Gene-Expression Pattern and Differentiation Potential of Synovial Sarcoma Suggest its Cellular Origin as Neural Crest-Derived Cells

1
Gene-Expression Pattern and Differentiation
Potential of Synovial Sarcoma Suggest its
Cellular Origin as Neural Crest-Derived Cells
Tatsuya Ishibe, Tomitaka Nakayama, Satoshi
Nagayama, Takeshi Okamoto, Tomoki Aoyama, Koichi
Nishijo, Takashi Nakamura, and Junya Toguchida
Department of Orthopaedic Surgery, Institute
for Frontier Medical Sciences, Kyoto University,
Japan
12th CTOS meeting at Boca Raton, FL.
2
Why synovial sarcoma ?
1865 First report Simon G., Arch Klin Chir
arises in para-articular region microscopic
resemblance to synovium 1934 Synovialsarcome
Sabrazes J. Gaz. Hebd. Sc. Med. Bordeaux
rarely arises in joint cavities. differs
from normal synovium by immunohistochemical
and ultrastructural study 1984 Synovial
sarcoma is a misnomer. Miettinen M. Am J
Pathol
The cell-of-origin of synovial sarcoma is still
unknown.
3
Backgrounds
We previously performed gene expression
profiling of 47 soft tissue sarcomas by cDNA
microarray (23,040 genes) , and reported that
synovial sarcoma was clustered into the same
group with malignant peripheral nerve sheath
tumor which is one of the neural crest-derived
tumor.
Nagayama et al. Cancer Res. 2002
Same cluster
Hypothesis Synovial sarcoma is neural
crest-derived tumor
4
Neural crest (stem) cells NCSC - unique cell
population of the vertebrate embryo -
arise from the neural ectoderm and recognizable
after neural tube closure
Head and neck
Cartilage Bone
Neuron
Neural tube
Trunk
Glia
Joseph NM Development, 2004
Melanocyte
NCSCs have multiple differentiation potential
Does synovial sarcoma have similar potential as
NCSC?
5
Objective
To estimate the origin of synovial sarcoma (SS)
by investigating its gene expression and
differentiation potential.
Materials and Methods
Fourteen cell lines including 5 SS
cells Twenty-nine tumor tissues including 18 SS
tumors
1) Gene expression of neural crest-related genes
by RT-PCR 2) Chondrogenic, osteogenic and
adipogenic inductions by the same methods as
mesenchymal stem cells (MSCs) 3) Neurogenic
induction by FGF2, ATRA, or BMP4
6
Differentiation markers in tumor tissues
Synovial sarcoma
Biphasic SYT-SSX1()
Monophasic SYT-SSX2()
Monophasic SYT-SSX1()
MPNST
PLS
MFH
LMS
chondro
SOX9
COL2A1
COL9A1
osteo
OSF2
ALP
neuronal
MASH1
NGFR
NFH
CRABP1
melano
EDN3
EDNRB
glial
HES1
S100b
adipo
Genes related to neural crest-derived cell
lineages are expressed in SS
PPARg
b actin
7
Chondrogenesis Pellet and matrix formation assay
5x105 cells
3D-culture for 7 days
Synovial sarcoma cells
HS-SY-II
SYO-1
Fuji
1273/99
YaFuSS
MSC
HE stain
loose
Alcian Blue
Pellet formation with positive Alcian blue was
observed in four SS cells
8
Chondrogenesis Gene expression before and after
induction
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
MSC
-
-
-
-
-
-






Induction
SOX9
COMP
COL2A1
COL9A1
COL11A1
COL11A2
AGC
b actin
COMP cartilage oligomeric matrix protein
Chondro-related genes were significantly
upregulated.
9
Osteogenesis Gene expression with/without
induction
Induction AA Dex b-GP
- Induction
for a week
or
MSC
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
-
-
-
-
-






Induction
OSF2
ALP
COL1A1
OC
b actin
Osteo-related genes were upregulated after
induction.
10
Osteogenesis ALPase activity and Alizarin Red
staining
HS-SY-II
SYO-1
Fuji
1273/99
YaFuSS
MSC
control
ALPase stain
Osteo induction
control
Alizarin red stain
Osteo induction
At least one cell line shows strong osteogenic
differentiation.
11
Adipogenesis
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
MSC
-
-
-
-
-
-






Induction
PPARg
AP2
ADP
b actin
HS-SY-II
SYO-1
Fuji
1273/99
MSC
YaFuSS
control
Oil-red-O stain
Adipo induction
SS cells have poor or no potential for adipogenic
induction
12
Neurogenesis neuron-related genes
NT2/D1 (neurogenic)
Without induction
NMS-2 (MPNST)
COLO205
HS-SY-II
HT1080
1273/99
YaFuSS
SW480
SaOS2
SYO-1
MSC
Fuji
NFH
NFM
NFL
MAP2
Nestin
b actin
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
ATRA
-
-
-
-
-





ATRA, 7days
MASH1
b actin
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99
BMP4
-
-
-
-
-





BMP4, 48hrs
MASH1
b actin
13
Immunostain of Neuron-specific beta III tubulin
(Tuj1)
YaFuSS
Fuji
1273/99
HS-SY-II
SYO-1
vehicle
PC12h (positive ctrl)
ATRA
FGF2
BMP4
Tuj1 / DAPI
14
Synovial sarcoma cells
Neural Crest Cell
YaFuSS
HS-SY-II
SYO-1
Fuji
1273/99




Gene exp. (growing phase)

Chondro





Gene exp. (after induc.)
Cartilage




Pellet formation





-
Alcian blue stain
-
-



Gene exp. (growing phase)


Osteo
Gene exp. (after induc.)



Bone
-
-


ALP stain

-
-
-

Alizarin red stain

Similar potential





Gene exp. (growing phase)




Tuj-1 stain (after FGF2)

Neuro



-
-
Gene exp. (after ATRA)

-
-

-
Neuron
Tuj-1 (after ATRA)
-
-
-

-
Gene exp. (after BMP4)
-
-
-

-
Tuj-1 (after BMP4)
Glial
-
-
-
-

Gene exp. (growing phase)
Glia

-



S100b (after 5-aza-dC)
Melano



-
Gene exp. (after 5-aza-dC)

-
-
-

-
Stress fiber reorgan.(FSK)
Melanocyte
-
-
-

-
-
Melanogenesis.(FSK)
-
-
Gene exp. (growing phase)



Adipo
-
-
-
-
-
Gene exp. (after induc.)
Oil-red-O stain





These spectra of differentiation are similar to
that of neural crest
15
Discussions from pathological point of view
May form ossification or calcification About
30 of synovial sarcomas have calcified focus.
(Enzinger FM, Cancer, 1982) May
arise from cartilage in head and neck region,
but not arise from articular cartilage which is
not neural crest origin. Arytenoid-epiglotic
rim (Szuhai K, Cancer Genet Cytogenet, 2004)
Cricoid cartilage, Monophasic SS (Kikuchi I,
Acta Cytol, 2003) May arise from neural
tissue. C7 spinal root (Chu PG, Mod Pathol,
2004) Common digital nerve (OConnell JX, Mod
Pathol, 1996) Radial nerve (Cugola L, J Hand
Surg, 1985)
These data also support our hypothesis that the
cell-of-origin of synovial sarcoma is neural
crest-derived cells.
16
Conclusion
Synovial sarcoma expressed the genes related to
chondrocyte, osteocyte, neuron, glia, and
melanocyte which are known to originate from
neural crest stem cells. Synovial sarcoma cells
had potential to differentiate into chondrocyte,
osteocyte, and neuron to various extent, but not
into adipocyte. This spectrum of differentiation
is similar to that of neural crest stem cells,
supporting our hypothesis that the
cell-of-origin of synovial sarcoma is neural
crest-derived cells.