ARTICLE ABSTRACTPurpose: The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation, and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells.Experimental Design: We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a γ-secretase inhibitor (GSI), which inhibits Notch pathway activity.Results: Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival.Conclusions: Notch activity can identify lung cancer stem cell–like population and its inhibition may be an appropriate target for treating lung adenocarcinoma. Clin Cancer Res; 19(8); 1972–80. ©2013 AACR.