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Tatsuya Ishibe, Tomitaka Nakayama, Satoshi Nagayama,

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

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Tatsuya Ishibe, Tomitaka Nakayama, Satoshi Nagayama,

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  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. Billings SD, 2004 Mod Pathol Billings SD, 2004 Mod Pathol 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. SS ＋ MPNST 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-SSX1(+) Monophasic SYT-SSX2(+) MPNST MFH PLS 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 SW480 YaFuSS 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 HS-SY-II Fuji 1273/99 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 (O’Connell 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”.

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