medium resulted in a slight recovery of cell mobility (10%); after 7 days of rest, the recovery was even more apparent (Fig. 3C, bottom left and top right). Moreover, by immunofluorescence analysis and confocal microscopy, we observed elevated expression of E-cadherin, a pro-MET marker, and reduced levels of vimentin, a pro-EMT marker, in cells treated with MSC-CM (Fig. 3D). These data indicate that the MSC-mediated inhibition of LAC migration may be caused by certain molecule(s) released by MSCs into the medium and that removing the molecule(s) by replacing the medium can reverse the inhibition of cell mobility.

MSC-CM promoted MET in LAC cells

The EMT is strongly linked to cell mobility and cancer metastasis. Repressing EMT or enhancing MET has been proposed as a mechanism to inhibit cancer progression (13–15, 39). Upon incubation in MSC-CM, the mesenchymal-like CL1-5 cells adopted a more epithelial-like morphology (Fig. 4A, left). Western blot analysis also revealed increased cytokeratin-18 (CK-18) and E-cadherin expression, and decreased vimentin, Snail, and Slug expression in MSC-CM-treated LAC cells compared with control cells (Fig. 4A, right). However, the level of another EMT-related transcription factor, Twist, was not altered (data not shown). These data suggested that MSC-CM treatment of LAC cells promotes a MET process that may involve Snail and Slug but not Twist. Interestingly, although the stemness factor Oct4 was not affected, Nanog expression was suppressed by MSC-CM, indicating the involvement of Nanog stemness signaling in the crosstalk between MSCs and LAC cells. We previously reported that coexpression of Nanog and Oct4 can induce EMT and promote lung cancer metastasis (40). It is possible that Nanog plays a key role in the MSC-mediated regulation of lung cancer cell mobility. We therefore generated A549 and CL1-5 stable cell lines overexpressing Nanog, Snail, or Slug (A549/Nanog, A549/Snail, A549/Slug, CL1-5/Nanog, CL1-5/Snail, and CL1-5/Slug) using a lentiviral transfection system; empty vector-transfected control cell lines (A549/Vec and CL1-5/Vec) were also generated. The expression of Nanog, Slug, and Snail was confirmed by Western blotting (Fig. 4B). Notably, overexpression of Nanog enhanced the protein levels of Snail and Slug in A549 cells, whereas neither Snail nor Slug overexpression affected Nanog levels, suggesting that Nanog may be an upstream regulator of Snail and Slug expression in some LAC cells (Fig. 4B). The stable cell lines were treated with MSC-CM or control medium before a migration assay. Compared with the vector control, overexpression of Nanog increased the number of migrating cells in MSC-CM-treated cultures by more than 2-fold (Fig. 4C, left; from 11.7% to 27.1%), whereas Snail- and Slug-overexpressing lines presented a 50% increase in migration (from 11.7% to 18.0% and 17.4%, respectively). However, compared with control medium-treated cells, exposure to MSC-CM still induced a pronounced reduction in migratory cell number, making the slight rescue effect induced by Nanog, Snail, and Slug overexpression scarcely noticeable (Fig. 4C, left). These data suggested that enhancing Nanog, Snail, or Slug levels is not sufficient to render cells resistant to the MSC-CM-induced inhibition of mobility. Similar results were observed in the wound-healing assay. Though Nanog-overexpressing cell lines showed better mobility than control cell lines, a 24-hour treatment with MSC-CM still dramatically reduced their migratory capacity and abrogated the significant difference between control and Nanog-expressing cells (Fig. 4C, right). Western blot analysis of the CL1-5 stable cell lines showed that the ectopically expressed Nanog and Slug were suppressed by MSC-CM, whereas E-cadherin expression was elevated (Fig. 4D). Together, these data indicated that MSC-CM promotes the MET process in LAC cells by suppressing EMT markers and the Nanog stemness factor.

Figure 4.

MSC-CM inhibited the migratory ability of Nanog-, Snail-, and Slug-overexpressing LAC cells. A, CL1-5 cells were subjected to Western blotting analysis (right) after 24 hours of incubation in CM or control medium. Micrographs show the morphologic changes induced by CM treatment (left). B, Western blot analysis of Nanog, Snail, and Slug expression in A549 and CL1-5 stable cell lines. C, CL1-5 stable cell lines were subjected to the Transwell migration assay in the presence of MSC-CM or control medium. The results are presented as the percentage of migratory cells treated with MSC-CM relative to the number of migratory cells treated with control medium (left). CL1-5-Vec, CL1-5-Nanog, A549-Vec, and A549-Nanog were subjected to a wound-healing mobility assay for 24 hours in the presence of MSC-CM or control medium (right). D, the aforementioned CL1-5 stable cell lines were treated with CM or control medium for 24 hours, and Western blottings were carried out to analyze protein expression.

Figure 4.

MSC-CM inhibited the migratory ability of Nanog-, Snail-, and Slug-overexpressing LAC cells. A, CL1-5 cells were subjected to Western blotting analysis (right) after 24 hours of incubation in CM or control medium. Micrographs show the morphologic changes induced by CM treatment (left). B, Western blot analysis of Nanog, Snail, and Slug expression in A549 and CL1-5 stable cell lines. C, CL1-5 stable cell lines were subjected to the Transwell migration assay in the presence of MSC-CM or control medium. The results are presented as the percentage of migratory cells treated with MSC-CM relative to the number of migratory cells treated with control medium (left). CL1-5-Vec, CL1-5-Nanog, A549-Vec, and A549-Nanog were subjected to a wound-healing mobility assay for 24 hours in the presence of MSC-CM or control medium (right). D, the aforementioned CL1-5 stable cell lines were treated with CM or control medium for 24 hours, and Western blottings were carried out to analyze protein expression.

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Oncostatin M mediated the MSC-dependent inhibition of cell proliferation and migration

The inhibitory effects of MSC-CM on LAC cells suggested the involvement of a paracrine mechanism mediated by molecules secreted from MSCs. We used a cytokine array to determine the compositional differences between control medium and MSC-CM. Within the array, the anticancer cytokine OSM presented a more than 3-fold difference (Fig. 5A, left). An ELISA assay also detected secreted OSM in the CM derived from 3 MSCs compared with control medium (Supplementary Fig. 3A). Moreover, the OSM mRNA was detected in MSCs by quantitative real-time PCR and visualized in agarose gel (Supplementary Fig. 3B and C). OSM is a multifunctional cytokine that inhibits the growth of many types of tumor cells (25, 28, 41, 42). Quantitative real-time PCR analysis of OSM receptor expression in LAC cells indicated that the level of OSM-specific receptor subunits (LIFR and OSMR-β) was higher than that of IL6 (IL6R), a cytokine reported to mediate MSC-dependent regulation of cancer cells (Fig. 5A, middle). Treating LAC cells with CM further enhanced the expression of the 2 OSM receptor subunits, OSMR-β and LIFR (Fig. 5A, right), which may magnify cellular sensitivity to OSM.

Figure 5.

OSM contributed to the MSC-mediated inhibition of LAC migration and promoted the MET. A, MSC-CM and control medium were subjected to a cytokine array analysis using a commercial membrane-bound array kit (left). The expression levels of the OSM receptor (OSM-R) subunits (LIFR and OSMR-β), the IL-6 receptor (IL6-R) subunit (IL6R), and their common receptor subunit (gp130) were analyzed by quantitative real-time PCR. The data are presented as relative log2-fold change relative to the 18S RNA expression level (middle). CL1-5 cells were incubated in CM or control medium for 24 hours and then subjected to quantitative real-time PCR analysis to determine the OSM-R subunits expression levels. The relative mRNA levels of each subunit in CM-treated cells are presented relative to the level measured in control medium-treated cells (right). B, CL1-5 and A549 cells were subjected to the Transwell migration and invasion assays after a 24-hour treatment with or without OSM (5 or 20 ng/mL). The percentages of migratory or invasive cellsin OSM-treated cultures compared with untreated controls are shown in the graphs (top). The CM was pretreated with control IgG or various amounts of anti-OSM (aOSM; 0.5 and 2 μg/mL for A549, 2 and 5 μg/mL for CL1-5) antibodies for 2 hours before it was applied to CL1-5 and A549 cells. Migration and invasion activities were measured 24 hours after incubation, and the results are presented as percentages relative to control medium-treated cells (bottom). C, CL1-5 treated with control medium, CM, OSM, or OSM-neutralized CM were subjected to immunofluorescent staining of E-cadherin, vimentin, and nucleus, and observed by confocal microscope. D, the morphology of CL1-5 cells after 24 hours of treatment with or without OSM is shown (left). CL1-5 and A549 cells were treated with or without recombinant OSM (20 ng/mL) for the indicated durations and then subjected to Western blot analysis to determine the expression levels of EMT-related proteins (middle). CL1-5 cells with stable knockdown of STAT1 were treated with or without OSM (20 ng/mL) for 24 hours, followed by Western blot analysis of the indicated proteins (right).

Figure 5.

OSM contributed to the MSC-mediated inhibition of LAC migration and promoted the MET. A, MSC-CM and control medium were subjected to a cytokine array analysis using a commercial membrane-bound array kit (left). The expression levels of the OSM receptor (OSM-R) subunits (LIFR and OSMR-β), the IL-6 receptor (IL6-R) subunit (IL6R), and their common receptor subunit (gp130) were analyzed by quantitative real-time PCR. The data are presented as relative log2-fold change relative to the 18S RNA expression level (middle). CL1-5 cells were incubated in CM or control medium for 24 hours and then subjected to quantitative real-time PCR analysis to determine the OSM-R subunits expression levels. The relative mRNA levels of each subunit in CM-treated cells are presented relative to the level measured in control medium-treated cells (right). B, CL1-5 and A549 cells were subjected to the Transwell migration and invasion assays after a 24-hour treatment with or without OSM (5 or 20 ng/mL). The percentages of migratory or invasive cellsin OSM-treated cultures compared with untreated controls are shown in the graphs (top). The CM was pretreated with control IgG or various amounts of anti-OSM (aOSM; 0.5 and 2 μg/mL for A549, 2 and 5 μg/mL for CL1-5) antibodies for 2 hours before it was applied to CL1-5 and A549 cells. Migration and invasion activities were measured 24 hours after incubation, and the results are presented as percentages relative to control medium-treated cells (bottom). C, CL1-5 treated with control medium, CM, OSM, or OSM-neutralized CM were subjected to immunofluorescent staining of E-cadherin, vimentin, and nucleus, and observed by confocal microscope. D, the morphology of CL1-5 cells after 24 hours of treatment with or without OSM is shown (left). CL1-5 and A549 cells were treated with or without recombinant OSM (20 ng/mL) for the indicated durations and then subjected to Western blot analysis to determine the expression levels of EMT-related proteins (middle). CL1-5 cells with stable knockdown of STAT1 were treated with or without OSM (20 ng/mL) for 24 hours, followed by Western blot analysis of the indicated proteins (right).

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To investigate the effect of OSM on LAC cells, we first carried out a WST-1 cell proliferation assay on CL1-5 and A549 cells treated with different concentrations of recombinant OSM. In line with our previous findings, OSM treatment inhibited cell proliferation: 3 days of 20 ng/mL OSM treatment reduced CL1-5 and A549 cell numbers by 40% and 25%, respectively, compared with untreated controls (Supplementary Fig. 4). Although OSM has been reported to inhibit the growth of many types of tumor cells (25, 28, 41, 42), its role in metastasis is less well understood. We determined that addition of OSM to the culture medium significantly inhibited cell migration and invasion of both LAC cell lines (Fig. 5B, top), as well as H2170 and H520 squamous carcinoma cell lines (Supplementary Fig. 5), in a dose-dependent manner. The use of a specific antibody against OSM (aOSM, R&D Systems) to neutralize the OSM secreted into the MSC-CM blocked the inhibitory effect of MSC-CM and increased the migration and invasion of both LAC cell lines compared with control anti-IgG-treated MSC-CM (Fig. 5B, bottom). Immunofluorescent staining of the CL1-5 cells treated with CM, OSM or OSM-neutralized CM (CM+aOSM) indicated that OSM, like CM, elevated E-cadherin and suppressed vimentin expression, whereas antibody against OSM blocked these effects of MSC-CM (Fig. 5C). OSM also affected cell morphology: 24 hours of OSM treatment induced an epithelial-like phenotype in CL1-5 cells (Fig. 5D, left). Western blot analysis of OSM-treated LAC cells showed that OSM inhibited the expression of Nanog, Snail, and Slug but elevated E-cadherin after 24 hours of treatment, suggesting an enhanced MET process in both LAC cell lines (Fig. 5D, middle). STAT1 is an important downstream target of OSM-dependent pathways. Activation of STAT1 has been reported to be critical for the efficacy of anti-metastatic immunotherapies (43). The anticancer effect of OSM against chondrosarcoma is thought to work through the JAK3/STAT1 pathway (27). The activation of STAT1 upon OSM treatment was confirmed in the LAC cells (Supplementary Fig. 6A). We then specifically knocked down STAT1 or STAT3, another downstream target of OSM, using lentiviral expressed short hairpin RNAs. After puromycin selection for 2 weeks, the knocked down pool of cells were treated with or without OSM (Fig. 5D, right; Supplementary Fig. 6C). Western blotting indicated that without OSM treatment, knockdown of STAT1 had little effect on Nanog or Slug protein levels. However, with OSM treatment, knockdown of STAT1, but not STAT3, resulted in elevated levels of Nanog, Snail, and Slug compared with a scramble control (SC). These data indicated that the OSM-dependent inhibition of Nanog and elevation of MET signaling may be mediated by a STAT1-dependent pathway.

OSM reduced LAC tumor growth and metastasis in vivo

To investigate the anticancer effect and therapeutic potential of OSM in vivo, we pre-treated luciferase-labeled CL1-5 cells with control medium, CM, OSM, or OSM-preneutralized CM (CM+aOSM) and transplanted the cells into immunocompromised mice. As shown in Fig. 6A, tumors appeared 2 weeks after transplantation in mice transplanted with control medium-treated cells, whereas the luciferase signals in mice transplanted with CM- or OSM-treated cells were barely detectable or significantly lower. Interestingly, preneutralizing OSM in the CM with a specific antibody reduced the inhibitory effect of the CM and resulted in an elevated luciferase tumor signal compared with CM-treated mice (Fig. 6A). The same result was also observed in mice transplanted with A549 cells (Fig. 6B, left; Supplementary Fig. 7A). Immunofluorescent staining of the tumor sections indicated that CM reduced the population of proliferating cells (Supplementary Fig. 7B). Treatments of CM or OSM increased E-cadherin (Fig. 6B, right) but suppressed vimentin (Supplementary Fig. 7C) expression level in the subcutaneous tumors. To evaluate the anti-metastatic effect of OSM, we conducted a tail vein injection of pretreated CL1-5 and A549 cells in immunocompromised mice. Two weeks after transplantation, control medium, CM, OSM, or CM+aOSM was injected into the mice 4 times at 7-day intervals. The tumor nodules in the lungs were counted and shown in Fig. 6C. The data clearly showed that CM and OSM reduced the number and volume of metastatic tumor nodules, whereas exposure to CM+aOSM abrogated the inhibitory effect. Histochemical staining of the lung sections from CL1-5-injected mice showed that CM or OSM treatment reduced the spread of tumors in lung tissue (Fig. 6C, left). Quantitative real-time PCR analysis of the tumor tissues from the A549-injected mice showed that CM and OSM elevated E-cadherin and suppressed Slug mRNA levels, which were reversed by CM-aOSM (Supplementary Fig. 7D). Taken together, these data showed that OSM mediated the CM-dependent elevation of MET and suppression of tumor growth and metastasis in vivo.

Figure 6.

OSM suppressed xenograft tumor growth and metastasis in an animal model. A, immunocompromised mice were subcutaneously transplanted with CL1-5-Luc cells pew-treated with control medium, CM, OSM (20 ng/mL), or OSM-neutralized CM (CM+aOSM; CM preincubated with 2 μg/mL aOSM for 1 hour). On day 5 and day 10, the same treatments were applied through peritumoral injections. Tumor volumes were monitored with an IVIS Lumina Live Imager, and the graph shows the mean ± SD of luciferase signal intensity on day 14 (n = 3). B, a similar experiment as described in A was conducted with A549 cells. The tumor volumes were measured by a caliper on day 58 (left, n = 3). Tumor sections were stained with anti-E-cadherin and Hoechst33258, and observed under confocal microscope (right). C, immunocompromised mice were transplanted through tail veins with CL1-5 or A549 cells pretreated as indicated. Two weeks after transplantation, control medium (100 μL), CM (100 μL), OSM (20 ng in 100 μL of control medium), or OSM-neutralized CM (CM+aOSM; 100 μL of CM preincubated with 2 μg/mL aOSM for 1 hour) was injected into the mice 4 times at 7-day intervals through intraperitoneal injection. Mice were sacrificed 3 months after transplantation. Tumor formation and histochemical staining of the CL1-5 tumor sections in lung were photographed (left). The numbers of metastatic nodules in the lung was counted (top right), and the tumor volume in lung was calculated from the histographs using the formula (length × width2)/2 (bottom right).

Figure 6.

OSM suppressed xenograft tumor growth and metastasis in an animal model. A, immunocompromised mice were subcutaneously transplanted with CL1-5-Luc cells pew-treated with control medium, CM, OSM (20 ng/mL), or OSM-neutralized CM (CM+aOSM; CM preincubated with 2 μg/mL aOSM for 1 hour). On day 5 and day 10, the same treatments were applied through peritumoral injections. Tumor volumes were monitored with an IVIS Lumina Live Imager, and the graph shows the mean ± SD of luciferase signal intensity on day 14 (n = 3). B, a similar experiment as described in A was conducted with A549 cells. The tumor volumes were measured by a caliper on day 58 (left, n = 3). Tumor sections were stained with anti-E-cadherin and Hoechst33258, and observed under confocal microscope (right). C, immunocompromised mice were transplanted through tail veins with CL1-5 or A549 cells pretreated as indicated. Two weeks after transplantation, control medium (100 μL), CM (100 μL), OSM (20 ng in 100 μL of control medium), or OSM-neutralized CM (CM+aOSM; 100 μL of CM preincubated with 2 μg/mL aOSM for 1 hour) was injected into the mice 4 times at 7-day intervals through intraperitoneal injection. Mice were sacrificed 3 months after transplantation. Tumor formation and histochemical staining of the CL1-5 tumor sections in lung were photographed (left). The numbers of metastatic nodules in the lung was counted (top right), and the tumor volume in lung was calculated from the histographs using the formula (length × width2)/2 (bottom right).

Close modal

Tumor progression with metastasis is one of the major causes of mortality in lung cancer patients and therefore represents an important clinical challenge. EMT has been considered a key mechanism responsible for the metastatic progression of lung cancer (44). Enhanced EMT characteristics are associated with poor overall and metastasis-free survival in patients with non–small cell lung cancer (11). Targeting the EMT pathway or enhancing MET has been proposed as a promising therapeutic method to address cancer metastasis and improve patient survival (45). Slug and Snail, EMT-related transcription factors, have been reported to increase the metastatic risk of lung cancer (11, 12, 15). In addition, stemness factors like Nanog and Oct4 are also involved in EMT regulation and are correlated with a poor clinical outcome for LAC patients (40). In this report, we showed that BM-MSCs isolated from 3 different donors inhibited the migration, invasion, proliferation, and cell-cycle progression of LAC cells through a paracrine mechanism. The EMT regulators Nanog, Snail, and Slug were all inhibited and the MET markers CK-18 and E-cadherin were elevated by MSC-CM, resulting in elevated MET process in LAC cells. The inhibitory effect on cell migration was also observed in endometrium-derived MSCs (Supplementary Fig. 2), suggesting that this effect on LAC cells may share in different types of MSCs. In an animal model, both MSCs and MSC-CM suppressed LAC tumor growth and metastasis; the treatment of MSC-CM enhanced MET and suppressed proliferation in the xenograft tumor tissues. Our data suggested that molecules secreted from MSCs may have a therapeutic potential for lung cancer treatment.

OSM is a regulator of stem cell pluripotency and modulates the differentiation of specific lineages. OSM induces osteogenesis in MSCs (23) and is involved in the differentiation of hepatocytes from human ESCs (22, 46, 47). OSM has also been reported to inhibit the proliferation of several tumor cell lines, including melanoma, glioblastoma, and breast cancer cells (21, 24–28). Yamashita and colleagues further showed that OSM renders liver cancer stem cells sensitive to chemotherapy by inducing hepatocyte differentiation (21). However, the effect of OSM on cancer metastasis is less understood. Lacreuseette and colleagues reported that some metastatic melanoma cells lost the expression of OSMR-β (48), implying a role for OSM in cancer metastasis. In the present study, we identified OSM as a mediator of the MSC-dependent inhibition of LAC tumorigenicity. Interestingly, MSC-CM treatment increased the mRNA expression of OSMR-β, which could be a mechanism to magnify the OSM pathway. The increased OSMR-β might be possibly due to the OSM in MSC-CM, as Blanchard and colleagues reported that OSM triggers the synthesis of OSMR-β (49), though we did not rule out the involvements of other soluble factors in regulating OSMR-β expression. We showed that OSM inhibited LAC cell proliferation and reduced xenograft tumor size in animal model. Moreover, OSM suppressed cell mobility and tended to induce MET in LAC cells; mice transplanted with OSM-incubated LAC cells and treated with OSM showed reduced metastatic tumor nodules in the lungs, along with increased pro-MET parkers and suppressed pro-EMT markers in tumor tissues. More importantly, preneutralizing OSM in MSC-CM reduced the effects of MSC-CM on LAC cells. It should be noted that there may be other cytokines also contributing to the MSC-CM-inhibited tumor progression, as neutralizing OSM in MSC-CM did not completely block this inhibition. Given that there is a gap in OSM concentrations between high-CM and the recombinant OSM we used, we suspected a synergistic cooperation of cytokines in MSC-CM, presuming that natural and recombinant OSM have equal efficacy. Potential candidates are currently under investigation. The downstream effector of OSM, STAT1, has been linked to anti-metastasis signaling in melanoma cells (43). We showed that knockdown of STAT1, but not STAT3 (Supplementary Fig. 6C), hampered the OSM-mediated inhibition of Nanog and Slug, suggesting a role for STAT1 in the OSM-regulated induction of MET involved in the metastasis of LAC cells. We also showed that knockdown of STAT1 resulted in increased cell mobility in LAC cells (Supplementary Fig. 6B). Further studies are needed to dissect the signaling pathways in LAC cells that are activated or inactivated in response to extracellular OSM stimulation.

The relationship between MSCs and tumor cells is still controversial (50). For example, BM-MSCs have been reported to promote colon cancer growth by enhancing angiogenesis and inhibiting tumor cell apoptosis (33). Direct coculture of BM-MSCs with breast cancer cells suggested that BM-MSCs stimulate the EMT in breast cancer cells and enhance cancer metastasis (32). On the contrary, studies of liver cancer indicated that human MSCs inhibited the cell-cycle progression, tumorigenesis, and metastasis of hepatocellular carcinoma (34, 35), possibly through downregulated TGF-β or Wnt signaling (34, 35). These discordant studies suggest a cancer type-dependent mechanism for MSC-mediated pro- or antitumor effects. MSCs are the source of many chemokines and cytokines that can be anti- or pro-tumorigenic and contribute to a state of immunotolerance (50). The complexity of their interactions with tumor cells, the large range of cytokines and growth factors they produce, and the receptors expression levels of the tumor cells might also explain the inconsistent effects of MSCs on tumorigenicity. Bergfeld and colleagues have stated that MSCs can be the friends or foes of cancer cells, depending on their origin, degree of differentiation, and the type of tumor cells with which they interact (50). Our investigation of the interactions between MSCs and LAC cells not only revealed inhibitory effects of MSCs on lung cancer growth and metastasis but also provided insight into how this crosstalk is mediated. We proposed a model in which MSC-derived OSM signals in a paracrine manner to LAC cells, resulting in increased MET, and thereby suppress tumorigenicity. Further studies are needed to evaluate the prospect of the extracellular cytokine-MET signaling pathway as a target for therapeutic development.

No potential conflicts of interest were disclosed.

Conception and design: M.-L. Wang, S.-H. Chiou

Development of methodology: M.-L. Wang, C.-M. Pan, S.-H. Chiou, O.K.-S. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.-L. Wang, C.-M. Pan, W.-H. Chen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.-L. Wang, C.-M. Pan, W.-H. Chen, O.K.-S. Lee

Writing, review, and/or revision of the manuscript: M.-L. Wang, C.-M. Pan, S.-H. Chiou, C.-W. Wu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.-L. Wang, C.-M. Pan, H.-Y. Chang, H.-S. Hsu

Study supervision: C.-W. Wu

We thank Dr. Oscar Kuang-Sheng Lee and Dr. Shih-Hwa Chiou for providing bone marrow- and endometrium-derived MSCs, respectively. We acknowledge the Taiwan Mouse Clinic funded by the National Research Program for Biopharmaceuticals (NRPB) at the National Science Council (NSC) of Taiwan for the animal core facility; the National RNAi Core Facility in Academia Sinica (NSC 97-3112-B-001-016) for lentiviral shRNA clones.

This research was supported by the Institute of Biomedical Sciences, Academia Sinica, the National Yang-Ming University, the Department of Health (DOH101-TD-C-111-007), and the National Science Council (NSC100-2321-B-010-020; NSC100-2325-B-010-011; NSC100-2321-B-010-021), Executive Yuan, Taiwan, R.O.C.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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