CANCER STEM CELLS Synovial Sarcoma Is a Stem Cell Malignancy NORIFUMI NAKA,a,b SATOSHI TAKENAKA,b,c NOBUHITO ARAKI,a TOSHITADA MIWA,d NOBUYUKI HASHIMOTO,c KIYOKO YOSHIOKA,b SUSUMU JOYAMA,a KEN-ICHIRO HAMADA,a YOSHITANE TSUKAMOTO,e YASUHIKO TOMITA,f TAKAFUMI UEDA,g HIDEKI YOSHIKAWA,c KAZUYUKI ITOHb Musculoskeletal Oncology Service, bDepartments of Biology, and fPathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan; cDepartment of Orthopedics, Osaka University Graduate School of Medicine, Osaka, Japan; dDepartment of Orthopedic Surgery, Osaka Koseinenkin Hospital, Osaka, Japan; e Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Japan; gDepartment of Orthopedic Surgery, Osaka National Hospital, Kinki-Block Comprehensive Cancer Center, Osaka, Japan a
Key Words. Synovial sarcoma • SS18-SSX • Cancer initiating ability • Cell of origin • Mesenchymal stem cell
ABSTRACT Synovial sarcoma (SS) is a malignant soft tissue tumor characterized by its unique t(X;18)(p11;q11) chromosomal translocation leading to the formation of the SS18-SSX fusion gene. The resulting fusion protein product is considered to play as an aberrant transcription factor and transform target cells by perturbing their gene expression program. However, the cellular origin of SS is highly debated. We herein established two novel human SS cell lines, named Yamato-SS and Aska-SS, and investigated their biological properties. We found the self-renewal ability of these cells to generate sarcospheres, to form tumors in serial xenotransplantation and reconstitute the tumor phenotypes without fractionation by any surface markers. Both SS cells as well as clinical tissue specimens from 15 patients expressed the marker genes-associated stem cell identity, Oct3/4, Nanog, and Sox2. We also found that
both SS cells displayed limited differentiation potentials for mesenchymal lineages into osteocytes and chondrocytes albeit with the expression of early mesenchymal and hematopoietic lineage genes. Upon SS18-SSX silencing with sequence-specific siRNAs, these SS cells exhibited morphological transition from spherical growth in suspension to adherent growth in monolayer, additional expression of later mesenchymal and hematopoietic lineage genes, and broader differentiation potentials into osteocytes, chondrocytes, adipocytes, and macrophages in appropriate differentiation cocktails. Collectively, these data suggest that a human multipotent mesenchymal stem cell can serve as a cell of origin for SS and SS is a stem cell malignancy resulting from dysregulation of self-renewal and differentiation capacities driven by SS18-SSX fusion protein. STEM CELLS 2010;28:1119–1131
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Synovial sarcoma (SS) accounts for 7–10% of all soft tissue sarcomas (STS), frequently affecting adolescents and young adults [1]. Clinically, it appears as deep-seated slowly growing mass, and more than half of the cases develop metastases mainly to the lungs and also sometimes to the lymph nodes and bone marrow [1]. SS is characterized by a recurrent chromosomal translocation [t(X;18)(p11;q11)] that leads to the formation of a fusion protein [2]. In the majority of SS, the SS18 (previously called as SYT) gene on chromosome 18q11 is fused to either SSX1, SSX2, or rarely SSX4 gene located on chromosome Xp11 [2–4]. As this translocation is quite spe-
cific to SS, the presence of SS18-SSX transcripts is clinically useful as a reliable diagnostic molecular maker for SS, and SS18-SSX fusion protein is considered to play a critical role for oncogenesis and development of SS. Although the transforming potential of the SS18-SSX fusion protein has been reported by ‘‘gain of function’’ studies using rat fibroblasts and murine myoblasts [5, 6], the molecular mechanisms that underlie SS sarcomagenesis on human cellular background are still unknown. The name ‘‘synovial sarcoma’’ was initially coined for tumors arising adjacent to joints and having some microscopic resemblance to developing synovial tissue. However, this is a historically based misnomer; SS is known to occur in a wide variety of organs, such as lung, kidney, and heart, and
Author contributions: N.N.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.T., N.A., T.M., N.H., K.Y., S.J., K.H., Y.T., Y.T.: collection and assembly of data; T.U., H.Y.: provision of study material; K.I.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Correspondence: Norifumi Naka, M.D., Ph.D., Musculoskeletal Oncology Service, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan. Telephone: 81-6-6972-1181; Fax: 81-6-6973-5691; e-mail:
[email protected]; or Kazuyuki Itoh, M.D., Ph.D., Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-2 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan. Telephone: 81-6-6972-1181; Fax: 81-6-6973-5691; e-mail:
[email protected] Received December 2, 2009; accepted for publication May 12, 2010; first published online in STEM C AlphaMed Press 1066-5099/2009/ CELLS EXPRESS June 1, 2010; available online without subscription through the open access option. V $30.00/0 doi: 10.1002/stem.452
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ultrastructural and immunohistochemical analyses have clearly excluded a synovial origin for this type of tumor. Thus, SS is currently classified as a miscellaneous tumor of uncertain histological origin [7] and the cellular origin of SS remains to be resolved. The hallmark traits of all cancers, that is, unlimited proliferative capacity and high phenotypic plasticity, are mirrored by self-renewal and differentiation potential of normal stem cells. Recent studies indicate that stem-like cells with dysregulated self-renewal and differentiation potentials have been significantly implicated in the pathogenesis of leukemia, breast tumors, and brain tumors [8–11]. Here, we established two novel human SS cell lines, named Yamato-SS and AskaSS. These SS cell lines possessed stem cell-like traits including the potentials to form sarcospheres, to express several stemness-related key molecules, to reestablish tumorigenicity following xenografting, and to differentiate into osteocyte and chondrocyte. We observed that SS18-SSX silencing caused both SS cells to alter the morphological characteristics from three-dimensional spherical growth to adherent monolayer growth. Further, we demonstrated that both SS18-SSX-silenced SS cells showed additional differentiation abilities into adipocytes and macrophage-like cells. On the basis of these results, we propose that a multipotent mesenchymal stem cell (MSC) is a potential cellular origin of SS, and SS is a stem cell disorder dysregulating the self-renewal and differentiation potentials mediated by SS18-SSX chimeric fusion protein.
MATERIALS
AND
METHODS
Establishment of Two Independent Cell Lines of SS Tumor cells were isolated from the surgically resected tissues at the excision of the primary tumor (patient 1) or the pulmonary metastatic tumor (patient 2) with the patients’ informed consent and under the guidelines of our institution’s ethical committee. Clinical courses of these two independent patients with SS were described in Supporting Information (patient1 and 2). The tumor tissues were minced and incubated with 1 mg/ml of collagenase (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) for 1 hour at 37 C. Cell suspensions were passed through a 40-lm nylon mesh (Becton Dickinson, Franklin Lakes, NJ, http:// www.bdbiosciences.com) and the tumor cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 10 or 20% fetal bovine serum (FBS; MP Biomedicals, Aurora, OH, http://www.mpbio. com). The adherent cells were maintained for over 24 months in culture, and the cells passed more than 200 times, fulfilling the criteria of a cell line. Throughout establishment of both cell lines, attached cells continuously expressed SS18-SSX1 transcripts (data not shown) and exhibited spindle-shaped morphology with the cluster formation of tightly packed small round cells (Supporting Information Fig. S1E, S2E). Both SS cells were cultured in serum-containing or serumfree medium at 37 C with 95% air, 5% CO2, and 100% humidity. The serum-free medium was composed of DMEM, 10 ng/ml basic fibroblast growth factor (bFGF; Invitrogen), 20 ng/ml epidermal growth factor (EGF; Invitrogen), and 10 ll/ml N2 supplement (Invitrogen).
was centrifuged at 200g for 5 min, and the cell pellet was resuspended in fresh medium (with or without serum). The resultant suspension was filtered through a 40-lm mesh, and 1 106 cells were plated on 100-mm low attachment plates (Terumo, Tokyo, Japan, http://www.terumo.com). For single-cell suspension assay, the cell pellet was resuspended in DMEM-20% FBS into a density of 10 cells/ml. Then, 100 ll single cell suspension was dispensed into each well of a 96-well culture plate (BD). Wells containing only a single cell were marked, checked daily, and maintained in DMEM-20% FBS.
Immunocytochemistry Cells were grown on coverslips coated with laminin (10 lg/ml) in four-well plates. They were fixed with 4% paraformaldehyde for 20 min at room temperature. After permeabilization with PBS containing 0.2% TritonR X-100 (Sigma-Aldrich) for 10 min, they were blocked with PBS containing 0.5% bovine serum albumin, 1% FBS, and 0.1% TritonR X-100 for 30 min, and incubated with primary antibodies for 1 hour at room temperature. The primary antibodies used were as follows; Oct3/4 (1:50, ab19857, Abcam, Cambridge, U.K., http://www.abcam.com), Nanog (1:50, ab21624, Abcam), CD44 (1:50, BBA10, R&D systems, Minneapolis, MN, http://www.rndsystems.com), CD133 (1:50, CD133/1 Pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), bIII-tubulin (1:50, MAB1195, R&D systems), Stro-1 (1:50, MAB1038, R&D systems), c-Kit (1:50, A4502, Dako, Glostrup, Denmark, http://www.dako.com). After three washes with PBS, coverslips were incubated with Alexa-conjugated second antibodies (1:1000, Invitrogen) for 30 min, washed and counterstained for 5 min with DAPI (1:25,000, Invitrogen). After mounting, the stained coverslips were observed with a fluorescence microscope (BX60; Olympus, Tokyo, Japan, http://www.olympus.co.jp) equipped with a CCD camera (VB7010; Keyence, Osaka, Japan, http://www.keyence.co.jp).
Immunohistochemistry Immunoperoxidase procedure (avidin-biotin complex method) was carried out on paraffin-embedded sections of primary SS. Antigen retrieval was done with heating the sections in 10 mmol/ l citrate buffer for 5 min. Anti-Oct3/4 (1:100, ab19857, Abcam) and anti-Nanog (1:100, ab21624, Abcam) antibodies were applied for 1 hour at room temperature. Sections were counterstained lightly with methyl green.
Transient RNA Interference Detailed experimental methods of RNA isolation, reverse transcription and PCR analysis were described in Supporting Information. siRNA-A was designed to target at the coding region of the SSX part in SS18-SSX1, whereas siRNA-B was designed to target at the 30 -untranslated region in SS18-SSX1 (Supporting Information Fig. S3A, Table S2). siRNA-control was designed for Photinus Pyralis GL3 luciferase as described previously (supporting information Table S2) [13]. Similar to secondary spheroid formation assay, 2 105 cells were resuspended in 35-mm low attachment plates (BD) and transfected with siRNA-A, siRNA-B, or siRNA-control to a final concentration of 50 nmol/l using Lipofectamine 2000 (Invitrogen). Cell lysates of Yamato-SS were prepared for immunoblot analysis 1st, 4th, 7th, 10th, 13th, and 16th days after transfection. Silencing effect of SS18-SSX fusion protein was continuously observed until day 7 (Supporting Information Fig. S3B).
Nude Mice Xenografting In Vitro Replating Assay To determine self-renewal ability, secondary spheroid formation assay and single-cell suspension assay were carried out as described previously [12], with slight modification. Briefly, for secondary spheroid formation assay, adherent cells (80% confluent) were harvested with trypsin (0.25%)/EDTA and resuspended in phosphate-buffered saline (PBS) buffer. The suspension
Five-week-old athymic nude mice (BALB/c nu/nu; SLC, Shizuoka, Japan, http://jslc.co.jp) were housed at the animal care unit of Osaka Medical Center for Cancer and Cardiovascular Diseases, in accordance with the guideline approved by the local animal ethical committee. To determine tumorigenicity, Yamato-SS or Aska-SS (2 102 to 1 107 unfractionated cells) was injected subcutaneously into the left side of back. It was difficult
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to separate the spheroids composed of highly compacted cells into single cells. To distinguish single cells from aggregates efficiently and count the number of single cells more accurately, we used trypsinized adherent cells in these experiments. Mice were inspected daily and were sacrificed when the total tumor burden reached 2 cm3. Tumor size was measured with a caliper and tumor volume calculated by the formula (a b2)/2, with a being the longest diameter and b the shortest diameter of the tumor.
In Vitro and In Vivo Interleukin-6 (IL-6) Measurement Resuspension of 2 105 cells were plated in DMEM-20% FBS on 35-mm low attachment plates (BD). At 4th, 8th, and 12th day, culture medium was collected and centrifuged at 500g for 10 min to remove floating cells, and then the supernatant was stored at 80 C until assay. Similarly, the supernatants were prepared for IL-6 measurement fourth and eighth days after SS18-SSX silencing with siRNA-A, siRNA-B, and siRNA-control. When Yamato-SS transplanted tumor reached 2 cm3, whole blood samples were collected by intracardiac puncture, allowed to clot at 4 C for 12 hour, and centrifuged at 2,000g for 10 min. The serum obtained was divided into aliquots and stored at 80 C until use. The concentrations of IL-6 were determined using the chemiluminescent enzyme immunoassay (CLEIA) kits (Fujirebio, Tokyo, Japan, http:// www.fujirebio.co.jp) in accordance with the manufacturer’s instructions.
Mesenchymal Differentiation Assay Mesenchymal differentiation assays were performed as previously described [14], with some modification. To promote osteogenic differentiation, cells were grown in DMEM, 10% FBS, 0.1 lM dexamethasone, 0.15 mM L-ascorbic acid (Sigma), 2 mM b-glycerophosphate (Sigma), and 1 mM NaH2PO4 for 10–21 days. To measure alkaline phosphatase (ALP) activity, cells were washed twice with PBS and lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc., Rockford, IL, http:// www.piercenet.com) following protocol. ALP activity was assayed using p-nitrophenylphosphate as a substrate by alkaline phosphatase test (Wako, Wako Pure Chemicals Industries, Osaka, Japan, http:// www.wako-chem.co.jp) and the protein content was measured using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc.). At day 21, paraformaldehyde-fixed calcium depositions were stained by the Von-Kossa protocol including incubation with 5% silver nitrate (Sigma) for 30 min, extensive washing with PBS, exposition for 20 min to UV and incubation in 5% sodium thiosulfate for 5 min. For chondrogenic differentiation, cells were grown in pellet as previously described [15], with some modification. Briefly, 5 105 cells centrifuged at 150g for 5 min in a 15-ml polypropylene tube were grown in pellet and cultured in 0.5 ml of DMEM with 10 ng/ml transforming growth factor b3 (TGF-b3, R&D systems), 0.1 lM dexamethasone, 50 mg/ml ascorbic acid, 100 mg/ml sodium pyruvate, 40 mg/ml L-proline and 50 mg/ml ITSþ premix (6.25 mg/ml insulin, 6.25 mg/ml transferin, 6.25 ng/ ml selenious acid, 1.25 mg/ml BSA, and 5.35 mg/ml linoleic acid) for 21 days. To assess acidic mucopolysaccharides present in cartilage tissue, pellets were formalin fixed and paraffin embedded for histological processing. Thin sections were slide-mounted and stained with alcian blue 8GX solution. For adipogenic differentiation, cells were plated and grown for 3 days at 37 C, 5% CO2 in adipogenic induction medium composed of DMEM containing 10% FBS, 0.5 mM Isobutylmethylxanthine (Sigma), 1 lM dexamethasone (Sigma), and 10 lg/ml Insulin (Sigma). Medium was then replaced by Adipogenic Maintenance Medium composed of DMEM, 10% FBS, with 10 lg/ml insulin and cell were grown for 1–3 days. After three cycles of induction/maintenance, cells were grown in Adipogenic Maintenance Medium for seven additional days. The extent of adipogenic differentiation was examined by microscopic observation of lipid vacuoles in the induced cells. To document the adipogenic differentiation, cells were fixed in 4%
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paraformaldehyde (Sigma) for staining with 0.3% w/v Oil-Red-O (Sigma) in 60% isopropanol.
Hematopoietic Differentiation Assay For hematopoietic differentiation, the colony-forming cell hematopoietic assay was performed on the SS18-SSX-silenced cells or control cells following the manufacturer’s instructions. Briefly, 2 104 cells were plated in triplicate and cultured in Methocult GF H4434 (Stemcell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) containing 1% methylcellulose in Isocove’s Modified Dulbecco’s medium (IMDM), 30% FBS, 1% bovine serum albumin, 104 M mercaptoethanol, 2 mM L-glutamine, 3 U/mL recombinant human (rh) erythropoietin (EPO), 50 ng/mL rh stem cell factor (SCF), 10 ng/mL rh granulocyte-macrophage colony stimulating factor (GM-CSF), and 10 ng/mL rh interleukin-3 (IL-3). Plates were incubated at 37 C in a humidified atmosphere with 5% CO2 and, after 14 days, each culture dish was examined under an inverted microscope (IX-70 Olympus). To confirm the hematopoietic colonies, we picked up the suspicious colonies, stained them with May-Gru¨nwald-Giemsa stain solution and immunostained with hematopoietic markers, anti-CD34 Abs (1:50, 1185, Immunotech, Marseille, France, http://www.immunotech.cz) and anti-CD68 Abs (1:1,000, PG-M1, Dako) for hematopoietic stem cell (HSC) and macrophage, respectively. We also examined the phagocytic activity of SS18-SSX-silenced cells by exposing to medium containing carbon particles (Indian ink) for 1 hour. Additionally, neurogenic differentiation assays were performed in accordance with the manufacturer’s instructions (Lonza, Basel, Switzerland, http://www. lonza.com), with some modification (see Supporting Information for details).
Statistical Analysis All data are presented as mean 6 SD. When two groups were compared, the Student’s t test was used (p < .05 was considered significant).
RESULTS Self-Renewal Capacities of Yamato-SS and Aska-SS In Vitro When cultured in the serum-containing medium (DMEM with 20% FBS) on the adhesive plates, Yamato-SS showed adherent growth pattern with spindle-shaped morphology. These adherent cells did not grow strictly in monolayer and presented a tendency to pile up on top of the first cell layer (Fig. 1A). A similar phenomenon was observed with Aska-SS (Supporting Information Fig. S4A). To study growth under nonadherent conditions, we performed secondary spheroid formation assay. Adherent cells were trypsinized, resuspended and then replated in the serum-free medium with bFGF and EGF on the low attachment plates, and their growth characteristics and morphology were monitored (Fig. 1B). Within 24 hours of replating, the single Yamato-SS cells began to form loose clumps that continued to increase in density. At day 4, there were no spheroids present, but only loose cell aggregates. At day 8, spheroids began to take shape, but the cells were still loosely organized. By day 12, spheroids (‘‘sarcosphere’’) formed completely and became well-rounded structures composed of numerous, highly compacted cells in which it is hard to distinguish individual cells from each other. Tumorigenic cancer cells with stem cell properties can form floating spheres and be enriched in the spheres only when cultured in the serum-free medium with proper mitogens such as bFGF and EGF [16–18]. Interestingly, similar time course of spheroid formation was observed even when replated in the serum-containing medium (DMEM with 20% FBS) on the
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Figure 1. Self-renewal ability retained in Yamato-SS. (A): Phase-contrast image of Yamato-SS cultured in the serum-containing medium (DMEM with 20% FBS). Adherent and spindle-shaped cells showed a tendency toward piling up over the attached cell layer. Scale bar ¼ 100 lm. (B, C): Secondary spheroid formation assay. Scale bar ¼ 100 lm. Phase-contrast images of Yamato-SS cultured in the serum-free medium supplemented with bFGF and EGF (B) or in the serum-containing medium (DMEM with 20% FBS) (C) on the low attachment plates at day 4 (left), day 8 (middle), and day 12 (right). (D): Single-cell suspension assay. Phase-contrast images of a single Yamato-SS cell cultured in the serum-containing medium (DMEM with 20% FBS) at day 1 (top left), day 4 (top middle), day 8 (top right), day 12 (bottom left), and day 20 (bottom middle). Scale bar ¼ 50 lm. With time in culture, a single Yamato-SS cell divided, clustered, formed a cellular aggregate, and eventually generated a sarcosphere.
low attachment plates (Fig. 1C). Sarcospheres could be passaged multiple times by mechanical dissociation of large spheres and reseeding in fresh medium every 7 days and could be maintained for 16 weeks or more. We also obtained similar findings in secondary spheroid formation assay with Aska-SS (Supporting Information Fig. S4B). We further investigated sphere forming capability of both SS cells using single-cell suspension assay in the serum-containing medium on the low attachment plates (Fig. 1D). First day, only individual Yamato-SS cells and no clusters could be observed.
Within 3 weeks, these single cells generated sarcospheres with an efficiency of 1/50, despite the fact that those assays were carried out without fractionation by any surface markers such as CD133, higher than that reported by others for breast and brain malignancies [10, 11]. By contrast, sarcosphere formation in single-cell suspension assay was observed in AskaSS cells with a lower efficiency of 1/400 (Supporting Information Fig. S4C). Despite imperfect, in vitro sphere formation assays have been used to evaluate self-renewal potential [19– 21]. Thus, these findings suggested that both SS cells
Figure 2. Expression of stemness-related genes in Yamato-SS, Aska-SS cells, and 15 clinical SS samples. (A): Reverse transcription polymerase chain reaction (RT-PCR) analysis of stemness-related genes in Yamato-SS and Aska-SS under adherent and nonadherent condition. (B): Immunofluorescence analysis with antibody against Oct3/4 and Nanog in adherent Yamato-SS cells. Nuclei of Yamato-SS cells were counterstained with DAPI (top left and bottom left). Nuclei of all Yamato-SS cells were stained for anti-Oct3/4 (top middle) and anti-Nanog antibodies (bottom middle). Phase-contrast image of Yamato-SS cells (top right and bottom right). Scale bar ¼ 25 lm. (C): Immunofluorescence analysis with antibody against Oct3/4 and Nanog in suspended (spheroid) Yamato-SS cells. Nuclei of Yamato-SS cells within a spheroid were counterstained with DAPI (top left and bottom left). Nuclei of nearly all Yamato-SS cells were stained for anti-Oct3/4 (top middle) and anti-Nanog antibodies (bottom middle). Phase-contrast image of Yamato-SS cells (top right and bottom right). Scale bar ¼ 25 lm. (D): RT-PCR analysis of stemnessrelated genes in clinical samples from 15 patients with SS. (E): Immunohistochemical staining for Oct3/4 and Nanog in sections from tumor biopsies of a representative SS clinical sample (case No 5; Supporting Information Table S3). H&E staining (top left). Multiple nuclear staining for anti-Oct3/4 (top right) and scattered nuclear staining for anti-Nanog antibodies (bottom left). Scale bar ¼ 25 lm. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Table 1. Comparison of tumorigenic cell doses between Yamato-SS and Aska-SS Cell doses and tumor formationa
SS cell lines Yamato-SS Aska-SS
100,000 6/6 6/6
10,000 6/6 6/6
1,000 6/6 6/6
200 6/6 2/6
a Cell dose, number of cells per injection; tumor formation, number of tumors formed/number of injections; tumor takes was considered unsuccessful when no tumor mass was visible during 5 months follow up. Abbreviation: SS, synovial sarcoma.
contained enriched individual stem-like cells to form sarcosphere colonies and self-renew in vitro, albeit with the higher ability in Yamato-SS compared to Aska-SS cells.
Both Yamato-SS and Aska-SS Express Stemness-Related Genes Current evidence indicated that some of key regulators of stem cell identity such as Oct3/4, Nanog, and Sox2 were expressed in several human cancer types, which could be implicated in cancer pathogenesis and stemness-related phenotype [22–25]. Using reverse transcription polymerase chain reaction (RTPCR) analysis, we found that both Yamato-SS and Aska-SS cells expressed all of those stemness-related genes tested under both adherent and nonadherent (spheroid) conditions (Fig. 2A). To determine whether these genes were expressed at the protein level, two representative pluripotent markers, Oct3/4 and Nanog were examined by fluorescent immunocytochemistry. Most of all adherent Yamato-SS cells showed strong fluorescent signals of Oct3/4 and Nanog (Fig. 2B). Further, nearly all SS cells within a spheroid showed a compact morphology and strong fluorescent signals of Oct3/4 and Nanog similar to adherent cells (Fig. 2C). We also assessed mRNA expression of stemnessrelated genes in 15 clinical cases with SS treated in our institute (Supporting Information Table S3). Interestingly, RT-PCR analysis revealed that all 15 clinical samples expressed Oct3/4, Nanog, and Sox2 as two SS cell lines did (Fig. 2D). Additionally, paraffin sections from four SS patients were evaluated using immunohistochemistry. Oct3/4 and Nanog nuclear staining were observed in all four tumors studied, although the number of positive cells varied considerably among tumor specimens (Fig. 2E).
Both Yamato-SS and Aska-SS Are Enriched for Cancer Initiating Ability Most of xenograft models using human sarcoma cell lines require 106–107 cells for tumor engraftment and formation. Transplantation of 107 Yamato-SS cells formed tumors in the subcutis of nude mice within 2 weeks, reaching a size of about 1 cm3 within 4 weeks (Supporting Information Fig. S5A). Inoculation of 107 Aska-SS cells also formed tumors albeit with slower growth compared to Yamato-SS (Supporting Information Fig. S5B). To determine whether small numbers of Yamato-SS or Aska-SS were able to initiate tumor formation, nude mice were transplanted with 104, 103, and 2 102 of adherent Yamato-SS or Aska-SS cells. For facility in separating into single cells, we used in this experiment the adherent SS cells instead of the spheroid cells, while mounting evidence suggested that adherent cells were less tumorigenic than spheroid counterparts when grafted to mice [26]. The cancer stem cell model has suggested that only a rare, phenotypically distinct subset of cancer cells that express surface markers such as CD44 or CD133 had the capacity to sig-
nificantly proliferate and form new tumors. By contrast, almost all adherent SS cells showed strong fluorescent signals of CD44 but little cells expressed CD133 (Supporting Information Fig. S14 and data not shown). Thus, we carried out these xenografting experiments using single SS cells without fractionation by any surface markers. The results showed a substantial difference in tumorigenic properties between two SS cell lines. Tumors were consistently generated even after injection of 2 102 unfractionated Yamato-SS cells in all six mice that were analyzed at 6–10 weeks postinoculation. By contrast, injection of 2 102 Aska-SS cells resulted in tumor growth in two out of six mice (Table 1). Histological examination revealed that the tumors in mice were composed of three morphologically different types of cells: lymphoma-like round cells, pseudoglandular epithelial cells, and fibrosarcoma-like spindle cells, corresponding to poorly differentiated type, biphasic type, and monophasic fibrous type of SS histological classification, respectively (Supporting Information Fig. S5C). These findings indicated that both SS cells had the ability to give rise to a heterogeneous cell population in vivo. To examine the self-renewal capacity in vivo of these SS cells, we evaluated the ability of these cells to generate tumors after serial transplantation in nude mice [27]. For these experiments, primary tumor xenografts that arose from injection of 104 Yamato-SS or Aska-SS cells were obtained at sacrifice and dissociated into single-cell suspensions. Subsequently, unfractionated 104 SS cells were reinjected into new recipient mice. Secondary tumor formation was observed with all mice and the tumor histology was similar to their respective primary tumors (Supporting Information Fig. S5D). Secondary xenografts were excised and reinjected, resulting in tertiary tumors that recapitulated the phenotypes of the primary tumors (Supporting Information Fig. S5E). Together, these results provided evidence for the enriched cancer (sarcoma)-initiating ability and in vivo self-renewal capacity of both Yamato-SS and Aska-SS cells.
SS18-SSX Silencing Causes Both SS Cells to Shift from Spherical Growth to Adherent In Vitro and to Reduce Their Self-renewing Abilities SS18-SSX fusion protein resulting from the SS-specific t(X;18)(p11;q11) chromosomal translocation is considered to play a pivotal role in SS development and progression. To investigate the cellular origin of SS, we performed SS18-SSX silencing on Yamato-SS using two sequence specific siRNAs, siRNA-A, and siRNA-B. Until day 3, no morphological difference was observed among the untreated cells, treated cells with siRNA-control, and two siRNAs, showing the gradual formation of spheroid aggregate in suspension culture on low attachment plates. At day 4, both SS18-SSX-silenced cells dramatically changed their appearance from spherical growth in suspension to adherent growth in monolayer (Fig. 3A). Compared to the fine spindle morphology of unsilenced cells under adherent condition (Supporting Information Fig. S1E), these SS18-SSX-silenced Yamato-SS cells exhibited slightly larger, flattened, and fibroblastoid (MSC-like) appearance. We also observed similar morphological changes with silenced AskaSS cells (Supporting Information Fig. S6). Collectively, these findings suggest that SS18-SSX fusion protein enhances the sphere-formation ability and may involve the self-renewal potential of a potential source of SS in vitro.
Yamato-SS Expresses IL-6 In Vitro and In Vivo, and Increases Its Production by SS18-SSX Silencing The patient from whom Yamato-SS was derived had presented the paraneoplastic syndrome including leukocytosis,
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Figure 3. SS18-SSX silencing using sequence-specific siRNAs. (A): Phase-contrast images of Yamato-SS cells growing in the serum-containing medium on low attachment plates at day 7. siRNA-mediated silencing of SS18-SSX causes YamatoSS cells to flatten (bottom left; siRNA-A, bottom right; siRNA-B) whereas no treated (top left) and control transfected cells (top right) continue to grow as sarcospheres. Scale bar ¼ 100 lm. (B): Secretion of IL-6 from Yamato-SS cells in vitro. SS18-SSX silencing significantly enhanced the secretion of IL-6 into the culture medium at day 4 and day 8 (siRNA-A, siRNA-B). Results showed the mean 6 SD of triplicate samples, (*p < .0005 compared to siRNA control at day 4 and day 8, respectively).
elevated C-reactive protein (CRP) induced by IL-6. In addition, MSCs were reported to constitutively express IL-6 [28, 29]. Thus, we examined whether Yamato-SS expressed IL-6 in vitro and in vivo, or regulated its expression by SS18-SSX silencing. The concentration of IL-6 was increased in a timedependent manner in the culture media of Yamato-SS cells (Supporting Information Fig. S7A). Also in vivo, the serum human IL-6 concentrations in the inoculated mice (50.2 6 12.4 pg/ml, mean 6 SD) were markedly higher compared to those in the control mice (0.3 6 0.1 pg/ml) (Supporting Information Fig. S7B). Upon SS18-SSX silencing with sequencespecific siRNA, these Yamato-SS cells extremely elevated the production of IL-6 into the culture media (Fig. 3B). We also found a similar response of IL-6 production in Aska-SS cells upon SS18-SSX silencing. The IL-6 concentrations were elevated in the culture media of silenced Aska-SS cells, though those levels were relatively low compared to those of silenced Yamato-SS cells (Supporting Information Fig. S8). These www.StemCells.com
findings suggested that the cellular origin of these SS cell lines might be a mesenchymal stem-like cell expressing IL-6, and SS18-SSX fusion protein rendered the SS cell of origin to downregulate IL-6 expression.
Both Yamato-SS and Aska-SS Cells Express Multiple Mesenchymal Lineage Genes and Start to Express More Committed Mesenchymal Lineage Genes by SS18-SSX Silencing We next examined whether Yamato-SS expressed the transcripts of mesenchymal lineage genes with or without SS18SSX silencing. RT-PCR showed that the unsilenced cells expressed numbers of transcripts, including runt-related transcription factor 2 (RUNX2) and Osterix (to osteogenic lineage), Sox9 (to chondrogenic), and peroxisome proliferatoractivated receptor c (PPARc) (to adipogenic), those were early stage key molecules for specific differentiation in each
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Figure 4. Mesenchymal differentiation potentials of SS18-SSX-silenced Yamato-SS cells. (A): Reverse transcription polymerase chain reaction analysis of mesenchymal lineage genes in Yamato-SS with or without SS18-SSX silencing. (B): ALP activity in Yamato-SS upon combined effects of SS18-SSX silencing and osteogenic (OS) inducer. Results showed the mean 6 SD of triplicate samples, (*p < .05; **p < .01 compared to the control). (C): Von-Kossa staining of the mineralized matrix formation in Yamato-SS upon combined effects of SS18-SSX silencing and osteogenic inducer. Scale bar ¼ 100 lm. (D): Alcian blue staining of the acid mucopolysaccharides formation in pellets of Yamato-SS upon combined effects of SS18-SSX silencing and chondrogenic inducer. Scale bar ¼ 60 lm. (E): Phase contrast images of Yamato-SS cells upon combined effects of SS18-SSX silencing and adipogenic inducer. Unsilenced cells cultured in standard medium (top left), silenced cells cultured in standard medium (top right), unsilenced cells cultured in adipogenic medium (bottom left), silenced cells cultured in adipogenic medium (bottom right) with bright vacuoles in the cytosol (arrow). Scale bar ¼ 100 lm. Abbreviations: ALP, alkaline phosphatase; ALPL, alkaline phosphatase, liver/bone/kidney; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPL, lipoprotein lipase; OCN, osteocalcin; OPN, oteopontin.
mesenchymal lineage (Fig. 4A). Further, SS18-SSX silencing induced these cells to express the transcripts of Osteocalcin (to osteogenic), Aggrecan and COL10A1 (to chondrogenic), and fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL) (to adipogenic), those were late stage key molecules of each specific mesenchymal differentiation (Fig. 4A). We also observed the mesenchymal expression profile of Aska-SS with or without SS18-SSX silencing similar to that of Yamato-SS (Supporting Information Fig. S9). Additionally, we analyzed the expression of neural-related genes with or
without SS18-SSX silencing in Yamato-SS and Aska-SS cells. While both unsilenced SS cells expressed all of neural-related genes tested including Nestin, neurofilament, light polypeptide (NFL), neurofilament, medium polypeptide (NFM), neurofilament, heavy polypeptide (NFH), bIII-tubulin, and mammalian achaete-scute homologue 1 (MASH-1), SS18-SSX silencing reduced the expression of bIII-tubulin and MASH-1 (Supporting Information Fig. S10A, B). Subsequently, we examined the expression of bIII-tubulin in Yamato-SS cells with or without SS18-SSX silencing by fluorescent
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Figure 4.
immunocytochemistry. On treatment with neurogenic inducer for 7 days, unsilenced Yamato-SS cells showed dendrite-like processes with strong bIII-tubulin staining, while silenced cells exhibited weak and disassembled fluorescent signals (Supporting Information Fig. S10C). www.StemCells.com
(Continued)
SS18-SSX-Silenced Cells Differentiate Along the Osteogenic, Chondrogenic, and Adipogenic Lineage To confirm that Yamato-SS arose from a MSC, we next tested whether this cell line could differentiate along terminal
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differentiation in the mesenchymal lineages (osteogenic, chondrogenic, and adipogenic) upon combined effects of SS18-SSX silencing and treatment with appropriate differentiation cocktails. First, we examined the activity of ALP, an early phase marker of osteogenic differentiation, in SS18-SSX-silenced or -unsilenced cells cultured in either standard or osteogenic differentiation media. At day 4, when morphological change was observed, SS18-SSX-silenced cells showed increased ALP activity compared to unsilenced cells. At day 8, when cultured in the respective condition for another 4 days, unsilenced cells cultured in differentiation media exhibited modest increase in ALP activity compared to those in standard media. SS18-SSX silencing of Yamato-SS cells grown in standard or differentiation media led to a much stronger induction of ALP activity (Fig. 4B). We also conducted similar experiments for longterm osteogenic differentiation and evaluated by Von-Kossa staining. Cultured in differentiation medium for 3 weeks, SS18-SSX-silenced cells formed dense mineralized nodules (Fig. 4C-d), but unsilenced cells did slightly sparse nodules (Fig. 4C-c), while neither silenced nor unsilenced cells formed mineralized deposition in the standard medium (Fig. 4C-a, 4C-b). Second, chondrogenic differentiation of Yamato-SS cells was examined in the pellet culture with either standard or specific differentiation medium. Similar to osteogenic differentiation assay, SS18-SSX-silenced cells cultured in the chondrogenic differentiation medium for 21 days showed positive signals of acidic mucopolysaccharides formation on Alcian Blue staining (Fig. 4D-d), while unsilenced cells cultured in the same condition exhibited weaker signals (Fig. 4D-c). Irrespective of SS18-SSX silencing, these SS cells incubated in standard medium could not show any acidic mucopolysaccharides formation (Fig. 4D-a, b). These data indicated that the Yamato-SS by itself possessed the capability of osteogenic and chondrogenic differentiation, and that these potentials were enhanced by SS18-SSX silencing. Finally, adipogenic potential was also assessed after three cycles of adipogenic induction or culturing in standard medium. When incubated in standard medium, Yamato-SS cells did not show any cell vacuolation with or without SS18-SSX silencing (Fig. 4E-a, 4E-b). Interestingly, unsilenced cells cultured in inductive condition disrupted adhesive growth and could not exhibit adipogenic differentiation (Fig. 4E-c). By contrast, exclusively SS18-SSX-silenced cells cultured in adipogenic medium demonstrated bright stained vacuoles in the cytosol (Fig. 4E-d, arrow), which were confirmed as lipid droplets by Oil red O staining (Supporting Information Fig. S11). These results indicated that the commitment into adipogenic lineage was presented to Yamato-SS only when both SS18-SSX silencing and incubation in specific inductive condition are combined. Likewise, SS18-SSX silencing enhanced the potential of Aska-SS cells to differentiate into osteocyte, chondrocyte, and adipocyte when incubated in appropriate differentiation media (Supporting Information Fig. S12). In addition, almost all adherent Yamato-SS cells showed strong fluorescent signals of CD44, Stro-1 and c-Kit, which were cell surface proteins associated with MSCs (Supporting Information Fig. S14). Taken together, these findings suggested that the cellular origin of SS might be a MSC.
SS18-SSX-Silenced Cells Differentiate into Macrophage-Like Cells IL-6 is mainly produced by several types of hematopoietic cells including T-cells, B cells, and monocytes, and this cytokine has also been reported to exert positive effect on hematopoiesis [30, 31]. As Yamato-SS and Aska-SS cells secreted IL-6 and upregulated its production by SS18-SSX silencing,
Synovial Sarcoma Is a Stem Cell Malignancy
we next asked whether these SS cell lines could differentiate along the hematopoietic lineage. We first examined whether Yamato-SS expressed the transcripts of hematopoietic lineage genes with or without SS18-SSX silencing. RT-PCR demonstrated that the unsilenced cells expressed early hematopoietic lineage genes, such as c-Kit and stem cell leukemia (SCL) (Fig. 5A). Along with SS18-SSX silencing, these cells started to express the transcripts of granulocyte macrophage colonystimulating factor receptor, which was a late stage marker of myelomonocytic differentiation in hematopoietic lineage (Fig. 5A). Second, we stained the smear specimen of these cells with or without SS18-SSX silencing using May-Gru¨nwaldGiemsa staining. Unsilenced cells showed considerably homogeneous appearance with large round nucleus, which was reminiscent of myelomonocytic leukemia (Fig. 5B-a). By contrast, with the treatment of SS18-SSX silencing, these cells heterogeneously changed their morphology into large macrophage-like cells with abundant cytoplasm and small monocyte-like cells (Fig. 5B-b). Finally, we treated the SS18-SSXsilenced Yamato-SS cells with rh SCF, rh GM-CSF, rh IL-3, and rh EPO for 2 weeks. These cells showed mature macrophage-like morphology with abundant cytoplasm and clearly demonstrated anti-CD68 immunoreactivity as well as Indian ink phagocytic activity, which were specific to macrophage (Fig. 5C). These observations indicated that Yamato-SS could differentiate into macrophage-like cells. We also observed similar hematopoietic expression profile of Aska-SS with or without SS18-SSX silencing and the potential of SS18-SSXsilenced Aska-SS cells to differentiate into macrophage-like cells (Supporting Information Fig. S9, S13A, B). Additionally, neither silenced Yamato-SS nor silenced Aska-SS cells demonstrated anti-CD34 immunoreactivity, which were related to HSCs (data not shown). Collectively, these studies indicated that the cell of origin for SS might be a multipotent stem cell that was capable of differentiating along mesenchymal lineage and into a macrophage-like cell.
DISCUSSION The relationship between cancer cells and normal stem cells is a question of great current interest. Recent reports that histologically poorly differentiated tumors show preferential overexpression of genes associated stem cell identity raises the possibility that these stemness-related genes may contribute to stem cell-like tumor phenotypes and aggressive tumor behavior [22–25]. We demonstrated in this study that Yamato-SS as well as Aska-SS cells expressed Oct3/4, Nanog and Sox2, which were thought to form the interconnected autoregulatory loop and act coordinately to maintain the transcriptional program required for pluripotency [32]. Although highly restricted in their expression pattern to embryonic stem cells, cells of inner cell mass, and to cells of the germ line [33], these three factors were expressed only in specific human cancer types [22, 23, 34, 35]. Parallel to the results of both SS cell lines, a similar expression profile was observed in all 15 clinical SS samples, suggesting that SS may be one of the tumors inherently possessing stem cell-like traits. In this study, we showed that both unfractionated SS cells could form sarcospheres in the secondary spheroid formation assays and the single-cell suspension assay in vitro. We also found that as few as 200 Yamato-SS or Aska-SS cells were able to initiate the tumor formation in nude mice. Moreover, hematoxylin-eosin staining and microscopic analysis revealed that serial xenografts of either SS cells consistently reproduced the tumors with histologically heterogeneous
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Figure 5. Hematopoietic differentiation potential of SS18-SSX-silenced Yamato-SS cells. (A): Reverse transcription polymerase chain reaction analysis of hematopoietic lineage genes in Yamato-SS with or without SS18-SSX silencing. (B): May-Gru¨nwald-Giemsa staining of Yamato-SS with or without SS18-SSX silencing. Scale bar ¼ 50 lm. (C): SS18-SSXsilenced Yamato-SS cells cultured for 2 weeks in methylcellulose supplemented with hematopoietic inducers. May-Gru¨nwald-Giemsa staining (top left), Immunostaining for anti-CD68 Abs (top right), Indian ink phagocyte test (bottom left). Scale bar ¼ 20 lm. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GATA, GATA family of zinc finger transcription factor; GCSFR, granulocyte colony stimulating factor receptor; GMCSFR, granulocyte macrophage colony-stimulating factor receptor; SCL, stem cell leukemia.
populations mimicking to human SS, indicating that these SS cell lines maintain their tumorigenic potential with the ability to replicate the original tumor. Collectively, these results suggest that both SS cell lines contain enriched stem-like cells retaining the self-renewing capacity and strong sarcoma-initiating ability, thus they have a number of characteristics to be called ‘‘stem-like cells" without fractionation by any surface markers. In this context, further characterization of these two SS cell lines will provide paradigms of the biology of sarcoma-initiating cells and might also lead to improved therapeutics for many sarcomas. www.StemCells.com
Identification of a potential cell of origin for SS is a current challenge and critical step in understanding the tumorigenesis of this malignancy and discerning its molecular features. SS is a relatively frequent, highly aggressive soft tissue sarcoma marked by a characteristic t(X;18) translocation. Although the resulting SS18-SSX chimeric fusion protein is thought to play a crucial role for transcriptional regulation and trigger SS development, the ‘‘cell of origin’’ for SS is still unknown. In an attempt to solve the tumorigenesis of SS, we have silenced SS18-SSX expression on two SS cell lines by sequence-specific siRNAs. Our study clearly showed that both
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SS18-SSX-silenced cells transformed their appearance from spherical growth in suspension to adherent growth in monolayer, indicating that SS18-SSX fusion protein may involve the ability of self-renewal in the ‘‘cell of origin’’ for SS. Previous studies indicated that Ewing sarcoma breakpoint region 1-Friend leukemia integration 1 (EWS-FLI1) and FUS-CHOP could transform murine MSCs and induce the formation of tumors resembling human Ewing’s sarcoma and myxoid liposarcoma, two other well-studied translocation-related sarcomas, respectively [36, 37]. In addition, recent evidence suggested that silencing EWS-FLI1 caused at least some Ewing’s sarcoma cell lines to exhibit MSC-like properties [38]. We also found in this study that SS18-SSX-silenced SS cells could exhibit differentiation characteristics of mesenchymal lineages. Long-term osteogenic or chondrogenic induction showed that terminal differentiation could be observed with the production of calcified matrix or acidic mucopolysaccharides in both silenced and unsilenced SS cells, albeit with stronger induction in the former. These data are consistent with several clinical reports that SS sometimes exhibited calcification/ossification or chondroid change in the tumor mass [1, 39]. By contrast, an evident commitment toward the adipogenic lineage was exclusively found in SS18-SSX-silenced SS cells, suggesting that the latent powers to differentiate into adipocytes was intrinsically present in the ‘‘cell of origin’’ for SS but attenuated by SS18-SSX fusion protein. On the contrary, the present study showed that the expression of some neural tissue-related genes was downregulated upon SS18-SSX silencing at the transcriptional and translational levels, suggesting that SS18-SSX expression itself may be responsible, at least in part, for neuronal phenotype of SS through the regulation of neural tissue-related genes. These experimental data are consistent with the findings of sequential studies that SS was clustered into one group with malignant peripheral nerve sheath tumor and several SS cell lines can undergo neural differentiation upon treatment with various differentiation-inducing agents [40, 41]. Moreover, those data may be parallel to the growing evidence that expression of the neural-specific markers of Ewing’s sarcoma is a result of upregulation by EWS-FLI1 fusion protein rather than dependent on the cellular origin of Ewing’s sarcoma [42, 43]. One of the most surprising outcomes of this study is that both Yamato-SS and Aska-SS cells can also differentiate into macrophage-like cells upon SS18-SSX silencing. These findings suggest that a MSC, a suspected ‘‘cell of origin’’ for SS, may exert another differentiation potential into a cell of hematopoietic lineage. Previous reports demonstrated the evidence for the presence of a multipotent MSC with differentiation capacity into a macrophage-like cell in experimental mouse model systems [44, 45]. Further, a recent work has shown that only two transcription factors could induce the differentiation of mouse fibroblast into macrophage [46]. Addi-
REFERENCES 1 2 3 4 5
Weiss SW, Goldblum JR. Enzinger and Weiss’s Soft Tissue Tumors, 5th ed. St. Louis MO: Mosby Inc, 2008:1161–1182. Clark J, Rocques PJ, Crew AJ et al. Identification of novel genes, Syt And SSX, involved in the t(X;18)(p112;q112) translocation found in human synovial sarcoma. Nat Genet 1994;7:502–508. Crew AJ, Clark J, Fisher C et al. Fusion of SYT to two genes, SSX1 And SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. EMBO J 1995;14:2333–2340. Skytting B, Nilsson G, Brodin B et al. A novel fusion gene, SYTSSX4, in synovial sarcoma. J Natl Cancer Inst 1999;91:974–975. Nagai M, Tanaka S, Tsuda M et al. Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1
tionally, human monocytes were reported to be capable of differentiating into fibroblast-like cells [47]. Those studies suggested a close relationship between mesenchymal and hematopoietic lineages, in particular fibroblast and macrophage lineage, and also support the proposal that human multipotent MSCs with potential to differentiate into macrophage-like cells are presumably in existence. Taken together, these results lead us to hypothesize that SS may arise from a stillunidentified human multipotent MSC and SS18-SSX chimeric fusion protein may transform a potential cell of origin for SS through alteration of self-renewal activity and perturbation of the differentiation program. Further, extensive comparison between SS18-SSX stably silenced SS cells and various MSC preparations would help to make definitive answers to these challenging issues.
CONCLUSIONS In summary, we report here the establishment of two human SS cell lines with different stem cell-like properties and demonstrate that human multipotent MSCs are a potential source of SS. These cell lines will lead to a considerable increase in our understanding of the sarcomagenesis in SS, raise the possibility that SS is a stem cell malignancy resulting from dysregulation of self-renewal and multilineage differentiation capacities by SS18-SSX fusion protein, and provide key insight into more efficient drug design and therapy in future.
ACKNOWLEDGMENTS We thank Akemi Takenaka, Kikuichi Nakagawa, and Katsuhiko Yoshizato for technical support and Naoko Shimatani, Chieko Naka, and Mayumi Obori for administrative support. This work was supported in part by the Japan Orthopedics and Traumatology Foundation, Inc., Osaka Cancer Research Foundation, Osaka Foundation for the Prevention of Cancer and Cardiovascular Diseases, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Foundation for Promotion of Cancer Research in Japan, and a Grant-in-Aid for Health Labor Sciences Research (11-6) from the Ministry of Health, Labor and Welfare, Japan.
DISCLOSURE
OF OF
POTENTIAL CONFLICTS INTEREST
The authors indicate no potential conflicts of interest.
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