e activation of canonical Wnt signaling. After we treated the mice bearing Hep3B xenografts with HS20, tumor growth was reduced by around 50%. Moreover, HS20 also showed an inhibitory effect on HepG2 tumor growth; after GPC3 was knocked down, tumor growth was no 10 / 13 Antibody Targeting the Heparan Sulfate Chains of Glypican-3 longer inhibited. These results showed that the HS20 antibody had significant anti-tumor activity against HCC and other GPC3-positive liver tumors in mice. Interestingly, HepG2 cells have constitutively activated -catenin signaling. Therefore, the inhibitory effect of HS20 on HepG2 tumor growth is unlikely to be attributed to the inhibition of Wnt signaling. In our current study, we show that GPC3 coordinates with HGF signaling in liver malignancy through its HS chains as well. The HS chains of GPC3 affect HGF binding but do not seem necessary for Wnt binding. This difference suggests that the HS chains of GPC3 are more relevant to the function of HGF. However, once cells are treated with HS20, both Met and Wnt activation are blocked efficiently. HS20 inhibits Wnt3a-dependent cell proliferation and HGF-induced cell migration, motility, and spheroid formation in HCC cells. Due to the limited understanding of HS structure, it is still a challenge to identify the specific binding motif on HS for either HGF or Wnt. It is possible that the function of HS chains relies more on the specific surface microenvironment of tumor cells. The HS chains could preferentially bind to certain types of factors or recruit whatever molecules carry the opposite charge, increasing the surface concentration of these factors and facilitating their receptor binding. In this case, Wnt and HGF may not be the only pathways blocked by HS20. Since most of these growth factors and their receptors are usually ubiquitously expressed, normal tissues could also be affected if we directly target them. To avoid this, targeting tumor-specific HSPGs to indirectly disturb these signaling pathways with an HS-specific antibody like HS20 would be a more desirable strategy. Many early studies on HSPGs focus on the composition, biosynthesis, and binding properties of the HS chains. Due to the high diversity of HS fine structures and their biological functions, studies in this field are limited by the lack of specific methodology to distinguish certain types of HS with others. HS20 TSU68 web recognizes a conserved HS structure of glypican proteins. In this regard, HS20 may be useful for multiple tumor types by targeting different glypicans, such as GPC1 in pancreatic cancer, GPC2 in pre-B cell ALL, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19735248 and GPC5 in rhabdomyosarcoma. In conclusion, we reported 
the role that PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19734877 the HS chains of GPC3 play in liver cancer cell migration and motility. This work may support a rationale for neutralizing HS in tumors for cancer therapies. Acknowledgments We thank Yanlin Yu for critical suggestions and the NIH Fellows Editorial Board and Yvonne Ye for editorial assistance.
