Wnt9a

Short term, primary cell lines were established from resected colorectal tumor specimens, classified and characterized by the attending surgical pathologist on each case. The tumors were obtained under an institutional review board approved protocol, from five patients (age range = 40–64 years) who were undergoing treatment for colorectal adenocarcinoma. Short term, primary CRC cell lines (n = 5) were established from resected tumors from patients with metastatic and/or recurrent disease. CRC were treated with LiCl (Sigma-Aldrich Corp., St. Louis, MO, USA), an activator of the canonical Wnt pathway (CWP) in the presence and absence of various Wnt pathway modulators including: IWP-2 (Sigma-Aldrich Corp.), a pan inhibitor of CWP and NCWP Wnt ligand secretion; conditioned media (CM) from LiCl ± IWP-2 treated cells; a specific antibody against Wnt ligand, Wnt9A (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA); and recombinant Wnt9A protein (Genemed Biotechnologies, Inc., San Francisco, CA, USA). Cell proliferation and apoptosis assays and RNA isolation for quantitative PCR gene expression array analysis were performed at 72 h. ELISA was performed on cell lysates at 24, 48 and 72 h.