CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells
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Tumor microenvironment conferred by stromal (mesenchymal) stem cells (MSCs) plays a key role in tumor development, progression, and response to therapy. Defining the role of MSCs in tumorigenesis is crucial for their safe utilization in regenerative medicine. Herein, we conducted comprehensive investigation of the cross-talk between human MSCs (hMSCs) and 12 cancer cell lines derived from breast, prostate, colon, head/neck and skin.
Human bone marrow-derived MSC line expressing green fluorescence protein (GFP) (hMSC-GFP) were co-cultured with the following cancer cell lines: (MCF7, BT-20, BT-474, MDA-MB-468, T-47D, SK-BR-3, MDA-MB-231, PC-3, HT-29, MDA-MB-435s, and FaDu) and changes in their morphology were assessed using fluorescent microscopy. For cellular tracking, cells were labeled with Vybrant DiO, DiL, and DiD lipophilic dyes. Time-lapse microscopy was conducted using Nikon BioStation IM-Q. Stable expression of mCherry, and luciferase genes was achieved using lentiviral technology. IL1-Beta neutralizing experiments were conducted using soluble recombinant IL-1R (srIL-1R). Changes in gene expression in sorted hMSCs were assessed using Agilent microarray platform while data normalization and bioinformatics were conducted using GeneSpring software.
We observed a dynamic interaction between cancer cells and hMSCs. High CDH1 (E-cadherin) and low IL1-Beta expression by cancer cells promoted reorganization of hMSCs into a niche-like formation, which was dependent on direct cell-cell contact. Our data also revealed transfer of cellular components between cancer cells and hMSCs as one possible mechanism for intercellular communication. Global gene expression analysis of sorted hMSCs following co-culturing with MCF7 and BT-20 cells revealed enrichment in signaling pathways related to bone formation, FAK and MAPKK signaling. Co-culturing hMSCs with MCF7 cells increased their growth evidenced by increase in Ki67 and PCNA staining in tumor cells in direct contact with hMSCs niche. On the other hand, co-culturing hMSCs with FaDu, HT-29 or MDA-MB-231 cells led remarkable decline in their cell growth.
Dynamic interaction exists between hMSCs and cancer cells. CDH1 and IL1-Beta expression by cancer cells mediates the crosstalk between hMSCs and cancer cells. We propose a model where hMSCs act as the first line of defense against cancer cell growth and spread.
KeywordsMCF7 Cell Focal Adhesion Kinase Magnetic Activate Cell Sorting FaDu Cell Niche Formation
RAC-alpha serine/threonine-protein kinase
Adipose tissue-derived mesenchymal (stromal) stem cell
Bone morphogenetic protein 2
bovine serum albumin
Epithelial cadherin type 1
Dulbecco’s modified Eagle’s medium
Ethylenediamine tetraacetic acid
Epidermal growth factor
Epithelial specific antigen
Fluorescence-activated cell sorting
Focal adhesion kinase
Fetal bovine serum
Green fluorescent protein
Human bone marrow-derived mesenchymal (stromal) stem cell
Marker of proliferation Ki-67
Mitogen-activated protein kinase kinase
Mesenchymal (stromal) stem cell
Urokinase-type plasminogen activator
Quantitative real-time polymerase chain reaction
Zinc finger protein
SRY (sex determining region Y) Box 9
Secreted protein, acidic, cysteine-rich (osteonectin)
Soluble recombinant IL-1 receptor
Telomerase reverse transcriptase
Transforming growth factor beta
Vascular endothelial growth factor A
Carcinogenesis is a complex process that involves transformed cells interacting with the microenvironment containing extracellular matrix, carcinoma-associated fibroblasts (CAFs), pericytes, endothelial cells, and immune cells . Cross-talk between transformed cells and the microenvironment contributes to tumor growth, invasion, and metastasis. Among tumor microenvironment components, growing evidence suggests that CAFs are derived from mesenchymal (stromal) stem cells (MSCs), which are multipotent stem cells present within the stroma of bone marrow and probably other organs . The precise role of CAFs or MSCs in cancer development and progression is an area of intensive investigation and remains controversial (for a review see ). For instance, Karnoub et al.  reported that MSCs in a breast cancer xenograft model promoted breast cancer invasion and metastasis via the chemokine (C–C motif) ligand/C–C chemokine receptor CCL5/CCR5 cytokine network. Similarly, Liu et al.  reported that MSCs promoted breast cancer stem cell expansion via interleukin (IL)-6 and chemokine (C–X–C motif) ligand 7 signaling. In another study, Huang et al.  demonstrated that activation of caspase 3 by tumor or stroma cells triggers tumor repopulation during radiation therapy. While these reports suggest a pro-tumorigenic role for MSCs, a number of other studies revealed an anti-tumor effect of MSCs. For example, Cooke et al.  have shown that targeted depletion of pericytes (which are part of the MSC lineage) in vivo promoted tumor metastasis, which was mediated via hypoxia-induced epithelial to mesenchymal transition. Also, Khakoo et al.  have reported a strong inhibitory effect of human bone marrow-derived MSCs (hMSCs) against Kaposi sarcoma in vitro and in vivo through inhibition of AKT signaling in tumor cells. The precise role of MSCs in tumorigenicity and the conditions under which MSCs exert pro-tumor or anti-tumor effects therefore need to be determined.
In the majority of previous studies, a single or a few tumor models were studied, which limits the generalizability of their findings to other tumor models. In the present study, we conducted a comprehensive investigation to characterize the cellular and molecular phenotype of hMSCs co-cultured with 12 cancer cell lines derived from the breast, colon, prostate, head and neck, and melanoma. Our data revealed that the outcome of MSC–tumor interaction is dependent on the nature rather than the type of tumor cells and that epithelial cadherin type 1 (CDH1) and IL-1β expression by tumor cells are key factors in determining the outcome of hMSC–tumor cross-talk.
Cell lines and culture
Tumor cell lines used in this study (breast: MCF7, MDA-MB-231, BT-20, BT-474, MDA-MB-468, T-47D, and SKB-R3; melanoma: MDA-MB-435S; prostate: PC-3; head and neck: FaDu; and colon: HT-29 and COLO-320) were purchased from Cell Lines Service GmbH Eppelheim, (Germany) or were obtained from other sources and subsequently authenticated by Genetica DNA Laboratories, Inc. Burlington, (NC, USA). As a model for primary hMSCs, we employed a well-characterized telomerized hMSC line (hMSC-TERT) that has been created through overexpression of the human telomerase reverse transcriptase gene (TERT) . The hMSC-TERT cell line expresses all known markers of primary hMSCs [10, 11] and exhibits hMSC “stemmness”, evidenced by being able to form bone and the bone marrow microenvironment following in-vivo implantation . hMSC-TERT cells were also engineered to express enhanced green fluorescent protein (GFP) gene . All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4500 mg/l d-glucose, 4 mM l-glutamine, and 110 mg/l sodium pyruvate, 10 % fetal bovine serum (FBS), 1 % penicillin–streptomycin and nonessential amino acids. The normal, nontransformed human mammary epithelial cell line (MCF10A) was maintained in universal medium (DMEM-F12 + 20 ng/ml human epidermal growth factor (EGF), 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5 % FBS). Primary normal adipose tissue-derived MSCs (AT-MSCs) were obtained and cultured as described previously .
For co-culture experiments, hMSCs were trypsinized, counted, and seeded at 0.5 × 105/well, and 1 × x105 tumor cells were added to the same well in 24-well culture plates (Falcon, Franklin Lake, NJ, USA). Co-cultures were subsequently monitored and images were taken at the indicated time points using a Nikon® ECLIPSE Ti-U inverted fluorescence microscope, (Nikon, Tokyo, Japan). Cells were either imaged directly or were washed with 1× phosphate-buffered saline (PBS), followed by staining with Hoechst 33342 (10 μg/ml) in PBS for 10 minutes at 37 °C. Recombinant human IL-1β was purchased from (Invitrogen, Carlsbad, CA, USA). Focal adhesion kinase (FAK) inhibitor (PF-573228), mitogen-activated protein kinase kinase (MAPKK) inhibitor (PD98059), and cytochalasin D were purchased from Sigma (St. Louis, MO, USA) and were reconstituted in dimethyl sulfoxide (DMSO). For experiments involving IL-1β or the abovementioned small molecule inhibitors, the agents were added from day 0 of the experiment as indicated in each figure.
Vybrant® multicolor cell labeling
Cells were harvested and suspended at a density of 1 × 106/ml in serum-free culture DMEM followed by fluorescence-labeling with the relevant Vybrant® Cell-Labeling Solution (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s protocol. AT-MSCs were labeled with green fluorescent probe (DiO), while the MCF7 cell line was labeled with DiL or DiD probes. Subsequently, direct co-culturing was performed by seeding the labeled primary hMSCs and the labeled MCF7 cells into 24-well culture plates at density of 1 × 105/ml/well for each cell type. Co-cultures were visualized and images were taken on the indicated days.
Lentiviral transduction and time-lapse microscopy
Lentiviral particles encoding for mcherry or firefly luciferase were purchased from Genecopoeia Inc. (Rockville, MD, USA). Then 100,000 tumor cells were seeded in complete DMEM in a 24-well plate. Forty-eight hours later (~80 % confluency), the medium was removed and then 20 μl crude lentiviral particles in 500 μl DMEM + 5 % heat-inactivated serum (Invitrogen), 1 % penicillin–streptomycin supplemented with polybrene (8 μg/ml; Sigma) were added to the cells. Seventy-two hours later, the medium was removed and transduced cells were selected with puromycin (1 μg/ml; Sigma) for 1 week until stably-transduced cells were generated. Time-lapse microscopy was conducted using Nikon® BioStation IM-Q.
Magnetic activated cell sorting
Direct co-culture of MCF7 (seeding number = 1.0 × 105/well) or BT-20 (seeding number = 1.0 × 105/well) cells and hMSCs (seeding number = 0.5 × 105/well) was conducted using BD Falcon 24-well culture plates. On day 7 when the niche-like structure was visible, co-cultured cells were trypsinized from three replicas, and washed once with 1× PBS. Cells were subsequently resuspended in sorting buffer (500 ml Ca2+/Mg2+ free PBS supplemented with 2 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 % bovine serum albumin (BSA)), which was subsequently filtered using a 0.22 μM filter. Sorting of hMSCs from MCF7 or BT-20 co-cultures was performed using the CD326 (EpCAM) MicroBeads kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as per the manufacturer’s recommendations. Purity of sorted cells was confirmed using the BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Sorted cells were then washed with 1× PBS and were kept at −80 °C. Cells from the same batch and passage number of hMSCs were used as control.
Gene expression microarray
Total RNA was isolated using total RNA Purification Kit (Norgen-Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. The concentrations and purity of total RNA were measured using NanoDrop 2000 (Thermo-Scientific, Wilmington, DE, USA). Extracted RNA was labeled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies, Santa Clara, CA, USA). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine, Riyadh, Saudi Arabia) as described previously . Data analyses were conducted using GeneSpring GX software (Agilent Technologies) as described [16, 17]. Microarray data were deposited in the Gene Expression Omnibus [GEO:GSE70103].
Quantification of gene expression using qRT-PCR
Expression levels of selected genes were assessed using qRT-PCR. Reverse transcription was performed on 500 ng total RNA using High Capacity Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s specifications. The qRT-PCR was carried out using FAST-SYBR Green Master Mix (Applied Biosystems) and the ViiA™ 7 Real-Time PCR Detection System (Applied Biosystems). Primers used for gene expression analysis are presented in Additional file 1 and were either published previously or were designed using NCBI Primer-BLAST . The 2∆CT value method was used to calculate relative expression of miRNAs and mRNAs .
Alkaline phosphatase staining
hMSCs were co-cultured with MCF7 or HT-29. On day 7, the cells were washed in PBS, fixed in 10 mM acetone/citrate buffer (1.5:1) at pH 4.2 for 5 minutes at room temperature, and incubated with alkaline phosphatase (ALP) substrate solution (naphthol AS-TR phosphate (Sigma) prepared 1:5 in water plus 10 mg Fast red TR (Sigma), in 24 ml of 0.1 M Tris buffer, pH 9.0) for 1 hour at room temperature. Cells were rinsed with water, stored in PBS, and photographed using a Nikon® ECLIPSE Ti-U inverted fluorescence microscope.
Day 7 MCF7–MSC co-cultures were fixed with methanol/acetone 1:1 (vol/vol) for 30 minutes at −20 °C. After fixation, cells were dried for 15 minutes and rehydrated with PBS for 15 minutes. Cells were blocked with 2 % BSA (Sigma) for 1 hour, followed by incubating with rabbit primary antibodies against Ki67 (1:200, ab15580; Abcam, Cambridge, MA, USA) or Proliferating cell nuclear antigen (PCNA) (1:100, ab2426; Abcam) in blocking solution at 4 °C overnight. After removal of primary antibodies, cells were washed three times with PBS, and then Alexa Fluor® 488 conjugated goat anti-rabbit IgG (H + L) secondary antibody (1/1000, A11008; Lifetechnologies, Carlsbad, CA, USA) was added and incubated for 2 hours at room temperature. Cells were washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye, mounted on slides in CC/Mount (C9368; Sigma), and were observed under a Nikon®ECLIPSE Ti fluorescence microscope.
All flow cytometry experiments were conducted using the BD FACSCalibur flow cytometer (BD Biosciences).
For selected experiments, the relative number of hMSCs was determined as follows. hMSCs 0.5 × 104 were cultured alone or with MCF7 (0.5 × 103) or HT-29 (0.5 × 103) cells in Corning® polystyrene flat-bottomed 96-well TC-treated black microplates Cambridge, MA, USA. The fluorescence signal was measured using a SpectraMax/M5 fluorescence spectrophotometer plate reader (Molecular Devices Co., Sunnyvale, CA, USA USA) using the bottom well-scan mode where nine readings per well were obtained using Ex (488 nm) and Em (509 nm) spectra. For luminescence measurements, hMSCs and tumor cells were cultured as above in Nunc™ F96 MicroWell™ white plates (Thermo-Scientific, Wilmington, DE, USA), and luminescence was measured using a BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, USA) with the ONE-Glo™ Luciferase Assay System (Promega, Madison, WI, USA).
HT-29 and MCF7 cells were cultured alone or were co-cultured with hMSCs, and on day 7 the scratch assay was performed using a p200 pipette tip. The scratch area was imaged on day 0 and day 2 using 4× magnification with the Nikon® ECLIPSE Ti-U inverted fluorescence microscope. Data are representative of at least three replicates.
Statistical analyses and graphing were performed using Microsoft Excel 2010 (Microsoft, Mountain View, CA) and Graphpad Prism 6.0 software (Graphpad Software, San Diego, CA, USA). P values were calculated using the two-tailed t test and P <0.05 was considered statistically significant.
Changes in hMSC morphology when co-cultured with cancer cell lines
The change in hMSC morphology requires direct contact with tumor cells
Gene expression analysis of hMSCs sorted from MCF7 and BT-20 co-cultures revealed enrichment for pathways related to bone formation
Pharmacological inhibition of FAK, MAPKK, and actin polymerization completely abrogated niche formation by hMSCs
High expression of CDH1 and low expression of IL-1β by tumor cells are associated with hMSC niche-like formation
Transfer of cellular components from cancer cells to hMSCs with no evidence of cell fusion during co-culture
Bidirectional influence on cell growth between hMSCs and cancer cells in co-cultures
Recent years have witnessed increased interest in studying the cancer tumor microenvironment and its contribution to tumor progression, invasion, and metastasis. However, the precise role of MSCs—an important component of the cancer tumor microenvironment—remains not fully understood. In the current study, we have utilized several cellular and molecular approaches to investigate the dynamic interaction between hMSCs and cancer cells. We employed several cancer cell lines representing breast, prostate, colon, head and neck, and melanoma. Our data revealed that hMSC interaction with cancer cells results in cytoskeletal and morphological reorganization leading to the formation of niche-like structures that exert variable effects on cancer cell growth. We also observed that cancer cell expression of CDH1/IL-1β is predictive for the presence of significant biological effects of hMSCs on tumor cells.
We observed that hMSCs exerted anti-tumor effects, evident by a significant decline in the number of tumor cells when co-cultured with hMSCs (Fig. 7a). Cell tracking and live imaging microscopy revealed a rapid increase in the numbers of hMSCs when they encountered cancer cells (Fig. 7a, c, d; Additional file 6), suggesting that hMSCs may act as the first line of defense against the transformed cells. Concordant with our hypothesis, one previous study has demonstrated a protective role for skin-derived fibroblasts in regulating transformed keratinocytes in vivo .
When hMSCs were co-cultured with cancer cells with aggressive phenotype (such as FaDu, PC-3, or MDA-MB-231), we observed a rapid decline in the number of hMSCs suggesting that the behavior of hMSCs is largely dependent on the nature of cancer cells, in particular their inflammatory status. Our data revealed that the decline in hMSC number occurred when co-cultured with cancer cells expressing high levels of IL-1β.
We previously reported that when hMSCs are exposed to IL-1β they acquire a proinflammatory phenotype and exhibit a decline in their multilineage differentiation potential . In the current study, when hMSCs were co-cultured with IL-1β producing cancer cells, hMSCs became proinflammatory cells and their number declined. The role of IL-1β was demonstrated by the complete reversal in hMSC niche formation when IL-1β signaling was blocked during hMSC–FaDu co-cultures (Fig. 5d).
We observed that hMSCs formed niche-like structures when co-cultured with cancer cells expressing high levels of CDH1 and lacked IL-1β (Fig. 7h). Interestingly, our data are in line with a published report suggesting that the inhibitory ability of hMSCs on tumor cell growth is mediated through binding to E-cadherin present on tumor cells . Concordant with this, niche formation was not observed when hMSCs were co-cultured with the COLO-320 cells (which lack CDH1 expression) compared with the HT-29 cells (which express high levels of CDH1), suggesting a possible role for CDH1 in promoting niche formation, possibly through facilitating homotypic cell adhesion.
While we have not characterized the nature of the hMSC niche-like formation under all co-culture conditions, the hMSC niches formed in the presence of MCF7 and BT-20 cells revealed differentiation into bone-forming osteoblastic cells, evident by microarray analysis and positive ALP expression (Fig. 3c, e). hMSC niche-like formations were dependent on FAK and MAPKK signaling because pharmacological inhibition of both pathways completely abrogated the niche-like formations. Interestingly, Navab et al.  have recently identified a prognostic gene expression signature derived from CAFs isolated from lung cancer patients and reported that FAK and MAPK are two major intracellular signaling pathways activated in CAFs, which collectively suggest the clinical relevance of our findings.
One interesting finding in our study is the transfer of cellular components from cancer cells to hMSCs. The transfer of membrane-derived vesicles has been reported previously in various biological systems as a mode of communication between adjacent cells [24, 25]. It is plausible that changes in hMSC phenotype when co-cultured with cancer cells are mediated via tumor-derived factors that induce hMSC differentiation. Interestingly, while morphologically similar, the niche-like formations in hMSCs co-cultured with HT-29 did not stain positive for ALP (Additional file 7) and exerted negative effects on cancer cell growth (Fig. 7a, e). Similarly, both HT-29 and MCF7 co-cultured with hMSCs had less ability to close the gap during the wound healing assay (Additional file 8). Our current hypothesis is that the niche-like formation of hMSCs plays a role in controlling cancer cell growth and spread. Our data are in agreement with recently published data implicating pericytes, which are related ontologically to MSCs, in preventing metastasis in vivo .
We observed that cell growth of MCF7 increased when co-cultured with hMSCs. Similar to our data, Karnoub et al.  showed that MCF7 is the only breast cancer cell line to have significant increase in tumor growth when implanted with MSCs in vivo. The biological differences between niche-like formation of hMSCs in response to MCF7 versus that induced by HT-29 cells are not known. However, this may be related to differences in multiple activated genetic pathways in MCF7 versus HT-29 as revealed by differences in their molecular signature (Additional file 9). We propose a working hypothesis in which MSCs interact with cancer cells and the outcome of this dynamic interaction is dependent on the expression of CDH1/IL-1β by cancer cells (Fig. 8c). MSCs exert inhibitory effects on cancer cell growth. However, it is possible that this interaction may also exert selection pressure on certain types of cancer cells leading to increased cancer cell growth and possibly metastases (Fig. 8c). Detailed molecular analysis of this hypothesis remains to be determined.
Our data revealed dynamic bidirectional interaction between hMSCs and tumor cells. Data also show that this interaction is dependent on the nature rather than the type of tumor cells and that CDH1 and IL-1β expression by tumor cells are key factors in determining the outcome of hMSC–tumor cross-talk.
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, award number (11-MED-1942-02). The authors would like to thank Dr Mohamed Abumaree (King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia) for assistance with the time-lapse imaging experiment.
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