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Introduction

Mesenchymal stem cells (MSCs) are defined as cells possessing the ability of self-renewal, proliferation and differentiation into multiple lineages, including chondrocytes, osteoblasts and adipocytes (1). MSCs are known to have homing properties and can repair injured tissues; thus, MSCs demonstrate potential as therapy for organ defects, regeneration or certain other conditions, including bone defects (2,3), hair loss (4), liver fibrosis (5), pancreatic islet transplantation, immunomodulation (6) and neuronal regeneration (7). MSCs can be found in various tissues, including bone marrow, umbilical cord blood, placenta and fat (8). Among these sites, adipose tissue is accessible and abundant for the isolation of adult stem cells. Therefore, human adipose tissue-derived MSCs (hATMSCs) obtained by relatively less invasive procedures are an attractive source for cell therapy.

Our previous study indicated that systemic transplantation of hATMSCs did not induce tumor development (9). MacIsaac et al (10) also showed that no malignancies were detected in normal mice treated with hATMSCs. However, in tumor models, MSCs have been reported to play a role in the process of carcinogenesis, tumor growth and metastasis, although numerous studies have reported contradicting results; some investigators found that MSCs promote tumor growth and others report that MSCs inhibit tumor growth (11). Maestroni et al (12) reported that mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) inhibited the proliferation and growth of Lewis lung carcinoma and B16 melanoma. It was also reported that hBM-MSCs showed a tumor-suppressive effect in an experimental model of Kaposi's sarcoma (13). BM-MSCs also exhibited an inhibitory effect on A549 tumor cells both in vivo and in vitro experiments (14). In contrast, a promoting effect of MSCs on tumor growth was observed in other studies. Zhu et al (15) subcutaneously injected hBM-MSCs along with colon cancer cells, and promotion of angiogenesis and tumor growth were noted. Xu et al (16) showed that hBM-MSCs promoted the growth and metastasis of osteosarcoma. The tumor-promoting effect of BM-MSCs was also demonstrated by Tsai et al (17) in HT-29 cancer cells.

Some mechanisms of MSC-related tumor supporting effects have been reported, which include vascular (18) and tumor stromal support (19), immunosuppressive effects (20) and metastatic support (21). On the other hand, the tumor inhibitory mechanisms of MSCs could be due to cell cycle arrest (22), angiogenesis inhibition (23) and regulation of certain soluble factors, such as Wnt inhibitor, Dickkopf-related protein-1 (DKK-1), that may block tumor-related cell signaling pathways (24). Despite these explanations regarding the action of MSCs, the tumor modulation mechanisms of MSCs are extremely complicated and difficult to demonstrate. Thus, in order for the clinical use of MSCs as a novel cell therapy for various conditions, it is critical to understand the interactions between tumors and MSCs. In the present study, we investigated the tumor-modulatory effects of hATMSCs in several types of human cancer cells to clarify the roles of hATMSCs on human cancers and to determine the underlying mechanisms.

Materials and methods

Tumor cell lines

Human malignant melanoma cell line A-375, human lung adenocarcinoma cell line A549 and human colorectal adenocarcinoma cell line HT-29 were purchased from ATCC (Manassas, VA, USA). Human epidermoid carcinoma cell line A-431, human gastric carcinoma cell line NCI-N87 and human pancreatic adenocarcinoma cell line Capan-1 were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.03% antibiotic-antimycotic (all from Gibco, Grand Island, NY, USA) at 37°C in a 5% CO2 humidified chamber.

Isolation and characterization of hATMSCs

hATMSCs were prepared from surplus banked stem cells under good manufacturing practice conditions (RNL Bio, Seoul, Korea) and used as anonymized materials. In brief, human abdominal subcutaneous fat tissue was obtained by simple liposuction following informed consent from donors, digested with collagenase I (1 mg/ml), and filtered through a 100-μm nylon sieve to remove cellular debris and centrifuged at 470 g for 5 min. The pellet was resuspended in RCME cell attachment medium (RNL Bio) containing 10% FBS and cultured at 37°C. After 24 h, the cell medium was changed to RKCM cell growth medium (RNL Bio) containing 5% FBS. When the cells reached 80–90% confluency, they were subcultured and expanded in RKCM medium until passage 3. The immunophenotypes of the hATMSCs were analyzed using flow cytometric analysis. Trypsinized hATMSCs were suspended [1×106 hATMSCs/25 μl of phosphate-buffered saline (PBS)] and stained with FITC-conjugated CD31, CD34 and CD45 antibodies and PE-conjugated CD73 and CD90 antibodies (BD Pharmingen, San Diego, CA, USA).

In vivo tumor xenograft models

Severe combined immunodeficient (SCID) and nude mice can be used as human tumor xenograft models (25). Six-week old female BALB/c-nude mice (Orient Bio Inc., Seongnam, Korea) and hairless SCID mice (Charles River, Wilmington, MA, USA) were housed in an environmentally controlled room (22±2°C, 40–60% humidity and a 12-h light cycle). The present study was approved by the Institutional Review Board (IRB) and the Institutional Animal Care and Use Committee (IACUC) of the Biomedical Research Institute at Seoul National University Hospital. Cancer cells (5×106) were subcutaneously inoculated into the back of mice. The injection was made through the subcutaneous layer of the cervicodorsal part of the animals. When the tumor volume reached close to 100 mm3, the mice were intratumorally injected with 1×105 hATMSCs suspended in PBS or PBS once a week for 8 weeks (n=3–6/group). The tumor size was measured twice a week using a caliper (Mitutoyo, Tokyo, Japan), and the tumor volumes were calculated as π/6 × (volume × width × height) according to a previously described method (26). After sacrifice at 8 weeks, the tumor masses were removed, weighed and fixed in 10% neutral buffered formaldehyde solution for histological analysis or preserved at −70°C.

In vitro co-culture and trypan blue exclusion assay

A549 and HT-29 cancer cells (1×105) were co-cultured with hATMSCs in RPMI-1640 medium with 10% FBS in 6-well plates for 72 h. Indirect co-culture was conducted using Transwell inserts (0.4-μm pore size, polycarbonate membrane; SPL, Pocheon, Korea) in 6-well plates to prevent cell-to-cell contact. All of the experiments were conducted in triplicate. Total cell number was assessed by a trypan blue exclusion assay using a hemocytometer under a microscope.

Western blot analysis

The proteins were extracted using RIPA lysis buffer (Millipore, Bedford, MA, USA) containing 0.1% phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor (Roche, Indianapolis, IN, USA). The protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein samples were loaded for immunoblotting using antibodies against phosphorylated nuclear factor κB (NF-κB) p65, β-actin (Cell Signaling Technology, Beverly, MA, USA), p-JAK3, p-STAT3 and β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Densitometry of the target bands was analyzed using ImageJ [National Center for Biotechnology Information (NCBI)].

RNA isolation and microarray analysis

Total RNA from the tumors was isolated using Ambion mirVana miRNA Isolation kit (Ambion, Austin, TX, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and the quantity was determined by the ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Affymetrix GeneChip® Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) was used to analyze differential gene expression profiles as previously described (27). In order to determine whether genes were differentially expressed among the groups, the genes that showed >1.5-fold difference between the average signal values of the control and test groups were selected. Additionally, genes with a p-value <0.05 were extracted by Student's t-test (two-tailed, unpaired). After significant genes were identified, the web-based tool Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform the biological interpretation of the differentially expressed genes. The genes were classified based on the information of gene function in Gene Ontology (GO) (http://david.abc.ncifcrf.gov/home.jsp).

Flow cytometric analysis

Cancer cells were analyzed by a flow cytometer (FACSCalibur; Becton-Dickinson, San Jose, CA, USA) to assess levels of proliferation, apoptosis and NF-κB expression. Apoptosis and proliferation were evaluated using Annexin V-FITC Early Apoptosis Detection kit (Cell Signaling Technology) and BrdU Flow kit (BD Pharmingen), respectively, according to the manufacturer's instructions. Harvested tumor cells were centrifuged, resuspended in PBS and fixed in 2–4% formaldehyde. Phospho-NF-κB p65-Alexa Fluor 647 conjugate (Cell Signaling Technology) antibody was used for NF-κB analysis.

Statistical analysis

Statistical analysis of the results was performed using one-way ANOVA (SPSS, Inc., Chicago, IL, USA). p-values of <0.05 were considered to indicate statistically significant results. If the variance was significant, the data were analyzed by the multiple comparison procedure of Dunnett's test.

Results

Effects of the hATMSCs on the growth of various types of tumors in vivo xenograft models

For immunophenotypic characterization of the hATMSCs, surface marker expression was examined by flow cytometry. The hATMSCs expressed high levels of MSC markers CD73 and CD90, whereas the hATMSCs were negative for cell adhesion molecule marker CD31 and hematopoietic stem cell markers CD34 and CD45 (Fig. 1A).

Figure 1 Effects of hATMSCs on the growth of various types of tumors in in vivo xenograft models using BALB/c-nude mice. (A) Flow cytometric characterization of hATMSCs. hATMSCs were labeled with antibodies specific for hATMSC surface antigens. The number in parenthesis indicates the percentage of surface marker-positive cells. (B) Changes in tumor volume following hATMSC injection in the various tumor models. Data are expressed as means ± SE (*p<0.05 and **p<0.01). (C) Tumor weights at 8 weeks following hATMSC injection. Data are expressed as means ± SD (**p<0.01). (D) Representative images of tumor growth. hATMSCs, human adipose tissue-derived mesenchymal stem cells.

In Korea, the stomach, colon and lung have been reported to be the five leading primary sites of cancer both in males and females in 2013 (28). In addition to xenograft models for these cancers, three well-known xenografts, A-375 melanoma, skin squamous cell carcinoma A-431 and Capan-1 pancreatic xenografts (29–31), were used in order to investigate the dual effects of the hATMSCs on tumor growth. The tumor size was measured for 8 weeks after the first subcutaneous hATMSC injection in the BALB/c-nude mice subcutaneously inoculated with the cancer cells. The rates of growth of the A431, Capan-1 and A549 tumors were reduced in the hATMSC-injected groups compared with the respective control groups, although the differences did not reach statistical significance (Fig. 1B). In contrast, NCI-N87 and A375 tumors showed the tendency toward increased volume in the hATMSC-injected groups in comparison to the respective control groups. Particularly, the tumor weight of the HT-29 tumors in the hATMSC group was significantly greater than that in the control group. The differences in tumor weights between the hATMSC and the respective control groups showed consistent patterns with the changes in tumor volume (Fig. 1C and D).

Based on the changes in mean volumes and weights by the hATMSC injection in the BALB/c-nude mice, we selected the A549 and the HT-29 tumors for additional in vivo experiments using hairless SCID mice, and confirmed similar dual effects of the hATMSCs in these two types of tumors. The volume and weight of the A549 tumors were significantly reduced in the hATMSC group compared with the parameters in the control group, whereas the tumor volume and weight of the hATMSC-treated HT-29 tumors were significantly greater than the control group (Fig. 2).

Figure 2 Effects of hATMSCs on the growth of A549 and HT-29 tumors in in vivo xenograft models using hairless SCID mice. (A) Changes in the tumor volume following hATMSC injection in two tumor models. Data are expressed as means ± SE (*p<0.05 and **p<0.01). (B) Tumor weights at 8 weeks following hATMSC injection. Data are expressed as means ± SD (*p<0.05). (C) Representative images of tumor growth. hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Co-culture effect of the hATMSCs on the viability of cancer cell lines A549 and HT-29 in vitro

Tian et al (8) reported opposite effects of MSCs in vivo and in vitro; human BM-MSCs exerted a tumor inhibitory effect in vitro, yet surprisingly had a promoting effect on tumor growth in vivo. To confirm the effects of the hATMSCs on tumor growth in vivo, the A549 and HT-29 cells were co-cultured with the hATMSCs at different ratios of 10:0.5, 10:1 and 10:2 (Fig. 3A). The direct co-culture of the A549 cells with the hATMSCs at the ratios of 10:1 and 10:2 attenuated the proliferation rates of the cells by 9.78 and 19.82%, respectively, compared to the control group (10:0), whereas the direct co-culture of the HT-29 cells with the hATMSCs at the ratios of 10:1 and 10:2 increased the proliferation rates of the cells by 17.22 and 29.78%, respectively, compared to the control group (10:0). This probably resulted from the proliferation rates of the cancer cells changed by the hATMSCs. However, further evidence that excluded the confounding effects associated with changes in the proliferation rates of the hATMSCs by the cancer cells was needed. Thus, indirect co-culture of the cancer cells with the hATMSCs using cell inserts was conducted in order to prevent cell-to-cell contact. The same trends were observed in the indirect co-culture. The proliferation rates of the A549 cells were reduced by 9.72 and 18.57%, respectively, whereas those of the HT-29 cells were increased by 19.90 and 25.79%, respectively, compared to the control group (10:0).

Figure 3 Co-culture effects of hATMSCs on the A549 and HT-29 tumor cells in vitro. (A) The percentage of live cells following direct or indirect co-culture of tumor cells with hATMSCs. (B) BrdU staining and flow cytometry for the proliferation rates in the indirect co-culture of tumor cells with hATMSCs. (C) Annexin V/PI double staining and flow cytometry for the apoptotic rates in the indirect co-culture of tumor cells with hATMSCs. Apoptotic cells include Annexin V+/PI+ cells and Annexin V+/PI− cells. Data are expressed as the means ± SD (*p<0.05 and **p<0.01). hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Co-culture effect of the hATMSCs on the proliferation and apoptosis of cancer cells

The proliferation of the cancer cells indirectly co-cultured with the hATMSCs was measured by BrdU staining and flow cytometry. The percentage of BrdU-positive A549 cells was reduced by the hATMSC co-culture by 34 and 23% at the ratios of 10:1 and 10:2, respectively, whereas there was a significant induction of the percentage of BrdU-positive HT-29 cells co-cultured with the hATMSCs at the ratios of 10:2 (Fig. 3B). The apoptotic rates of the cancer cells co-cultured with the hATMSCs were analyzed by Annexin V/PI double staining and flow cytometry. While the percentage of live A549 cells was significantly diminished by co-culture with the hATMSCs, the percentage of apoptotic A549 cells was elevated by 40, 85 and 95% at the ratios of 10:0.5, 10:1 and 10:2, respectively (Fig. 3C). However, the hATMSCs had opposite effects on the HT-29 cells; there was an increase in live HT-29 cells and a decrease in apoptotic HT-29 cells of 23 and 26% at the ratios of 10:1 and 10:2, respectively. Collectively, the hATMSCs inhibited the proliferation, yet elevated the apoptosis in the A549 cancer cells, whereas it promoted the proliferation and reduced apoptosis in the HT-29 cancer cells.

Global differences in gene expression in the xenograft tumors in the presence or absence of the hATMSCs

In the microarray experiment with the A549 tumors of the hairless SCID mice, 4,022 probe sets were used to identify significant differences between the hATMSC and the control group in regards to expression levels of genes (fold-change >1.5) (Fig. 4A and B). Particularly, we found that the expression levels of 1,725 probes were increased and those of 2,297 probes were decreased in the hATMSC group. In contrast, in the microarray experiment with the HT-29 tumors of the hairless SCID mice, 164 probes were found expressed more highly in the hATMSC group, whereas 135 probes were found to be more lowly expressed in the hATMSC group. Based on more stringent statistical criteria (fold-change >1.5 and p<0.05), 568 genes were expressed at a higher level and 1,191 genes were expressed at a lower level in the hATMSC group compared with the control group in the A549 tumors (Table I). In contrast, 9 genes were expressed at a higher level and 14 genes were expressed at a lower level in the hATMSC group when compared with the control group in the HT-29 tumors (Table II).

Figure 4 Identification of genes exhibiting differential expression levels following hATMSC injection in the A549 and HT-29 tumor models using hairless SCID mice. (A) Genes differentially expressed (DEGs) in the microarray analysis among the groups. (B) 'Heat-map' representation of the genes differentially expressed (fold-change >1.5 and p<0.05). (C) The differences in HIST1H2AJ, NPY4R and LOC100505817 expression (intensity) in the microarray analysis among the groups. hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Table I Top 30 genes showing differential expression in the hATMSC-treated A549 tumors ranked by fold-change.

Table I

Top 30 genes showing differential expression in the hATMSC-treated A549 tumors ranked by fold-change.

Probe set	Gene	Gene description	Fold-changea
Genes expressed at a higher level in A549 tumors after hATMSC injection (p<0.05, fold-change >1.5)
16863393	IGFL2	IGF-like family member 2	3.68
16976821	PPBP	Pro-platelet basic protein [chemokine (C-X-C motif) ligand 7]	3.18
17061759	NRCAM	Neuronal cell adhesion molecule	2.77
16852322	MAPK4	Mitogen-activated protein kinase 4	2.47
16873513	LOC400706	Uncharacterized LOC400706	2.46
17083433	IL33	Interleukin 33	2.45
16745990	miR3167	MicroRNA 3167	2.17
16909319	DNER	Δ/notch-like EGF repeat containing	2.07
17022996	ROS1	c-ros oncogene 1, receptor tyrosine kinase	1.97
16713760	HNRNPA1P33	Heterogeneous nuclear ribonucleoprotein A1 pseudogene 33	1.97
16733325	KIRREL3-AS2	KIRREL3 antisense RNA 2	1.87
16704464	NPY4R	Neuropeptide Y receptor Y4	1.86
16742086	PGM2L1	Phosphoglucomutase 2-like 1	1.85
16710126	HTRA1	HtrA serine peptidase 1	1.79
17012392	RSPO3	R-spondin 3	1.69
16737590	SYT13	Synaptotagmin XIII	1.66
16997795	VCAN-AS1	VCAN antisense RNA 1	1.66
16852849	SERPINB11	Serpin peptidase inhibitor, clade B (ovalbumin), member 11 (gene/pseudogene)	1.63
16958382	MUC13	Mucin 13, cell surface associated	1.62
16713762	ANXA8L1	Annexin A8-like 1	1.61
16851950	miR3975	MicroRNA 3975	1.59
17060820	MUC12	Mucin 12, cell surface associated	1.58
16705934	CHST3	Carbohydrate (chondroitin 6) sulfotransferase 3	1.58
16677556	TGFB2	Transforming growth factor, β2	1.57
16704614	ANXA8	Annexin A8	1.55
16704516	ANXA8L2	Annexin A8-like 2	1.55
16957304	ZBED2	Zinc finger, BED-type containing 2	1.54
16960922	RARRES1	Retinoic acid receptor responder (tazarotene induced) 1	1.51
17067696	NRG1	Neuregulin 1	1.50
16697674	LINC00862	Long intergenic non-protein coding RNA 862	1.50
Genes expressed at a lower level in A549 tumors after hATMSC injection (p<0.05, fold-change >1.5)
16971272	EDNRA	Endothelin receptor type A	−4.75
17113448	IL13RA2	Interleukin 13 receptor, α2	−4.58
17112149	XIST	X inactive specific transcript (non-protein coding)	−4.44
16965377	SLIT2	Slit homolog 2 (Drosophila)	−4.32
16977241	NAA11	N(α)-acetyltransferase 11, NatA catalytic subunit	−4.22
17055447	MEOX2	Mesenchyme homeobox 2	−4.11
17101570	LOC100133123	Uncharacterized LOC100133123	−4.05
16870838	ZNF208	Zinc finger protein 208	−4.01
17001578	PDGFRB	Platelet-derived growth factor receptor, β polypeptide	−3.93
16732840	OR8G2	Olfactory receptor, family 8, subfamily G, member 2	−3.91
16736764	MUC15	Mucin 15, cell surface associated	−3.86
16870821	ZNF100	Zinc finger protein 100	−3.83
16909828	COL6A3	Collagen, type VI, α3	−3.72
16997383	F2RL2	Coagulation factor II (thrombin) receptor-like 2	−3.68
16979875	PCDH18	Protocadherin 18	−3.62
17058002	LOC650226	Ankyrin repeat domain 26 pseudogene	−3.56
16942958	EPHA3	EPH receptor A3	−3.52
17108003	BGN	Biglycan	−3.51
16852966	CCDC102B	Coiled-coil domain containing 102B	−3.51
17097661	TNC	Tenascin C	−3.45
17078870	MMP16	Matrix metallopeptidase 16 (membrane-inserted)	−3.32
16772888	RNU6-52P	RNA, U6 small nuclear 52, pseudogene	−3.29
16870854	ZNF676	Zinc finger protein 676	−3.21
16888610	COL3A1	Collagen, type III, α1	−3.13
16923842	COL6A1	Collagen, type VI, α1	−3.13
16666509	IFI44	Interferon-induced protein 44	−3.08
17107835	MAGEA4	Melanoma antigen family A, 4	−3.04
17107867	MAGEA6	Melanoma antigen family A, 6	−3.02
16673075	DDR2	Discoidin domain receptor tyrosine kinase 2	−3.00
16885596	POTEJ	POTE ankyrin domain family, member J	−2.97

a Each value represents the fold-change (in log₂ value) vs. the control group. hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Table II Genes showing differential expression in the hATMSC-treated HT-29 tumors ranked by fold-change.

Table II

Genes showing differential expression in the hATMSC-treated HT-29 tumors ranked by fold-change.

Probe set	Gene	Gene description	Fold-changea
Genes expressed at a higher level in the HT-29 tumors after hATMSC injection (p<0.05, fold-change >1.5)
16703429	RNA5SP307	RNA, 5S ribosomal pseudogene 307	0.97
17016490	HIST1H2AJ	Histone cluster 1, H2aj	0.91
16761541	PRB4	Proline-rich protein BstNI subfamily 4	0.85
17063776	TRBV6-8	T cell receptor β variable 6-8	0.77
16854216	miR133A1	MicroRNA 133a-1	0.74
16943350	GPR128	G protein-coupled receptor 128	0.67
17117463	LOC100509635	Uncharacterized LOC100509635	0.64
17063800	TRBV7-4	T cell receptor β variable 7-4 (gene/pseudogene)	0.63
16904484	SNORA70F	Small nucleolar RNA, H/ACA box 70F	0.61
Genes expressed at a lower level in the HT-29 tumors after hATMSC injection (p<0.05, fold-change >1.5)
16691574	RNA5SP56	RNA, 5S ribosomal pseudogene 56	−1.05
16798050	PAR-SN	Paternally expressed transcript PAR-SN	−0.74
17114740	SPANXN3	SPANX family, member N3	−0.74
16798146	SNORD116-5	Small nucleolar RNA, C/D box 116-5	−0.73
16798150	SNORD116-5	Small nucleolar RNA, C/D box 116-5	−0.73
16734339	miR4298	MicroRNA 4298	−0.72
16716369	miR4679-2	MicroRNA 4679-2	−0.70
16743207	TRIM51EP	Tripartite motif-containing 51E, pseudogene	−0.68
16853028	LOC100505817	Uncharacterized LOC100505817	−0.64
16767367	miR3913-1	MicroRNA 3913-1	−0.63
17114772	miR891B	MicroRNA 891b	−0.60
16696120	DPT	Dermatopontin	−0.59
17096623	BAAT	Bile acid CoA: amino acid N-acyltransferase (glycine N-choloyltransferase)	−0.59
16704464	NPY4R	Neuropeptide Y receptor Y4	−0.59

a Each value represents the fold-change (in log₂ value) vs. the control group. hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Among the genes differentially expressed in the xenograft tumors after the hATMSC injection, histone cluster 1, H2aj (HIST1H2AJ) was expressed in direct proportion to the xenograft tumor size. In other words, it was expressed at lower level in the A549 tumors and was expressed at a higher level in the HT-29 tumors after the hATMSC injection in comparison to the respective control groups. In addition, neuropeptide Y receptor Y4 (NPY4R) and LOC100505817 were expressed in inverse proportion to the xenograft tumor size. They were expressed at a higher level in the A549 tumors and were expressed at a lower level in the HT-29 tumors after the hATMSC injection in comparison to the respective control groups (Fig. 4A and C).

Functional classification of genes showing differential expression in the hATMSC-treated xenograft tumors

In order to identify the biological function of the genes differentially expressed following the hATMSC injection in the A549 and HT-29 tumor models using hairless SCID mice, GO analysis was performed. The statistically significant categories of GO terms [p<0.01; false discovery rate (FDR) <0.05] are shown in Table III. No enriched GO term was altered following treatment with the hATMSCs in the HT-29 tumors, whereas the genes involved in biological processes, such as 'Regulation of growth' and 'Regulation of cell growth' were upregulated in the hATMSC-treated A549 tumors. The genes involved in biological processes, such as 'Nucleosome assembly', 'Nucleosome organization', 'Protein-DNA complex assembly', 'Chromatin assembly or disassembly', 'Cell-cell adhesion' and 'Regulation of transcription (DNA-dependent)' were downregulated in the hATMSC-treated A549 tumors.

Table III GO terms associated with upregulated and downregulated trends in the hATMSC-treated A549 tumors (p<0.01, FDR<0.05).

Table III

GO terms associated with upregulated and downregulated trends in the hATMSC-treated A549 tumors (p<0.01, FDR<0.05).

GO ID	Biological process term	Gene count	P-value	Benjamini	FDR
Upregulated trend
GO:0040008	Regulation of growth	24	1.19E-05	0.0258	0.021
GO:0001558	Regulation of cell growth	17	2.37E-05	0.0256	0.041
Downregulated trend
GO:0006334	Nucleosome assembly	24	9.31E-11	2.44E-07	1.65E-07
GO:0034728	Nucleosome organization	25	1.41E-10	1.85E-07	2.51E-07
GO:0031497	Chromatin assembly	24	2.02E-10	1.77E-07	3.58E-07
GO:0065004	Protein-DNA complex assembly	24	5.37E-10	3.53E-07	9.52E-07
GO:0006323	DNA packaging	27	8.47E-10	4.45E-07	1.50E-06
GO:0006333	Chromatin assembly or disassembly	28	1.15E-09	5.02E-07	2.03E-06
GO:0006350	Transcription	175	9.61E-09	3.60E-06	1.70E-05
GO:0006355	Regulation of transcription, DNA-dependent	151	3.65E-08	1.20E-05	6.47E-05
GO:0051252	Regulation of RNA metabolic process	153	5.05E-08	1.47E-05	8.96E-05
GO:0034621	Cellular macromolecular complex subunit organization	45	5.14E-07	1.35E-04	9.12E-04
GO:0034622	Cellular macromolecular complex assembly	41	1.01E-06	2.41E-04	0.002
GO:0006325	Chromatin organization	46	1.01E-06	2.22E-04	0.002
GO:0016339	Calcium-dependent cell-cell adhesion	10	1.91E-06	3.85E-04	0.003
GO:0045449	Regulation of transcription	193	6.37E-06	0.0012	0.011
GO:0051276	Chromosome organization	52	7.69E-06	0.0013	0.014

[i] GO, Gene Ontology; FDR, false discovery rate.

Effects of the hATMSCs on the phosphorylation of NF-κB p65 in the A549 and HT-29 tumors in vivo and in vitro

The proteins extracted from the xenograft tumor masses of the hairless SCID mice were analyzed by western blot analysis to investigate whether there is a relationship between certain cell signaling pathways and the hATMSC-associated modulation of tumor growth. Among several proteins related to cell signaling pathways, the expression level of phosphorylated NF-κB p65 was reduced by 31% in the hATMSC-injected A549 tumor, whereas it was increased by 77% in the hATMSC-injected HT-29 tumors (Fig. 5A and B). The levels of other cell signaling-related proteins, such as p-JAK3 and p-STAT3, were not affected by the hATMSCs in the A549 and HT-29 tumors, and the β-catenin protein level was elevated by the hATMSCs in the HT-29 tumors, yet not affected in the A549 tumors, providing a possible correlation between the NF-κB pathway and hATMSC-related tumor modulation, at least in the A549 and the HT-29 tumors.

Figure 5 Effect of hATMSCs on phosphorylated NF-κB p65 in the A549 and HT-29 tumors in vivo and in vitro. (A) The expression levels of phosphorylated NF-κB p65, p-JAK3, p-STAT3, β-catenin and β-actin in A549 and HT-29 tumor masses of hairless SCID mice were detected by western blotting. (B) The mean of relative band intensities of phosphorylated NF-κB p65. (C) Flow cytometry for phosphorylated NF-κB p65 in A549 or HT-29 cells co-cultured with hATMSCs. Data are expressed as means ± SD (*p<0.05 and **p<0.01). hATMSCs, human adipose tissue-derived mesenchymal stem cells.

Changes in phosphorylated NF-κB p65 in the tumor masses in vivo were confirmed using flow cytometric analysis in the cancer cells indirectly co-cultured with the hATMSCs, which revealed significant reductions in phosphorylated NF-κB p65 by 12, 25 and 32% in the co-culture of the A549 cancer cells with the hATMSCs at the ratios of 10:0.5, 10:1 and 10:2, respectively, compared with the control (10:0) (Fig. 5C). However, there was an inverse effect in the HT-29 cancer cells, showing a profound increase in phosphorylated NF-κB p65 by 15% in the co-culture of HT-29 cancer cells with the hATMSCs at the ratio of 10:2.

Discussion

In the present study, we found conflicting results concerning the effects of the hATMSCs on tumor growth, particularly in the A549 and HT-29 tumors which were suppressed and supported, respectively, by the hATMSCs. It is possible that the dual effect of MSCs can be dependent on tumor type, at least partially, although these two cell lines do not represent the types of cancer that they are derived from. Thus, further analysis is needed to verify the actual factors in different microenvironment induced by different cancer cells rather than attempting to propose the type of tumor as the reason.

A recent study showed that direct cell-to-cell interaction between cancer cells and MSCs is responsible for the effects of MSCs on tumor growth (32). Although we also observed this result when hATMSCs were directly injected at the tumor site in the xenograft tumor models in vivo, our hypothesis is that this effect of hATMSCs on tumor growth could be indirect according to the results of previous studies (14,33). The hATMSCs showed dual effects such as a strong inhibitory effect on the A549 cancer cell viability and a dramatic promoting effect on the HT-29 cancer cell viability in vitro. In particular, in consistent with a study by Fierro et al (34), the hATMSCs showed a similar impact on cell viability between direct and indirect co-culture in our experiments using in vitro co-culture systems, indicating that the hATMSCs affected tumor cell growth even without direct cell-to-cell contact possibly due to certain soluble factors in the conditioned medium released from the hATMSCs. Among the many MSC-related soluble factors, proangiogenic factors, such as vascular endothelial growth factor (34), fibroblast growth factor (35), and angiopoietin-1 (36), promote endothelial and smooth muscle migration and proliferation at the tumor site, facilitating angiogenesis. In addition, there are factors such as interleukin 6 and tumor necrosis factor α (37) that are produced by MSCs and have immunosuppressive effects and eventually tumor supporting effects. Karnoub et al (21) reported that MSC-secreted chemokine ligand 5 induced a transient pro-metastatic effect on breast cancer cells. Meanwhile, ATMSCs were found to inhibit the proliferation in primary leukemia cells by secreting DKK-1, which exhibits an inhibitory effect on β-catenin signaling (15,24). Further studies are necessary to analyze which hATMSC-releasing factors contribute to the effect of hATMSCs on tumor growth.

The tumor growth was differentially mediated by hATMSCs between the A549 and the HT-29 tumors with in vivo xenograft models. For functional classification of the genes that were expressed differentially following treatment with the hATMSCs in the A549 and HT-29 tumors, GO and KEGG pathway analyses were performed. Noteworthy, 17 enriched GO categories were identified in the hATMSC-treated A549 tumors (p<0.01, FDR<0.05), whereas no GO terms were identified in the HT-29 tumors. The GO analysis clearly showed that the treatment of the A549 tumors with the hATMSCs upregulated the expression of many genes associated with 'Regulation of cell growth', which has been known to be induced by a potential anticancer drug (38). In contrast, 'Regulation of transcription (DNA-dependent)' and 'Cell-cell adhesion', which have been previously reported to be upregulated in xenograft tumor models and in cancer cell lines (39,40), were suppressed in the A549 tumors following treatment with the hATMSCs. Lo et al (41) showed that GO biological processes, including 'Nucleosome assembly', 'Nucleosome organization', 'Protein-DNA complex assembly' and 'Chromatin assembly or disassembly' were found to be altered in Asian and Caucasian lung cancer patients. 'Nucleosome assembly' has also been known as a process which alters the regulatory mechanisms of cancer cells (42). In the present study, the hATMSCs downregulated the above-mentioned biological processes in the A549 tumors. In particular, the KEGG pathway analysis indicated that 'Small cell lung cancer pathway' in the downregulated trend gene set was significant in the hATMSC-treated A549 tumors (p<0.05) (data not shown). These findings presented here, suggesting that biological processes possibly associated with the tumor modulation, are active in only A549 tumors, can provide important implications to verify the dual effects of the hATMSCs.

To identify possible genetic factors for hATMSC-induced tumor modulation, the differentially expressed genes in proportion to xenograft tumor size following hATMSC administration were identified. Histone proteins, which are basic nuclear proteins responsible for the nucleosome structure of the chromosomal fiber, were expressed from four different clusters in the human genome. In histone cluster 1, mainly histone 1 (e.g. HIST1H1T) and histone 2 (e.g. HIST1H2BC, HIST1H2AC and HIST1H2BD) genes were found to be overexpressed in the cancer tissues (43). Although there are few reports of HIST1H2AJ which is related to cancer directly, HIST1H2AJ (histone cluster 1, H2aj) was found to be expressed in direct proportion to the xenograft tumor size and is likely to be functionally relevant in tumorigenesis. We also found that NPY4R was differentially expressed in inverse proportion to the xenograft A549 and HT-29 tumor size after hATMSC administration. NPY family peptides, one of the most widely distributed peptides in the central and peripheral nervous system, regulate physiological processes associated with energy homeostasis such as energy expenditure, lipid metabolism and insulin secretion through their actions on Y receptors (44–47). Moreover, the NPY system is known to be involved in cancer development via effects on energy homeostasis, immune function, as well as direct actions on tumor biology (47), raising the possibility of the involvement of NPY4R expression in the regulation of tumor growth following treatment with hATMSCs.

Cell signaling pathways associated with translational changes have been referenced to explain the mechanisms of the conflicting effects of MSCs on tumors. PI3K/AKT was referred as a possible means related to the tumor-inhibitory effect of umbilical cord MSCs (48). WNT (24) and JAK/STAT pathways (17) were also suggested as cell signaling pathways involved in the effects of MSCs. We additionally investigated the expression levels of certain proteins to determine which cell signaling pathways are related to the effects of hATMSCs on tumor growth. First of all, the hATMSCs affected the levels of phosphorylated NF-κB p65, yet not p-JAK3, p-STAT3 or β-catenin that are also key players in the effects of MSCs on tumors. Second, the hATMSC-induced changes in phosphorylated NF-κB p65 levels were correlated with the hATMSC-associated modulation on A549 and HT-29 tumor growth, suggesting the NF-κB pathway as a potential mediator in the effects of hATMSCs on tumors. The NF-κB pathway is triggered in response to infection or exposure to pro-inflammatory cytokines, leading to inhibitor of κB (IκB) degradation and finally translocation of p50/p65 heterodimers into the nucleus. In general, NF-κB regulates a variety of downstream genes that govern immunity, cell growth and apoptosis (49). In the tumor environment, the NF-κB pathway is widely known to regulate the proliferation of tumor cells through the transcription of anti-apoptotic proteins, leading to the increased growth and metastasis of various types of tumors (50). Actually, in the A549 and HT-29 cancer cells, correlations were reported in which cell proliferation was promoted by activation of the NF-κB pathway and suppressed by blocking the NF-κB pathway (51,52), supporting our hypothesis on the possible link of the NF-κB pathway to the tumor growth modulated by hATMSCs.

Given the possibility of MSCs as a novel therapeutic tool for various diseases, it is critical to explain the conflicting findings in tumor-MSC studies. Despite previous reports on the possible factors of MSC-induced tumor modulation, the exact mechanisms remain unclear. The most direct outcome of the present study is that we confirmed the dual effects of hATMSCs in the two types of cancer cells in different micro-environments in vivo and in vitro. We also suggest the possible roles of the identified gene as profiled and the NF-κB pathway in the dual modulatory effects of hATMSCs on tumor growth. Our findings have profound implication in understanding the interaction between MSCs and tumors although further research is needed to confirm these findings using various types of cancer.

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References