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. 2013 Apr 15;19(8):1972-80.
doi: 10.1158/1078-0432.CCR-12-0370. Epub 2013 Feb 26.

Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma

Khaled A Hassan  1 , Luo Wang, Hasan Korkaya, Guoan Chen, Ivan Maillard, David G Beer, Gregory P Kalemkerian, Max S Wicha
Affiliations

Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma

Khaled A Hassan et al. Clin Cancer Res. .

Abstract

Purpose: 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.

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Figures

Figure 1
Figure 1. GFP-dim and GFP-bright sphere formation
GFP-dim sphere: (A) bright field; (B) GFP filter; (C) merged. GFP-bright sphere: D) bright field, E) GFP filter, F) merged. G) GFP-dim secondary sphere, H) GFP-bright secondary sphere. (All 40X magnification); (I) Average primary and secondary sphere formation of GFP-bright and GFP-dim cells. GFP-bright cells form more primary (p=0.00048) as well as secondary (p=0.0008) spheres as compared to GFP-dim cells.
Figure 2
Figure 2. GFP-bright and GFP-dim xenografts
Bioluminescence imaging of GFP-bright and GFP-dim secondary xenografts, showing the presence of tumor only in animals injected with GFP-bright cells. (A) Back and (B) Front of NOD/SCID mice injected with 10,000, 1000 or 100 GFP-bright cells subcutaneously showing tumor mass and increased bioluminescence after luciferin injection. (C) Back and (D) Front of NOD/Scid mice injected with 10,000, 1000 or 100 GFP-dim cells subcutaneously with absence of tumor formation. Hematoxylin and eosin staining of xenograft tumors showing similar histology at 10X and 40X. (E) Parental H1299; (F) Primary GFP-bright xenograft; (G) Secondary GFP-bright xenograft; (H) Single cell sphere xenograft tumors.
Figure 3
Figure 3. Annexin V flow cytometry apoptosis assay
(A) Control, untreated NCI-H1299 GFP- bright and -dim cells; (B) 2% of untreated GFP-dim cells are AnnexinV-APC positive; (C) 1.7% of GFP-bright cells are Annexin V-APC positive; (D) Docetaxel treated GFP-bright and -dim cells; (E) 61% of Docetaxel treated GFP-dim cells are Annexin V-APC positive; (F) 10% of Docetaxel treated GFP-bright cells are Annexin V-APC positive; (G) Cisplatin treated GFP-bright and -dim cells; (H) 25% of Cisplatin treated GFP-dim cells are Annexin V-APC positive; (I) 7% of Cisplatin treated GFP-bright cells are Annexin V-APC positive. Annexin V apoptosis assay after exposure to Docetaxel and Cisplatin in different cell lines (average of three experiments); (J) NCI-H1299; (K) NCI-H358; (L) NCI-H460; (M) A549; (N) NCI-H441. Annexin V expression was significantly higher in all treated GFP-bright cells as compared to treated GFP-dim cells except the docetaxel treated NCI-H358 cells where the difference wasn’t statistically significant ( p=0.38)
Figure 3
Figure 3. Annexin V flow cytometry apoptosis assay
(A) Control, untreated NCI-H1299 GFP- bright and -dim cells; (B) 2% of untreated GFP-dim cells are AnnexinV-APC positive; (C) 1.7% of GFP-bright cells are Annexin V-APC positive; (D) Docetaxel treated GFP-bright and -dim cells; (E) 61% of Docetaxel treated GFP-dim cells are Annexin V-APC positive; (F) 10% of Docetaxel treated GFP-bright cells are Annexin V-APC positive; (G) Cisplatin treated GFP-bright and -dim cells; (H) 25% of Cisplatin treated GFP-dim cells are Annexin V-APC positive; (I) 7% of Cisplatin treated GFP-bright cells are Annexin V-APC positive. Annexin V apoptosis assay after exposure to Docetaxel and Cisplatin in different cell lines (average of three experiments); (J) NCI-H1299; (K) NCI-H358; (L) NCI-H460; (M) A549; (N) NCI-H441. Annexin V expression was significantly higher in all treated GFP-bright cells as compared to treated GFP-dim cells except the docetaxel treated NCI-H358 cells where the difference wasn’t statistically significant ( p=0.38)
Figure 4
Figure 4
(A) Kaplan–Meier curve of overall survival for patients with high and moderate expression of Notch negative modulators compared to those with low expression. Hazard ratio 0.52 (95% CI, 0.39-0.70). (B) Kaplan–Meier curve of overall survival for patients with high and moderate expression of Hes-1 compared to those with low expression. Hazard ratio HR 2.44 (95% CI, 1.02 – 5.81).
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