Mesenchymal stem cells (MSCs) possess the potential for differentiation into multilineages. MSCs have been reported to play a role as precursors for tumor stroma in providing a favorable environment for tumor progression. Hyperthermia destroys cancer cells by raising the temperature of tumor-loaded tissue to 40°C to 43°C and causes indirect sensitizing effects when combined with chemo- and/or radiotherapy. However, how hyperthermia affects the tumor-supportive stroma is unknown. Here, the authors investigated the effects of hyperthermia-treated MSCs, from different sources, on the human ovarian cancer cell line SK-OV-3.
MSCs from adipose tissue and amniotic fluid were untreated or heat-treated (HS-MSCs). The culture supernatant of each treatment group was collected and transferred to the SK-OV-3 cells.
The morphological analysis and cell proliferation assay showed a reduced viability of the tumor cells in the conditioned medium with the HS-MSCs. Further investigations revealed that the conditioned medium of the HS-MSCs induced a higher nuclear condensation and a greater number of sub-G1 cells among the tumor cells. Analysis of the mRNA expression demonstrated that the conditioned medium of the HS-MSCs induced up-regulation or down-regulation of several tumor-associated molecules. Finally, the cytokine array of each conditioned medium showed that angiogenin, insulin-like growth factor binding protein 4, neurotrophin 3, and chemokine (C-C motif) ligand 18 are involved as main factors.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells. They can self-renew and have the capability of differentiating into a variety of cell types such as osteoblasts, chondrocytes, myocytes, adipocytes, and neuronal cells under certain conditions.1, 2 Although MSCs were initially found in the marrow stroma, they have also been obtained from several different tissues, including umbilical cord blood, adipose tissue, and amniotic fluid, as well as bone marrow.3–6
In addition to their properties of self-renewal and multilineage potential, MSCs have been reported to migrate to damaged tissue sites such as sites with inflammation, wounds, or cancer cells.7, 8 There is accumulating evidence that MSCs may be potential precursors to the tumor stroma that plays a fundamental role in tumor growth, invasion, and dissemination by interacting with carcinoma cells.9, 10 During tumor progression, stromagenesis occurs in parallel with tumorigenesis, and predisposes to the development of tumor metastasis by generating new vessels.11, 12 A recent study demonstrated that MSCs, within the tumor stroma, promoted tumor invasion and metastasis,13 clearly suggesting an interaction between MSCs and cancer cells in tumor progression and malignancy. In addition, the MSCs may be associated with tumor propagation or dissemination by preventing recognition of the tumor cells by the immune system as well as promoting tumor cell invasiveness.14, 15 Therefore, prior reports have shown a relationship between MSCs and cancer cells.
Hyperthermia is a therapeutic procedure that raises the temperature of tumor-loaded tissue to 40°C to 44°C.16 The effectiveness of hyperthermia can be strengthened by combination with radiotherapy and chemotherapy.17, 18 Such therapy has been used effectively for breast cancer, malignant melanoma, head and neck lymph node metastasis, glioblastoma, and cervical cancer.16, 19, 20
Cancer cells are vulnerable to hyperthermia, because of the high acidity caused by their inability to expel waste created by anaerobic metabolism.21 Hyperthermia can directly impair acidic cells by disrupting the stability of cellular proteins and the structures within cell membranes as well as the cytoskeleton. In addition, hyperthermia can act as an antiangiogenic agent by suppressing tumor-derived vascular endothelial growth factor production, thereby inhibiting endothelial-cell proliferation and extracellular matrix remodeling in blood vessels.22, 23 Furthermore, hyperthermia can sensitize cancer cells to the cytotoxic effects of radiotherapy and chemotherapy by enhancing membrane permeability, increasing intracellular accumulation of chemotherapeutic agents, inhibiting DNA repair, activating antiangiogenic protein production, or promoting immunologic host defenses in tumor tissue.24, 25
Although the effects of hyperthermia on cancer cells have been well documented, the influence of hyperthermia on the tumor stromal tissue or cells that interact with the cancer cells has not been studied. The goal of this study was to evaluate the effects of culture medium conditioned by heat-shocked MSCs, from adipose tissue and amniotic fluid, on the viability and growth of the human ovarian adenocarcinoma cell line SK-OV-3. The results of the experiments were used to determine whether hyperthermia was associated with the production of soluble factors secreted from the mesenchymal tumor stromal cells that sensitized the tumor cells to undergo cell death.
MATERIALS AND METHODS
Preparation and Culture of Human MSCs
Adipose-derived MSCs were obtained from lipoaspirates washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells.26, 27 The washed aspirates were treated with 0.075% collagenase (type I; Sigma Aldrich, St. Louis, Mo) in PBS for 60 minutes at 37°C with gentle agitation, followed by inactivation with an equal volume of Dulbecco modified Eagle medium (DMEM)/10% fetal bovine serum (FBS). After being centrifuged for 10 minutes at low speed, the cellular pellet was resuspended in DMEM/10% FBS and filtrated through a 100 μm mesh filter to remove debris. The filtrate was centrifuged as detailed above, plated onto conventional tissue culture plates in control medium (DMEM, 10% FBS, 1% antibiotic/antimycotic solution), and maintained at 37°C in a CO2 incubator. Amniotic fluid-derived MSCs were obtained by amniocentesis performed between 16 and 20 weeks of gestation for fetal karyotyping.28 For culturing the MSCs, nonadhering amniotic fluid cells from the supernatant medium were collected on the fifth day after initiating the primary amniocyte culture and kept until completion of the fetal chromosome analysis. The cells then were centrifuged and plated in 5 mL of α-modified minimum essential medium (Gibco, Carlsbad, Calif) supplemented with 20% FBS (Hyclone, Logan, Utah) and 4 ng/mL basic fibroblast growth factor in a 25-cm2 flask and incubated at 37°C in a CO2 incubator. MSCs after 3 to 5 passages were used throughout this study. Each donor provided consent after being fully informed at the Kangbuk Samsung Hospital. The study protocols have been approved by the institutional review board of Kangbuk Samsung Hospital.
Heat Treatment of MSCs and Conditioned Culture of SK-OV-3 cells
The MSCs were heat-shocked at 43°C for 45 minutes, followed by incubation for 24 hours at 37°C. Each culture supernatant was collected and filtrated through 0.2 μm pore-sized filters (Sartorius, Goettingen, Germany) and transferred to the SK-OV-3 cells, which were subsequently cultured for the indicated times.
Cell Proliferation Assay
The cells were cultured in normal culture medium (control) or each conditioned medium for 24, 48, and 72 hours. After the indicated times, the cells were washed with PBS and incubated with Wst-1 reagent (Roche Diagnostics, Indianapolis, Ind), which was premixed with media at a 1:10 ratio. One hour later, the optical density was measured at 450 nm by an enzyme-linked immunosorbent assay reader (Bio-Rad, Hercules, Calif). Statistical analysis was performed using the Student t test of Sigmaplot 2001 (Aspire International Software, Ashburn, Va). P values of less than .05 were regarded as statistically significant.
Nuclear Condensation Assay
The SK-OV-3 cells cultured in each conditioned medium for 48 hours were washed with PBS and then stained with 1 mL of 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution (Molecular Probes, Carlsbad, Calif) for 10 minutes. After washing with PBS, images were obtained by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).
The SK-OV-3 cells, having been cultured in each conditioned medium for 48 hours, were fixed with 70% ice-cold ethanol for 4 hours. The cells were then washed with PBS and incubated with 20 μL of 10 mg/mL RNase A (Sigma Aldrich) for 30 minutes at 37°C. After incubation, the cells were stained with 1 mL of 50 μg/mL propidium iodide staining solution (BD, Franklin Lakes, NJ) and analyzed by FACS Calibur (BD).
Reverse Transcription–Polymerase Chain Reaction
The SK-OV-3 cells cultured in each conditioned medium for 48 hours were used for RNA extraction. Total RNA was extracted using the Trizol method (Invitrogen, Carlsbad, Calif). Briefly, cells were vigorously lysed in Trizol, followed by chloroform. After centrifugation, the clear top phase was collected and mixed with isopropanol, followed by centrifugation. The pellet was washed with 75% ethanol solution and harvested. The final pellet was air-dried for 10 minutes and resuspended in diethyl pyrocarbonate-treated water. Next, 3 μg of RNA were used for synthesizing complementary DNA (cDNA) with M-MLV reverse transcriptase (Promega, Madison, Wis). One μg of the cDNA was used as a template for subsequent polymerase chain reaction (PCR) amplification using Taq polymerase premix (Takara Bio Inc., Shiga, Japan) with 35 cycles of 1 minute at 90°C, 1 minute at 55°C, and 1 minute at 72°C. The primers used in this study are the following: MDR1 5′-ACAGGAGATAGGCTGGTTTG-3′ (sense), 5′-GTTGCCATTGACTGAAAGAA-3′ (antisense); tumor necrosis factor receptor (TNF-R) 5′-TCAGTCCCGTGCCCAGTTCCACCTT-3′ (sense), 5′-CTGAAGGGGGTTGGGGATGGGGTC-3′ (antisense); Bax 5′-TTGGAATTCCGACGGGTCCGGGGAG-3′ (sense), 5′-GCCGAATTCCGCCCATCTTCTTCCAGAT-3′ (antisense); Bcl-2 5′-TTGGCCCCCGTTGCTT-3′ (sense), 5′-CGGTTGTCGTACCCCGTTCTC-3′ (antisense); Bcl-xL 5′-TTGGACAATGGACTGGTTGA-3′ (sense), 5′-GTAGAGTGGATGGTCAGTG-3′ (antisense); Bfl-1 5′-GGCAGAAGATGACAGACTGTGAA-3′ (sense), 5′-TGGTCAACAGTATTGCTTCAGGA-3′ (antisense); β-actin 5′-GTCCTCTCCCAAGTCCACAC-3′ (sense), 5′-GGGAGACCAAAAGCCTTCAT-3′ (antisense).
The culture supernatant, from the SK-OV-3 cells cultured in each conditioned medium for 48 hours, was collected and centrifuged to remove the cell debris. The cleared supernatant was added to the membrane, which was spotted with antibodies against various cytokines (AAH-CYT-6, Raybiotech, Norcross, Ga). The detection procedure used followed the manufacturer's protocols. Briefly, the membrane was blocked with blocking buffer for 30 minutes. After washing steps, each of the samples was reacted for 2 hours at RT, followed by subsequent incubation of biotin-conjugated antibodies and horseradish peroxidase-conjugated streptavidin. The labeled proteins were observed by enhanced chemiluminescence using detection buffer.
Characterization of Heat-treated MSCs
The MSCs cultured over 3 to 5 passages were either untreated or heat-treated, as described in the Materials and Methods section. First, we observed the morphology of the cells using phase-contrast microscopy. The results showed that normal MSCs (no shock) had a fibroblast-like structure, as previously reported (Fig. 1A).29 After the heat shock, the morphology of the MSCs had a mild morphological change of the cytosol region that appeared to be transient (Fig. 1B). To investigate whether a molecular change occurred with the heat shock, we performed flow cytometric analysis of both MSCs using antibodies against several marker proteins. The results demonstrated that both MSCs were negative for a typical hematopoietic marker CD34 and a lymphocytic marker CD45, whereas CD44, CD90, and CD105 were positive, as previously reported.30 Heat treatment resulted in no identifiable change in the molecules, suggesting that heat treatment at 43°C for 45 minutes induced neither serious structural damage nor molecular changes of the MSCs (data not shown).
Inhibition of Tumor Cell Proliferation by Conditioned Medium of Heat-treated MSCs
To determine whether the tumor stroma with hyperthermia influences tumor cells, the SK-OV-3 were cultured in normal culture medium (control), untreated (NS-MSCs), or heat-treated (HS-MSCs) conditions for 24, 48, or 72 hours, as illustrated in Figure 2A. Figure 2B shows the results of the cell proliferation assay performed at the indicated time points. The growth of the SK-OV-3 tumor cells was significantly decreased in the medium conditioned by HS-MSCs, whereas the medium conditioned by the NS-MSCs showed only mildly suppressed tumor cell proliferation. The same results were obtained with cells of another ovarian cancer, OV-CA-3, and cells of a cervical cancer C33A, as well as cells of 2 types of breast cancer, MCF-7 and MDA-MB-231 (Fig. 3). However, neither of the MSC-conditioned media affected the viability of normal fibroblast cells (Fig. 3A). These results suggest that soluble factors secreted from the MSCs have strong growth-inhibitory effects on tumor cells. In addition, hyperthermia may facilitate the production and/or secretion of these factors from the MSCs.
Condensation of the Tumor Cell Nucleus by the Conditioned Medium of the Heat-treated MSCs
As the proliferation of SK-OV-3 cells was inhibited by the conditioned medium of the HS-MSCs, as shown in Figure 2, we examined whether the decreased cell viability resulted from either simple cell growth arrest or apoptotic/necrotic cell death. As nuclear condensation and abnormal DNA content as the cell death indicators, we stained the tumor cells with DAPI or propidium iodide (PI), respectively, which can bind to and label intracellular DNA in the cellular nucleus. First, we performed DAPI staining of the SK-OV-3 cells cultured for 48 hours in each of the conditioned medium samples. The optical microscopy (Fig. 4, gray scale) showed increased cytoskeleton breakdown and detachment of the SK-OV-3 cells from substrate in the medium with the HS-MSCs compared with those in the medium with the NS-MSCs. In addition, the DAPI staining experiments analyzed by fluorescence microscopy showed that nuclear condensation occurred more prominently in tumor cells cultured in the medium with the HS-MSCs compared with the medium with NS-MSCs (Fig. 4, color scale).
Sub-G1 Analysis of the Tumor Cells in the Conditioned Medium With the Heat-treated MSCs
Further evaluation was performed by PI staining of the SK-OV-3 tumor cells in each conditioned medium. After 48 hours of incubation, the tumor cells were fixed and stained with the PI solution, followed by analysis using flow cytometry. As shown in Figure 5, the tumor cells in the conditioned medium with the HS-MSCs showed a greater sub-G1 population. The results were 2.28% and 2.78% for the NS-MSCs compared with 19.56% and 45.22% for the HS-MSCs. These findings indicate that greater apoptotic cell death was induced by the medium with the HS-MSCs, and suggest that the medium conditioned by the HS-MSCs had strong suppressive effects on tumorigenesis or growth of the tumor cells.
Changes of mRNA Expression of the Tumor-associated Molecules
On the basis of the data from the above experiments, the medium conditioned by HS-MSCs appeared to lead the tumor cells to undergo cell death. To determine the molecular changes that might have occurred, we hypothesized that the conditioned medium of the HS-MSCs might have increased expression of antitumorigenic proteins and/or decreased expression of protumorigenic factors in the tumor cells. To test these possibilities, the mRNA expression of several protumorigenic and antitumorigenic factors in the tumor cells was determined by reverse transcription–PCR (RT-PCR) (Fig. 6). The results showed a significant up-regulation of TNF-R, important for transmitting exogenous death signals from chemotherapeutic drugs into the cells. By contrast, the mRNA expression of the multidrug resistance protein MDR1 and antiapoptotic protein Bfl-1 were reduced. These data indicate that the conditioned medium with the heat-treated MSCs modulated the homeostatic balance of the tumor cells toward cell death. Therefore, our findings suggest that soluble factors secreted by the HS-MSCs may provide an antitumorigenic microenvironment that renders tumor cells more sensitive to chemotherapeutic drugs by effectively mediating exogenous cell death signals into the cells.
Cytokine Analysis of the Tumor Cells in Each Condition
The above data suggested that cytokines in the tumor microenvironment are critical in determining the tumor cell destiny. To identify the major factors responsible for the above results, we collected the culture supernatant of the SK-OV-3 tumor cells that was cultured for 48 hours under each condition and analyzed the results using a cytokine array system, as mentioned in the Materials and Methods section. The results shown in Figure 7 demonstrated the different cytokine profiles between adipose-derived mesenchymal stem cells (ADSCs) and amniotic fluid-derived mesenchymal stem cells (AFSCs). Nevertheless, the results also showed a few cytokines in common as indicated in bold in Figure 7: angiogenin and insulin-like growth factor binding protein 4 (IGFBP-4) were more abundant whereas neurotrophin 3 (NT-3) and chemokine (C-C motif) ligand 18 (CCL18) were less abundant in the medium conditioned by HS-MSCs compared with NS-MSCs, suggesting that the distinctive cytokines may cooperatively play a role in leading tumor cells to undergo death.
MSCs have “tropism migration” toward wounds, injury, and inflammation that result from various paracrine and endocrine signals. MSCs can also migrate to tumor sites considered to be “never-healing wounds” and compose tumor stroma.31 Because of such properties, MSCs are considered potentially efficient vehicles for cancer therapeutic genes.32 The tumor stroma plays a supporting role in the propagation or dissemination of tumors by producing protumorigenic cytokines.33 A prior study demonstrated that with the exposure to tumor cell-conditioned medium, migration of MSCs was promoted with an increase in stromal cell-derived factor-1 protein production.34 More importantly, a recently published study showed that MSCs within the tumor stroma promote breast cancer metastasis by CCL5 signaling through the chemokine receptor CCR5 expressed in the tumors.13 Therefore, the manipulation of the MSCs within the tumor stroma might improve conventional cancer therapy. The results of our study demonstrate the effects of hyperthermia on the tumor environment where the stromal cells and cancer cells interact.
Hyperthermia is a treatment for cancer that can be highly effective when combined with chemotherapy, radiotherapy, or immunotherapy.17, 35, 36 When tumor cells are heat-treated, their immunosuppressive properties are changed to an immunosusceptible status, rendering the tumor cells sensitive to immunotherapy or apoptotic signals.37 Considering the interaction between the MSCs and tumor cells in the tumor microenvironment, we studied whether the effect of hyperthermia on the MSCs provided an antitumorigenic environment that inhibited tumor propagation or dissemination. We observed how the tumor cells were affected by the soluble factors produced by the stromal stem cells by culturing the SK-OV-3 tumor cells with the conditioned medium of MSCs. In this study, the culture medium conditioned with the HS-MSCs induced significant tumor cell death as well as decreased expression of protumorigenic proteins and increased expression of proapoptotic proteins. The findings suggest that a protumorigenic environment can be changed to an antitumorigenic environment with hyperthermia treatment.
Cell-to-matrix as well as cell-to-cell attachment and communication are important in structural maintenance and information exchange.38 Adaptor and cytoskeletal proteins in particular have influence on a variety of cellular processes, including not only normal cell movement and differentiation but also tumor cell invasion and metastasis.39, 40 The result in Figure 4 demonstrated that the secreted factors from HS-MSCs induced cytoskeleton destruction and floating of adherent tumor cells (it was difficult to stain the tumor cells with DAPI because most of them were washed out in the washing steps of the DAPI staining experiment), finally causing cell death involving nuclear condensation and abnormal DNA content (Figs. 4 and 5).
The RT-PCR results (Fig. 6) provided another important clue as to how hyperthermia can synergistically make chemotherapy more effective when the 2 methods are combined. After repeated chemotherapy, tumor cells often become resistant to chemotherapeutic drugs because of induced defects in the delivery of exogenous death stimuli into the cells.41 MDR1 is a membrane protein that provides resistance to various drugs by facilitating excretion of the chemotherapeutic molecules out of the cells. TNF-R is also a plasma membrane protein that delivers exogenous cell death signals into the cells. Our results showed that the mRNA level of MDR1 was markedly decreased, and the mRNA level of TNF-R was highly increased in the SK-OV-3 cells after incubation with the conditioned medium of the HS-MSCs. These results suggest that the combination of hyperthermia with chemotherapy renders the tumor cells less able to become drug resistant. This may be because of mechanisms associated with the hyperthermia-treated tumor stromal cells.
Bfl-1 is a Bcl-2 family member that is involved in antiapoptotic activity and is known to be overexpressed in certain human epithelial and hematopoietic malignancies.42 Figure 6 illustrates that the conditioned medium from the HS-MSCs clearly reduced the expression of Bfl-1 in the tumor cells. This result implies that tumor cell progression might be markedly weakened by the contents released from the HS-MSCs, and suggests that identification of these contents may provide novel candidates for targeted cancer therapy.
The cytokine array shown in Figure 7 identified some of the contents in the conditioned medium of NS-MSCs and HS-MSCs. The result showed that several of the cytokines were distinctive in the same pattern: angiogenin, IGFBP-4 and NT-3, CCL18. Angiogenin is a small polypeptide implicated in angiogenesis in tumor growth.43 This protein functions as a transfer RNA (tRNA)-specific ribonuclease that abolishes cellular tRNA by specifically hydrolyzing cellular tRNAs in endothelial cells. Because angiogenins initiate their action by binding to actin on the surfaces of endothelial cells,44 we assumed that the angiogenin may play a role in the tumor cells by influencing the cytoskeleton. IGFBP-4 is a member of the insulin-like growth factor binding protein family. IGFBP-4 is a unique protein that consistently inhibits several cancer cells in vivo and in vitro, reduces the growth of cancer, and acts as an apoptotic factor.45 NT-3 is a neutrophic factor in the nerve growth factor family of neutrophins.46 This protein is a protein growth factor that helps to support the survival and differentiation of existing neurons and potentially to stimulate the growth and differentiation of new neurons and synapses. CCL18 is a small cytokine belonging to the CC chemokine family that was previously called PARC (pulmonary and activation-regulated chemokine).47 CCL18 is a chemotactic factor that attracts lymphocytes but not monocytes or granulocytes.48 We speculate that the decrease of NT-3 and CCL18 is associated with the lack of growth factor or signal for the tumor cell survival. Although this study discovered a few candidates responsible for tumor cell death, further investigation into their exact biological mechanisms is required.
In the study that demonstrated CCL5 as the inducer of tumor cell metastasis,13 CCL5 was not produced by either tumor cells or MSCs when separately cultured, whereas its production was prominently increased when cocultured. This means that the real actors in effects in such circumstances are from 2-way, not 1-way, communication between MSCs and tumor cells. This is why we put more focus on the contents produced by tumor cells under the conditioned medium. However, because the analysis of the conditioned medium as well as the tumor cell culture supernatant is certainly important in understanding the signaling between MSCs and tumor cells, further extensive studies on all aspects, including even direct cell-to-cell communication, are needed to reach clear conclusions.
Interestingly, the overall antitumor effects of the conditioned medium from HS-MSCs were even more significant by AFSCs than by ADSCs. We assume that the reason is because of their different characteristics. ADSCs have the typical characteristics of adult mesenchymal stem cells,49 whereas AFSCs have characteristics of both embryonic and adult stem cells.50 Therefore, it can be speculated that AFSCs may be genetically more primitive and versatile and have more potential possibilities than ADSCs.
Human diseases caused by tumors cannot be fully cured without destruction of the tumor stroma, which may support relapse of the tumor cells when present.51 The results of this study showed for the first time the importance of the effects of hyperthermia treatment on MSCs within the tumor stroma, not only the tumor cells. Furthermore, this study also provides new insights into epigenetic modification, targeting the MSCs within the tumor stroma, as another promising approach for cancer therapy.
Conflict of Interest Disclosures
Supported by a grant of the National R&D Program for Cancer Control, Ministry for Health, Welfare & Family Affairs, Republic of Korea(0820250) and the research and development fund of professor Choong Hyun Chang, a chairman of the Department of Plastic and Reconstructive Surgery, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine.