Pleiotropic effects of cancer cells’ secreted factors on human stromal (mesenchymal) stem cells
© Al-toub et al.; licensee BioMed Central Ltd. 2013
Received: 24 February 2013
Accepted: 12 September 2013
Published: 17 September 2013
Studying cancer tumors’ microenvironment may reveal a novel role in driving cancer progression and metastasis. The biological interaction between stromal (mesenchymal) stem cells (MSCs) and cancer cells remains incompletely understood. Herein, we investigated the effects of tumor cells’ secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs.
Morphological changes were assessed using fluorescence microscopy. Changes in gene expression were assessed using Agilent microarray and qRT-PCR. GeneSpring 12.1 and DAVID tools were used for bioinformatic and signaling pathway analyses. Cell migration was assessed using a transwell migration system. SB-431542, PF-573228 and PD98059 were used to inhibit transforming growth factor β (TGFβ), focal adhesion kinase (FAK), and mitogen activated protein kinase kinase (MAPKK) pathways, respectively. Interleukin-1β (IL1β) was measured using ELISA.
MSCs exposed to secreted factors present in conditioned media (CM) from FaDu, MDA-MB-231, PC-3 and NCI-H522, but not from MCF7 and HT-29, developed an elongated, spindle-shaped morphology with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK severely impaired the pro-inflammatory response of MSCs to tumor CM (approximately 80% to 99%, and 55% to 88% inhibition, respectively), while inhibition of the TGFβ pathway was found to promote the pro-inflammatory response (approximately 3-fold increase). In addition, bioinformatics and pathway analysis of gene expression data from tumor cell lines combined with experimental validation revealed tumor-derived IL1β as one mediator of the pro-inflammatory phenotype observed in MSCs exposed to tumor CM.
MSCs exhibited significant tropism toward secreted factors from the aforementioned tumor cell lines, while both normal and MSCs exposed to tumor CM were capable of attracting human peripheral blood mononuclear cells (PBMCs).
Our data revealed tumor-derived IL1β as one mediator of the pro-inflammatory response in MSCs exposed to tumor CM, which was found to be positively regulated by FAK and MAPK signaling and negatively regulated by TGFβ signaling. Thus, our data support a model where MSCs could promote cancer progression through becoming pro-inflammatory cells within the cancer stroma.
Stromal (mesenchymal) stem cells (MSCs), also referred to as stromal cells, are multipotent cells which are present within the stroma of bone marrow and probably other organs and capable of differentiating into the three canonical lineages: osteoblasts, adipocytes and chondrocytes. Aside from their differentiation potential, MSCs are also capable of migrating to injured tissues and contributing to tissue regeneration[2–4]. Emerging data suggest that MSCs possess immunomodulatory and regenerative properties as they can secrete a large number of growth factors and immune active molecules that can improve tissue survival and suppress the activity of various immune cells, such as alloantigen activated T and B lymphocytes[6, 7]. Moreover, MSCs can secrete a large number of paracrine factors, including chemoattractants for endothelial cells, monocytes and macrophages. Several recent studies have reported that bone marrow MSCs migrate to the stromal compartment of tumors[9, 10] and that a dynamic interaction between tumor cells and MSCs exists resembling what has been reported during inflammation and, thus, ‘tumors are wounds that never heal’.
Over the past several years, a significant amount of research has emerged documenting a role for MSCs in promoting epithelial-to-mesenchymal transition (ETM) and accelerating tumor growth and metastasis[9, 12–14]. In addition, MSCs are being introduced into therapy for a number of clinical indications and there is a concern of possible promoting effects on tumor growth by MSCs. On the other hand, several other studies reported that MSCs exert tumor suppressive effects[16–18]. Therefore, understanding the settings under which MSCs exert promoting versus inhibitory effects on tumor growth and metastasis is currently under intensive investigation.
Given this complex interplay between MSCs and tumor cells, the goal of this study was to assess the cellular and molecular changes in MSCs in response to secreted factors present in conditioned media (CM) from a panel of human tumor cell lines covering a spectrum of human cancers (breast, prostate, lung, colon, and head and neck). Integrated analysis of phenotypic changes, gene expression and bioinformatics revealed a pro-inflammatory response of MSCs when exposed to CM of several tumor cell lines. Interestingly, the biological responses of MSCs were not identical. MSCs responded mainly to tumor cell lines which express high levels of IL1β. We identified tumor-derived IL1β as the prominent cytokine responsible for induction of inflammatory response in MSCs and signaling via focal adhesion kinase (FAK) and, to lesser extent, mitogen activated protein kinase kinase (MAPKK), as key positive regulators of an inflammatory response, while transforming growth factor β (TGFβ) signaling was found to inhibit the response of MSCs to tumor CM. Our data further support a model where MSCs could drive tumorigenicity through induction of inflammation.
Experiments performed in this study do not need ethics committee approval.
Tumor cell lines used in this study (breast, MCF7 and MDA-MB-231; prostate, PC-3; lung, NCI-H522; head and neck, FaDu; and colon, HT-29) have been described previously[19–23]. The human telomerized hMSC-TERT-GFP cell line was developed by Dr Kassem, Odense, Denmark[24, 25]. All cell lines were maintained in (D)MEM 4.5g/L glucose (Invitrogen Corp., Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum, 1% NEAA, 1% L-glutamine, 100 mg/L penicillin and 100 mg/L streptomycin at 37°C and 5% CO2. For TGFβ inhibition experiments, MSC were cultured as described above and were exposed to MDA-MB-231 CM in the presence of 10 μM SB-431542 (Sigma, St. Louis, MO, USA). Control wells were treated with dimethyl sulfoxide (DMSO). CM plus SB-431542 or vehicle (DMSO, Sigma) was changed every three to four days for the duration of the experiment. Recombinant human IL1β and IL6 were purchased from Invitrogen. FAK inhibitor (PF-573228) and mitogen activated protein kinase kinase (MAPKK) inhibitor (PD98059) were purchase from Sigma and were reconstituted in DMSO.
Collection of tumor cell lines conditioned media
The tumor cell lines, MCF7, HT-29, MDA-MB-231, PC-3, NCI-H522 and FaDu were seeded in six-well plates at 1 × 106/well (4 ml total) in (D)MEM supplemented with 10% fetal bovine serum (FBS), 1% NEAA and 1% penicillin/streptomycin and incubated at 37°C and 5% CO2. Forty-eight hours later (cells were approximately 90% confluent), CM from the tumor cell lines were collected and spun down at 300 × g for 10 minutes to remove any cellular content and debris. In some experiments, CM was passed through a 0.45 μΜ filter to remove any remaining cellular content and debris. The hMSC-TERT-GFP cells were then seeded in 24-well plates at 8 × 104/ml in the collected CM (80% tumor CM + 20% fresh medium). The MSCs were exposed to fresh CM every two to three days for the duration of the experiment.
Quantification of secreted IL1β using ELISA
Quantification of secreted IL1β from tumor cell lines or from MSCs exposed to tumor CM was done using the LEGEND MAX™ Human IL-1β ELISA Kit (Biolegened Inc., San Diego, CA, USA) according to the manufacturer’s recommendations. CM from tumor cell lines were collected as described above and stored at −80°C for the ELISA. To measure secreted IL1β from control MSCs or MSCs exposed to tumor CM, MSCs were exposed to MCF7 or FaDu CM for seven days. Subsequently, the cells were washed three times with PBS and fresh culture medium was added. CM was collected for the ELISA 72 hours later.
Microscopy was performed on the indicated days using a Nikon® ECLIPSE Ti-U inverted fluorescence microscope. Cells were either imaged directly or were washed with 1x PBS, followed by staining with Hoechst 33342 (10 μg/ml) in PBS for 10 minutes at 37°C.
Human MSCs were exposed to FaDu tumor CM as described above. On day 7, when the spindle-shape phenotype was usually observed, the cells from three different replicates were harvested and RNA was extracted using the Roche MagNA Pure automated nucleic acid purification system (Roche Diagnostics GmbH, Mannheim, Germany). RNA quantity and quality were measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Control RNA was collected from the same batch of MSCs exposed to normal medium. Extracted RNA was labeled and then hybridized to the Agilent Human GE 4x44K v2 Microarray chip (Agilent Technologies, Santa Carla, CA, USA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine, Riyadh, Saudi Arabia). Data analyses were conducted using GeneSpring X software (Agilent Technologies) and the DAVID bioinformatic tool as described previously. Microarray data were deposited in the Gene Expression Omnibus (GEO) database (accession number GSE50722).
Quantitative real-time polymerase chain reaction
Primer sequences used for qRT-PCR
5’ AAGGACACGGGCAGCAGACA 3’
5’ AGCAGCAAGTGATGCAGAGAACTGG 3’
5’ TACATTGCCCGCCCACTGCC 3’
5’ TCGGGTGACAAAGACGACTGCT 3’
5’ GGGACTTGCTCAGCCAGATTCAGT 3’
5’ CAGCACAGACCTCCTTGCCCC 3’
5’ GGGGTTCGCCGTTCTCGGA 3’
5’ TGCGAGGAGGAGAGCTGGCAA 3’
5’ CGCCCAAACCGAAGTCATAGCCA 3’
5’ TGGTAAGGGCAGGGACCACCC 3’
5’ GTTGAGAGAGCTGCGTTGCGT 3’
5’ TCAGGGAGGCTACCACTTCCACC 3’
5’ GGTAAACTGCAGGTGTTCCCCGC 3’
5’ CCCGTTCTTCAGGGAGGCTACCA 3’
5’ CGAGCCCACCGGGAACGAAA 3’
5’ GGACCGAAGGCGCTTGTGGAG 3’
5’ AGGCACAAGGCACAACAGGCT 3’
5’ TGGCTGCTTCAGACACTTGAGCAAT 3’
5’ GCTCTGCCCCGTCGCACATT 3’
5’ TTGGTGTCTGGAAGCCGGCGA 3’
5’ GGCATGGGGTTGCCGGAGAG 3’
5’ CTGGAAACATTGCACGTGGAGTAGC 3’
5’ GACCTCCAGGAGCCGACCTGC 3’
5’ AGTTTCCGGGGTTGGAGGGCA 3’
5’ GGCTTTTGATGGGGCTCGGGA 3’
5’ CCCCCAGGTCCCCTTTTGGC 3’
5’ TGCCCGTGGAGCTCATGGAGAC 3’
5’ TGTGCATCCCCTCCAATGCCAG 3’
In vitro angiogenesis assay
An in vitro angiogenesis assay was conducted as we described previously. MSCs were seeded in a 24-well plate at 8 × 104/well in normal or CM from FaDu or MDA-MB-231 cell lines. On day 10, a 24-well plate was prepared for the matrigel assay by adding 250 μl of chilled Matrigel® (10 mg/mL, Basement Membrane Matrix, BD Biosciences, San Diego, CA, USA) for each well, and then the plate was incubated at 37°C for 30 minutes. MSCs exposed to CM or control were trypsinized and cultured in 24-well plates pre-coated with Matrigel® at 1 × 105 in 500 μl of media. Images were taken at 2 hours and 72 hours using a Nikon® ECLIPSE Ti-U inverted fluorescence microscope.
Adipogenic and osteoblastic differentiation
MSCs were seeded in a 24-well plate at 8 × 104/well in normal or CM from FaDu or MDA-MB-231 cell lines. On day 10, cells were switched to adipogenic ((D)MEM supplemented with 10% FBS, 10% horse serum (Sigma), 1% penicillin/streptomycin, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine ((IBMX) (Sigma)), 3 μg/mL insulin (Sigma) and 1 μM rosiglitazone ((BRL49653) (Novo Nordisk, Bagsvaerd, Denmark)) or osteogenic ((D)MEM containing 10% FBS, 1% penicillin/streptomycin, 50 μg/mL L-ascorbic acid (Wako Chemicals GmbH, Neuss, Germany), 10 mM β-glycerophosphate (Sigma), and 10 nM calcitriol ((1α,25-dihydroxy vitamin D3) (Sigma)), 10 nM dexamethasone (Sigma)) differentiation medium as we previously described. Medium was changed every three days. On day 6, adipocytic and osteoblastic differentiation was measured using Oil-Red-O and alkaline phosphatase (ALP) staining, respectively.
Transwell cell migration assay
On the day of the experiment, tumor cells were trypsinized and counted using an automated cell counter (Vi-Cell XR cell viability analyzer, Beckman Coulter Inc, Fullerton, CA, USA). Subsequently, 4 × 105 cells were seeded in 2 ml of low serum (D)MEM ((D)MEM + 1% FBS, 1% NEAA, 1% penicillin/streptomycin) in the lower chamber of a 12-well transwell migration system (BD Biosciences). Twenty four hours later, 1 × 105 hMSC were re-suspended in 1 ml of low serum (D)MEM in the upper chamber. MSC migration toward (D)MEM supplemented with 1% FBS was used as a negative control. Twenty four hours later, inserts were removed, and cells on the upper surface were scraped using a cotton swap, and, subsequently, were fixed with 4% Paraformaldehyde (PFA) for 20 minutes, followed by H & E staining. Stained inserts were subsequently cut and mounted on microscope slides. Digital slides were taken using a digital microscope and eight (1600 × 1000 mcM2) fields were counted from each insert. For leukocyte migration, MSCs were exposed to tumor CM for seven days. Subsequently, wells were washed and fresh (D)MEM + 0.5% BSA was added. CM from control MSCs ((D)MEM + 0.5% BSA) or MSCs exposed to FaDu CM ((D)MEM + 0.5% BSA) was collected 72 hours later and used in the migration experiment. Human peripheral blood mononuclear cells (PBMCs) (1 × 105) were seeded in the upper chamber, while control medium or MSC CM was placed in the lower chamber. Two hours later, images of migrating cells were taken using a Zeiss inverted microscope.
Statistical analyses and graphing were performed using Microsoft® excel 2007 and Graphpad Prism 6.0 software (Graphpad® software, San Diego, CA, USA). P values were calculated using the two-tailed t-test. Correlative analyses were done using Pearson’s correlation using Graphpad prism 6.0.
Effects of conditioned media on MSCs morphology and gene expression
This striking finding led us to hypothesize that secreted factors from FaDu tumor cells mediated biological changes in MSC phenotype and gene expression. To identifiy those genetic changes, we conducted global gene expression analysis of MSCs exposed to FaDu CM compared to control MSCs cultures. Microarray data and pathway analyses of the upregulated genes revealed significant enrichment for genes involved in inflammatory response-related cytokines and chemokines, for example, IL1β, CSF2, CSF3, IL6, CXCL2, CXCL1, IL13 and IL1α, as well as metalloproteinases (Figure 1b, c, and Additional file1: Table S1).
Effects of CM from tumor cell lines on MSC morphology and gene expression is cell line-dependent
Pro-inflammatory response of MSCs exposed to FaDu CM is mediated mainly through focal adhesion kinase signaling
Signaling via TGFβ negatively regulates the pro-inflammatory response of MSCs to FaDu CM
MSCs exposed to tumor CM have diminished multilineage differentiation potential
Clustering analysis of tumor cell lines gene expression profile
IL1β treatment induced a pro-inflammatory phenotype in MSCs similar to that induced by tumor CM
MSCs exhibited significant tropism toward different tumor cell lines in vitro
Control MSCs or MSCs exposed to tumor CM are capable of attracting human PBMCs
For several decades, the molecular changes within tumor cells were studied in order to understand factors responsible for promoting tumor progression and metastasis, while little attention was paid to the possible contributory role of tumor microenvironment. Recent evidence suggests that the tumor microenvironment, which is composed of a very complex network of extracellular matrix (ECM) proteins and many cell types, such as endothelial cells, stromal (mesenchymal) stem cells, pericytes, fibroblasts and immune cells, plays a critical role in tumor progression and metastasis[36, 37]. Among these components, MSCs have been the focus of intensive investigation[9, 17, 38–45].
In the present report, we examined the crosstalk between tumor cells and MSCs and we investigated the effect(s) of tumor secreted factors on MSCs at the cellular and molecular levels. As surrogates for malignant tumors, we employed a number of well characterized cancer cell lines. We reported that secreted factors from FaDu cells led to significant morphological and genetic changes in MSCs with enhanced expression of pro-inflammatory cytokines, and similar responses were also observed when additional tumor cell lines were evaluated. However, these effects were not universal for all malignant cell lines. For example, MCF7 and HT-29 did not exert these effects. Our findings corroborate recent findings of the presence of morphological and functional changes in mouse MSCs in response to cancer cell lines (MDA-MB-231, PANC-1 and U87) CM, which exhibit a carcinoma-associated fibroblast (CAF)-like myofibroblastic phenotype.
Interestingly, several of the pro-inflammatory molecules identified in the current study have been linked to cancer progression. For instance, cancer cells that overexpress CXCL1 and 2 were found to be more primed for survival at metastatic sites, and are capable of attracting CD11b(+)Gr1(+) myeloid cells into the tumor that enhance cancer cell survival and enhance their chemoresistance and metastatic ability. In addition to that, CXCL2 was also found to be involved in cancer-associated bone destruction. A recent study has reported differentiation of human MSCs into pericyte–like cells upon exposure to glioblastoma tumor CM. In our current study, we observed no evidence of differentiation of MSCs into pericytes or endothelial-like cells using an in vitro angiogenesis assay (Figure 6a). In fact, MSCs exposed to FaDu or MDA-MB-231 CM failed to form any vascular-like tubular networks compared to control MSCs, suggesting MSCs have lost their ability to support angiogenesis. Nonetheless, MSCs exposed to tumor CM also exhibited poor adipocytic and osteoblastic differentiation potential (Figure 6b), probably as a result of differentiation into pro-inflammatory cells. Glioblastoma are known for their high angiogenic capability and the secretion of high levels of VEGF, which might account for the variable effects of CM from breast, lung, prostate, and head and neck cancer models investigated in the current study compared to published glioblastoma data; hence, the response of MSCs to tumor secreted factors can vary depending on the tumor type.
Our gene expression data revealed significant correlation between the expression of a panel of genes involved in inflammation and the metalloprotease pathway (CCL8, CCL5, CXCL6, CXCL5, SAA1, MMP12, EHF, CCL3, CSF2, CXCL3, IL6, IGF2, CXCL2 and IL1b) in MSCs exposed to FaDu and to those exposed to MDA-MB-231, PC-3 and NCI-522 CM, while the expression of these genes was almost unchanged in MSCs exposed to MCF7 CM (Figure 3). These data support our hypothesis of the ability of tumor cells to recruit MSCs to their stroma and which in turn induce inflammation, either directly or through recruiting circulating immune cells (Figure 10b). It seems that this model does not apply to all cancer models since in the MCF7 model, MSCs seemed to promote tumorigenicity via direct interaction with tumor cells (Al-toub et al., in preparation).
Bioinformatics and pathway analysis of gene expression data from tumor cell lines revealed that the phenotypic changes were mostly observed in MSCs exposed to CM from cell lines with a pro-inflammatory nature (such as, FaDu and PC-3, Figure 7c). Indeed our investigation has identified tumor-derived IL1β to be the primary driver of the pro-inflammatory phenotype observed in MSCs exposed to tumor CM, whereas treating MSCs with recombinant IL1β mimicked the effects of tumor CM at the cellular and molecular level (Figures 7d-e and8a-b).
Nonetheless, we also identified signaling via FAK and, to lesser extent, MAPK to be critical for the induction of the observed phenotype (Figure 4). In contrast, pharmacological inhibition of TGFβ signaling in MSCs led to substantial enhancement in the observed changes in phenotype and gene expression in MSCs exposed to MDA-MB-231 CM (Figure 5a and b), which was also associated with a slight increase in cell proliferation [see Additional file5: Figure S3]. Treating MSCs with recombinant TGFβ1 and TGFβ3 in the presence of FaDu CM led to significant inhibition of the observed phenotype at the cellular and molecular level (Figure 5c and d), which further implicated TGFβ signaling in negatively regulating MSC differentiation in response to tumor CM. Thus, our findings corroborate previous studies suggesting a role for the TGFβ signaling pathway in regulating mesenchymal stem cell differentiation.
Our data support an evolving hypothesis that cancer cells secrete a large number of factors regulating biological characteristics of MSCs and transforming MSCs into pro-inflammatory cells. We identified tumor-derived IL1β as one potential mediator of the observed phenotype. Nonetheless, we also identified FAK and MAPK signaling to regulate positively, while TGFβ signaling was found to negatively regulate the response of MSCs to tumor CM. Taken together, our data support a model where MSCs contribute to tumorigenicity through their pro-inflammatory phenotype induced by cancer cell-derived factors, such as IL1β (Figure 10b).
M Al-toub performed the experiments and participated in preparing the manuscript; A Almusa, M Almajid performed the experiments; M Al-Nbaheen characterized MSCs phenotype; M Kassem, A Aldahamsh participated in study design, interpretation of data and preparation of the manuscript. M Kassem characterized and provided hMSC-TERT-GFP cell lines. NM Alajez was responsible for obtaining funding, study design, data interpretation, bioinformatics analysis and preparation of the manuscript. All authors read and approved the final manuscript.
Bovine serum albumin
Chemokine (C-C motif) ligand 3
Chemokine (C-C motif) ligand 5
Chemokine (C-C motif) ligand 8
Colony stimulating factor 2
Colony stimulating factor 3
Chemokine (C-X-C motif) ligand 1
Chemokine (C-X-C motif) ligand 2
Chemokine (C-X-C motif) ligand 3
Chemokine (C-X-C motif) ligand 5
Chemokine (C-X-C motif) ligand 6
(D)ulbecco’s modified Eagle’s medium
Ets homologous factor
Enzyme-linked immunosorbent assay
Epithelial mesenchymal transition
Focal adhesion kinase
Fetal bovine serum
Glyceraldehyde 3-phosphate dehydrogenase
- H & E:
Hematoxylin and eosin
Human mesenchymal stem cell-telomerized-green fluorescence protein
Insulin-like growth factor 2
Interleukin 1, Alpha
Interleukin 1, Beta
Mitogen activated protein kinase
Matrix metallopeptidase 12
Mesenchymal stem cells
Non-essential amino acids
Peripheral blood mononuclear cells
Quantitative real-time reverse-transcription PCR
Serum amyloid A1
Transforming growth factor beta
Vascular endothelial growth factor A.
This work was supported by grant No. 11-MED1582-02 from the National Plan for Sciences and Technology Program, King Saud University, kingdom of Saudi Arabia. We thank Dr Abdulkarim Alhetheel (King Saud University) for providing the PBMCs. We thank Radhakrishnan Vishnubalaji and Dana Hamam (Stem Cell Unit, King Saud University) for their assistance with the in vitro angiogenesis, and the Oil Red O/ALP staining, respectively. We thank Dr Amer Mahmood (Stem Cell Unit, King Saud University) for his assistance with TGFβ inhibition experiments.
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