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,Department of Orthopaedic Surgery,Institute for Frontier Medical Sciences,Institute for Frontier Medical Sciences,

Kyoto University, Japan

12th CTOS meeting at Boca Raton, FL.

Why “synovial” sarcoma ?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.

Billings SD, 2004 Mod PatholBillings SD, 2004 Mod Pathol

BackgroundsBackgrounds

Hypothesis: Synovial sarcoma is neural crest-derived tumor

MPNSTSS＋Same cluster

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

Neural crest (stem) cells: NCSCNeural crest (stem) cells: NCSC- unique cell population of the vertebrate embryo - - unique cell population of the vertebrate embryo -

Neural tube

Joseph NMDevelopment, 2004

Cartilage　 & Bone

Neuron

Glia

Melanocyte

arise from the neural ectoderm and recognizable after neural

tube closure

Trunk

Head and neck

Does synovial sarcoma have similar potential as NCSC?

NCSCs have multiple differentiation potential

ObjectiveObjective

To estimate the origin of synovial sarcoma (SS) by investigating its gene expression and differentiation potential.

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

Materials and MethodsMaterials and Methods

Fourteen cell lines including 5 SS cells

Twenty-nine tumor tissues including 18 SS tumors

PLS

LMS

MFH

MPN

ST BiphasicSYT-SSX1(+)

MonophasicSYT-SSX2(+)

MonophasicSYT-SSX1(+)

Differentiation markers in tumor tissuesSynovial sarcoma

actin

CRABP1

EDN3

EDNRB

MASH1

NGFR

SOX9

ALP

OSF2

HES1

PPARg

S100b

NFH

COL2A1

COL9A1cho

nd

ron

euro

nal

mel

ano

gli

alad

ip oo

ste

o

Genes related to neural crest-derived cell lineages are expressed in SS

YaFuSS HS-SY-II SYO-1 Fuji 1273/99 MSC

HE

sta

inA

lcia

n

Blu

e5x105 cells

3D-culture

for 7 days

Chondrogenesis: Pellet and matrix formation assay

loose

Synovial sarcoma cells

Pellet formation with positive Alcian blue was observed in four SS cells

actin

COMP

SOX9

AGC

YaFuSS

COL2A1

HS-SY-II SYO-1 Fuji 1273/99 MSC

COL11A1

COL11A2

COL9A1

Chondrogenesis: Gene expression before and after induction

- + - + - + - + - + - +Induction

Chondro-related genes were significantly upregulated.

COMP: cartilage oligomeric matrix protein

actin

ALP

OC

OSF2

COL1A1

Osteogenesis: Gene expression with/without induction

- +

YaFuSS

- +

HS-SY-II

- +

SYO-1

- +

Fuji

- +

1273/99 MS

C

Induction +

Osteo-related genes were upregulated after induction.

- Induction + Induction + AA + Dex +-GP for a week

or

YaFuSS HS-SY-II SYO-1 Fuji 1273/99

AL

Pas

e st

ain

MSCco

ntro

l

Osteogenesis: ALPase activity and Alizarin Red stainingA

lizar

in r

ed s

tain

Ost

eoin

du

cti

on

cont

rol

Ost

eoin

du

cti

on

At least one cell line shows strong osteogenic differentiation.

YaFuSS HS-SY-II SYO-1 Fuji 1273/99 MSC

actin

ADP

PPARg

AP2

Oil

-red

-O s

tain

cont

rol

Ad

ipo

ind

uct

ion

- +

YaFuSS

- +

HS-SY-II

- +

SYO-1

- +

Fuji

- +

1273/99

- +

MSC

Induction

Adipogenesis

SS cells have poor or no potential for adipogenic induction

BMP4

actin

MASH1

- +YaFuSS

- +HS-SY-II

- +SYO-1

- +Fuji

- +1273/99

MASH1

actin

ATRA

MAP2

NFL

NFMNFH

Nestin

YaFu

SSH

S-SY

-IISY

O-1

NM

S-2

(MPN

ST)

HT1

080

SW48

0M

SC

Fuji

1273

/99

SaO

S2C

OLO

205

NT2

/D1

(neu

roge

nic)

actin

ATRA, 7days

- +YaFuSS

- +HS-SY-II

- +SYO-1

- +Fuji

- +1273/99

BMP4, 48 ｈｒｓ

Neurogenesis: neuron-related genes

Without induction

Tuj1 / DAPI

SYO-1YaFuSS Fuji 1273/99ve

hic

leA

TR

AF

GF

2B

MP

4

PC12h(positive ctrl)

HS-SY-II

Immunostain of Neuron-specific beta III tubulin (Tuj1)

YaFuSS HS-SY-II SYO-1 Fuji 1273/99

Gene exp. (growing phase) ++ ++ ++ ++ ±Gene exp. (after induc.) ++ ++ ++ ++ ++Pellet formation + + + ++ ±Alcian blue stain + + + ++ -

Gene exp. (growing phase) - - + + +Gene exp. (after induc.) ± ± ± + +ALP stain - - + ± ++Alizarin red stain - - - ± ++

Gene exp. (growing phase) ++ ++ ++ ++ +Tuj-1 stain (after FGF2) ++ + + ± ++Gene exp. (after ATRA) + + + - -Tuj-1 (after ATRA) + - - + -Gene exp. (after BMP4) - - - + -Tuj-1 (after BMP4) - - - ++ -

Gene exp. (growing phase) - - - - +S100 (after 5-aza-dC) + -

Gene exp. (after 5-aza-dC) ± + + + -

Gene exp. (growing phase) ± - ± - ±Gene exp. (after induc.) - - - - -Oil-red-O stain ± ± ± ± ±

Chondro

Osteo

Neuro

Glial

Melano

Adipo

Stress fiber reorgan.(FSK) - - - + -Melanogenesis.(FSK) - - - + --

Cartilage　

Neuron

Glia

Melanocyte

Bone 　

NeuralCrest Cell

Synovial sarcoma cells

Similarpotential

These spectra of differentiation are similar to that of neural crest

+ + +

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, butnot arise from articular cartilage which is not neuralcrest 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 (O’Connell JX, Mod Pathol, 1996)

・ Radial nerve (Cugola L, J Hand Surg, 1985)

Discussions from pathological point of view

These data also support our hypothesis that “the cell-of-origin of synovial sarcoma is neural crest-derived cells.”

ConclusionConclusion

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”.