Molecular Mechanisms Regulating the Phenotype of Tumor
Initiating Cells of Osteosarcoma 1,3Fujiwara T; 1Kosaka N;
1Takahashi R; 1Takeshita F; 2Kawai A; 3Ozaki T; +1Ochiya T
+1 Division of Molecular and Cellular Medicine, National Cancer
Center Research Institute, Tokyo, 2 Department of Musculoskeletal
Tumor Surgery, National Cancer Center Hospital, Tokyo,
3 Department of Orthopedic Surgery, Okayama University Graduate
School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama
[email protected]
INTRODUCTION:
There are growing evidences that tumors contain a subset of
cells with stem like properties. These cells are referred as
“cancer stem cells (CSCs)” or “tumor initiating cells (TICs)” that
are responsible for forming the bulk of tumor1). These cells
possess both self renewal and differentiation capabilities and are
believed to give rise to tumor heterogeneity2). Furthermore, they
have been shown to be associated with the most lethal
characteristics of tumors—drug resistance and metastasis2)3).
Osteosarcoma is the most common primary bone malignancy and
accounts for 60% of all malignant childhood bone tumors. Despite
intensive efforts to improve both surgical and medical management,
the long-term survival has not improved over the last 30 years4).
There is a great need for developing new osteosarcoma
treatments.
Since the proposal of the cancer stem cell hypothesis, several
studies have been performed to identify cancer stem cells of
osteosarcoma5). These cells have been detected in spherical clones
under anchorage-independent, serum-starved culture conditions, as
side population (SP) cells based on efflux of Hoechst 33342 dye or
as CD117 and stro-1 cells sorted using cell surface marker6). To
date, however, the molecular mechanisms underlying the phenotype of
CSCs remain obscure. In this study, we aimed to detect a subset of
cells showing the phenotype of cancer stem cells and to analyze the
molecular mechanisms including microRNAs which have enormous
possibilities as new hopes for diagnostic and therapeutic strategy
against osteosarcoma. METHODS: Cells and cell culture:
Institutional Review Board Approval was obtained before all
experimental studies. The human osteosarcoma cell lines SaOS2,
U2OS, MG63, HOS, MNNG/HOS, 143B were purchased from the American
Tissue Culture Collection.
Flow cytometric analysis: Cell sorting by flow cytometry was
performed on a JSAN cell sorter (Baybioscience) using PE-conjugated
monoclonal mouse anti-human CD133/2 (Miltenyi Biotec), and
APC-conjugated monoclonal mouse anti-human CD44 (BioLegend).
Spheroid formation assay: 5,000cells/well were cultured in
serum-free DMEM/F12 medium, supplemented with EGF, bFGF, insulin,
B27, penicillin and streptomycin in ultra-low-attachment 24-well
plates. Invasion assay: Cells were seeded at 100,000cells/well on
BD Bio Coat Matrigel Invasion chambers (BD Biosciences). After
incubation for 24 h at 37°C, cells that passed through the filters
into the lower wells were fixed, stained with Hema-3, counted and
photographed.
RT-PCR and quantitative real-time PCR: Total RNA was isolated
with QIAzol (QIAgen). cDNA was synthesized using the High Capacity
cDNA Reverse Transcription Kit (Applied Biosystems). For each qPCR
reaction, equal amounts of cDNA were mixed with Power SYBR Green
PCR master mix and following primers: CD133, Oct3/4, Nanog, ABCB1,
ABCG2, MMP13 and CXCR4. β-actin was amplified as an internal
control.
MiRNA Profiling: MiRNA microarrays were manufactured by Agilent
Technologies. Labeling and hybridization of total RNA samples were
performed according to the manufacturer's protocol.
Transfection with synthetic miRNAs: Synthetic hsa-miRNAs and
miRNA control were transfected into cell lines using DharmaFECT (GE
Healthcare).
Statistical analyses: Statistical analyses were conducted using
the Student’s t-test. P-value of 0.05 or less was considered to
indicate a significant difference.
RESULTS:
Osteosarcoma cell lines contained a rare population of CD133high
cells showing cancer stem-like phenotype: All cell lines contained
small fraction of CD133high cells and large fraction of CD44high
cells. SaOS2 and HOS had relatively large fraction of CD133high
cells among these cell lines. CD133 high fraction showed asymmetric
devision, more potential to form spheroid colony formation in the
anchorage-
independent environment (1AB), more invasive potential (1CD).
CD133high cells showed higher tumorigenicity in vivo.
CD133high cells showed high expression of several marker
genes:
CD133high fraction showed higher expression of Oct4, Nanog
(associated with stemness), ABCB1, ABCG2 (associated with drug
resistance), MMP13 and CXCR4 (associated with metastasis) than
CD133low fraction (Figure 2). Several microRNAs were up-regulated
in CD133high cells and altered the phenotype of CD133low cells
synergically: Microarray analysis identified several miRNAs were
up-regulated in CD133high cells compared to CD133low cells. These
miRNAs promoted chemoresistance and matrigel invasion of SaOS2
(Figure 3) and MNNG/HOS CD133low cells. Interestingly, the
combination of these miRNAs highly promoted matrigel invasion.
Moreover, the exposure of 143B cells to doxorubicin (DOX) increased
the expression of CD133 (4A) and related miRNAs (4B).
DISCUSSION:
The present study demonstrated that osteosarcoma contained a
small population of CD133high cells showing TIC-like phenotype.
Moreover, several microRNAs revealed to act as regulators of the
phenotype in light of asymmetric division, chemoresistance, and
invasiveness. In addition, chemotherapeutics might induce the
phenotype of cellular signaling pathways involved in the phenotype.
Our results, with the analysis of the target of the miRNAs, not
only will allow for better understanding of this specific
population of cells but also will provide insight into the gradual
improvement of more effective therapies against osteosarcoma.
SIGNIFICANCE:
Our results demonstrated that several microRNAs regulated the
phenotype of tumor initiating cells of osteosarcoma. This study
would contribute to achieve a new clinical technique through a
perspective of RNA interference. REFERENCES: 1) Clarke MF, Dick JE,
et al. Cancer Res. 2006; 66: 9339-44. 2) Visvader JE, Lindeman GJ,
et al. Nat Rev Cancer. 2008; 8: 755-68. 3) Liu C, Kelnar K, et.al
Nat Med. 2011; 17: 211-5. 4) Bielack SS, Kempf-Bielack B, et al. J
Clin Oncol 2002; 20: 776–90. 5) Siclari VA, Qin L, et al. J Orthop
Surg Res. 2010; 27; 5: 78. 6) Adhikari AS, Agarwal N, et al. Cancer
Res. 2010; 70: 4602-12.
Poster No. 1469 • ORS 2012 Annual Meeting
Main MenuSearchProgramAuthor IndexKeyword IndexCopyrightSession
Number 001: Stem Cells and ProgenitorsSession Number 002: Tendon
and Ligament: Biology & DevelopmentSession Number 003:
Molecular Influences on Cartilage BiomechanicsSession Number 004:
Inflammation in Osteoarthritis and Immune-Medicated
ArthritisSession Number 005: Cell and Molecular Biomechanics:
OsteocytesSession Number 006: Intervertebral Disc: Stem Cells &
Cell LineSession Number 007: Tendon and Ligament:
RegenerationSession Number 008: Cell and Molecular Biomechanics:
Physical EffectsSession Number 009: Experimental Osteoarthritis
Models: Lubrication and PainSession Number 010: Fracture Healing -
QualitySpotlight Session 011: SpineSpotlight Session 012: ACL
ReconstructionSpotlight Session 013: Non-Coding RNA and
Posttranscriptional Regulation in OASpotlight Session 014: Tissue
Reactions to WearSpotlight Session 015: OsteoporosisSession Number
016: Intervertebral DiscSession Number 017: ACL Injury
MechanicsSession Number 018: Cartilage and Meniscus RepairSession
Number 019: Cell SignalingSession Number 020: Bone - AgingSession
Number 021: Bone and Spine BiomechanicsSession Number 022: Tendon
BiomechanicsSession Number 023: Bone MechanicsSession Number 024:
Cartilage Degradation Biomarkers and ImagingSession Number 025:
Fracture Healing ModulationSpotlight Session 026: Current Trends in
Joint ReplacementSpotlight Session 027: Tendon and Ligament:
Translational ResearchSpotlight Session 028: microRNASpotlight
Session 029: Regenerative Approaches for OsteoarthritisSpotlight
Session 030: Muscle/BoneSession Number 031: Hip and
Femoroacetabular ImpingementSession Number 032: Knee and Hip Finite
Element ModalitySession Number 033: The Post-Traumatic Joint
Experimental ModelsSession Number 034: Muscle and NerveSession
Number 035: Tissue Engineering I: Cartilage and BoneSession Number
036: Biomaterials Stem Cells and Growth FactorsSession Number 037:
MeniscusSession Number 038: Osteolysis and Implant FixationSession
Number 039: Tissue Engineering II: TendonSession Number 040:
Polymeric and Nano-BiomaterialsSession Number 041: Foot and
AnkleSession Number 042: Growth Factors and Bone FixationSession
Number 043: Knee, ACL, Patello-Femoral JointSession Number 044:
Tumors and DiseasesSession Number 045: Orthopaedic InfectionSession
Number 046: Cartilage MechanicsSession Number 047: ShoulderSession
Number 048: SpineSession Number 049: New Polyethylene Implant
WearSession Number 050: Knee ArthroplastyNIRA 1: Cartilage and
SpineNIRA 2: Soft TissueNIRA 3: Cartilage Biology and
OsteoarthritisNIRA 4: Fracture RepairNIRA 5: BonePS1 Bone
MechanicsPS1 Skeletal Growth and Development - Developmenal
BiologyPS1 Cell and Molecular Biomechanics - Physical Effects on
CellsPS1 Bone FracturePS1 Fracture FixationPS1 ImagingPS1
Cancer/TumorsPS1 Bone BiologyPS1 Bone - Growth FactorsPS1 Bone -
Osteoporosis & Metabolic Bone DiseasePS1 Progenitors & Stem
CellsPS1 Biomaterials - BioactivePS1 Tissue Engineering - Soft
TissuePS1 Cartilage/Meniscus/Synovium - Genetics/GenomicsPS1
Cartilage/Meniscus/Synovium - Cartilage & Matrix ProteinsPS1
Cartilage/Meniscus/Synovium - Cartilage Matrix DegradationPS1
Cartilage/Meniscus/Synovium - CytokinesPS1
Cartilage/Meniscus/Synovium - Growth Factors & AgingPS1
Cartilage/Meniscus/Synovium - Cartilage RepairPS1
Cartilage/Meniscus/Synovium - Cartilage Repair - Cell Based
ApproachesPS1 Cartilage/Meniscus/Synovium - MeniscusPS1 Cell &
Molecular Imaging of CartilagePS1 Cartilage/Meniscus/Synovium -
OsteoarthritisPS1 Cartilage/Meniscus/Synovium - Cartilage
MechanicsPS1 Cartilage/Meniscus/Synovium - MechanobiologyPS1
Disease Process Knee - Joint MechanicsPS1 Disease Process HipPS1
Gait & KinematicsPS1 Foot & AnklePS1 Infection &
InflammationPS1 TraumaPS1 Total Hip Replacement - GeneralPS1 Total
Knee Replacement - General/WearPS1 Arthroplasty - Osteolysis Wear
DebrisPS1 Arthroplasty - Implant FixationPS1 Total Knee Replacement
- KinematicsPS1 Arthroplasty - Finite Element Analysis/HipPS1
Polyethylene Wear - UHMWPEPS1 Arthroplasty - Finite Element
Analysis - KneePS1 Total Hip Replacement - ImplantPS1 Implant -
Uni-Knee ReplacementPS1 Hip/Knee Arthroplasty - Surgical Navigation
& RoboticsPS1 Implant Wear - Vitamin E PolyethylenePS1 Implant
Wear - Metal on Metal & Ceramic BearingsPS1 SpinePS1 Spine
BiomechanicsPS1 Spine - Intervertebral Disc BiomechanicsPS1 Spine -
Intervertebral Disc BiologyPS1 Spinal TherapeuticsPS1 Upper
Extremity - Shoulder & ElbowPS1 Upper Extremity - Hand &
WristPS1 Muscle/NervePS1 Ligament & Tendon MechanicsPS1
Ligament & Tendon BiologyPS2 Bone MechanicsPS2 PublicationsPS2
Cell & Molecular Biomechanics - Physical Effects on CellsPS2
Bone FracturePS2 Fracture FixationPS2 ImagingPS2 Cancer/TumorsPS2
Bone BiologyPS2 Bone - Growth FactorsPS2 Bone - OsteoporosisPS2
Progenitors and Stem CellsPS2 Biomaterials - BioactivePS2 Tissue
Engineering - Soft TissuePS2 Cartilage/Meniscus/Synovium - Gene
TherapyPS2 Cartilage/Meniscus/Synovium - Cartilage & Matrix
ProteinsPS2 Cartilage/Meniscus/Synovium - Immune-Mediated
ArthritisPS2 Cartilage/Meniscus/Synovium - Cartilage Matrix
DegradationPS2 Cartilage/Meniscus/Synovium - CytokinesPS2
Cartilage/Meniscus/Synovium - Growth Factors & AgingPS2
Cartilage/Meniscus/Synovium - Cartilage RepairPS2
Cartilage/Meniscus/Synovium - Cartilage Repair - Cell-Based
ApproachesPS2 Cartilage/Meniscus/Synovium - MeniscusPS2
Cartilage/Meniscus/Synovium - Cell and Molecular Imaging of
CartilagePS2 Cartilage/Meniscus/Synovium - OsteoarthritisPS2
Cartilage/Meniscus/Synovium - Cartilage MechanicsPS2
Cartilage/Meniscus/Synovium - MechanobiologyPS2 Disease Process
Knee - Joint MechanicsPS2 Disease Process HipPS2 Gait and
Kinematics, KinesiologyPS2 Foot and AnklePS2 Infection and
InflammationPS2 Total Hip Replacement - Resurfacing and Metal on
MetalPS2 Total Knee Reconstruction - General/WearPS2 Arthroplasty -
Osteolysis Wear DebrisPS2 Arthroplasty - Implant FixationPS2 Total
Knee Replacement - KinematicsPS2 Arthroplasty - Finite Element
Analysis - HipPS2 Polyethylene Wear - UHMWPEPS2 Arthroplasty -
Finite Element Anlysis KneePS2 Total Hip Replacement ImplantPS2
Implant - Uni-Knee ReplacementPS2 Arthroplasty Hip & Knee -
Surgical Navigation and RoboticsPS2 Implant Wear - Vitamin E
PolyethylenePS2 Implant Wear - Metal on Metal and Ceramic
BearingsPS2 SpinePS2 Spine BiomechanicsPS2 Spine - Intervertebral
Disc BiomechanicsPS2 Spine - Intervertebral Disc BiologyPS2 Spine -
Spinal TherapeuticsPS2 Upper Extremity - Shoulder and ElbowPS2
Upper Extremity - Hand and WristPS2 MusclePS2 Ligament and Tendon
MechanicsPS2 Ligament and Tendon Biology
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