Abstract 4253: Wnt signaling pathway reporter: A dual fusion construct to isolate and image colon cancer initiating cells
Authors: Shenoy Anitha , Carpentino Joseph E. , Fisher Robert C. , Goluguri Tata R. , Chang Lung-Ji , Scott Edward W. , Huang Emina H. 
Published: February 2011

Introduction: Recent developments in colon cancer pathogenesis imply that colon cancer can form from a rare population of colon cancer initiating cells (CCICs). We recently demonstrated that similar to sporadic colorectal cancer (CRC), CCICs are present in colitic tissues which may be propagated both in vivo as xenografts and in vitro as non-adherent spheroid colonies. Mutations in the Wnt (Wingless) signaling pathway common in the pathogenesis of sporadic CRC are implicated in the perpetuation of CCICs. We hypothesize that Wnt signaling in CCICs is involved in mediating colitis to cancer transformation.

Methods: We designed a Wnt signaling pathway-dependent dual fusion reporter construct TTLG, [TCF enhancer-TK promoter-firefly luciferase (FL)-enhanced green fluorescent protein (eGFP)], to identify and monitor CCICs. The TTLG contains FL and eGFP genes under the control of a T cell factor (TCF) enhancer array upstream of a minimal thymidine kinase (TK) protmoter. Activation of the canonical Wnt signaling pathway leads to the formation of β-catenin-TCF complexes on the TCF enhancer array and subsequent expression of the FL and eGFP reporter genes. The eGFP and FL expressing cells were readily isolated using flow cytometry and visualized by bioluminescent imaging. The functional activity of TTLG was evaluated by transfecting the SW480 colon cancer cell line (constitutively active canonical Wnt pathway) and the HEK293T cell line (lacks canonical Wnt signaling). A TTLG with mutated Wnt promoter, denoted as TLG, which bears a minimal TK promoter-FL-eGFP, but lacks the TCF enhancer array, was used as a negative reporter construct. A dual fusion reporter construct containing renilla luciferase and red fluorescent protein regulated by a ubiquitous enhancer and promoter served as a transfection control. The TTLG and TLG dual reporter cassettes were packaged into a lentiviral vector to efficiently introduce the dual reporter expression constructs into the non-adherent sphere cultures from chronic colitis and CRC patients. Flow cytometry was used to quantify the transduced colitis-derived and the CRC-derived spheres for eGFP expression.

Results: SW480 cells transfected with TTLG showed a 17-fold increase in Wnt activity when compared to TLG transfected cells. Conversely, HEK293T cells showed weak Wnt activity that was further confirmed by imaging for firefly luciferase and eGFP activities. In our sphere cultures, we found a considerable level of Wnt activity in both colon cancer and colitis spheres: one of our cancer sphere lines expressed 19% Wnt activity while two of the colitis sphere lines demonstrated 25% Wnt activity.

Conclusions: TTLG is a functional dual fusion Wnt reporter construct. The level of reporter gene expression for the cancer and colitic CCICs is indicative of canonical Wnt signaling, and further supports a role for the Wnt signaling pathway in the maintenance and propagation of CCICs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4253.