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. 2010 Dec 1;70(23):9937-48.
doi: 10.1158/0008-5472.CAN-10-0881. Epub 2010 Nov 30.

Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling

James P Sullivan  1 , Monica Spinola, Michael Dodge, Maria G Raso, Carmen Behrens, Boning Gao, Katja Schuster, Chunli Shao, Jill E Larsen, Laura A Sullivan, Sofia Honorio, Yang Xie, Pier P Scaglioni, J Michael DiMaio, Adi F Gazdar, Jerry W Shay, Ignacio I Wistuba, John D Minna
Affiliations

Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling

James P Sullivan et al. Cancer Res. .

Abstract

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

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Figures

Figure 1
Figure 1. Survival analysis of NSCLC TMA samples expressing ALDH1A1, ALDH3A1 and CD133
A. Kaplan-Meier plot (left panel) of patient overall survival based on tumors expressing high (red: n = 142) and low (black: n = 140) ALDH1A1 show high ALDH1A1 scores are associated with poor overall survival. B. Survival analysis of patients with tumors expressing high (red: n = 141) and low (black: n = 141) ALDH3A1 indicates no association between ALDH3A1 expression and overall patient survival. C. Similar analysis of patient samples expressing detectable CD133 (red: n = 56) and no CD133 (black: n = 151) indicates no association between CD133 expression and overall patient survival. Images taken from tumors with high (upper panel) and low (lower panel) protein expression are shown to the right of each corresponding survival curve.
Figure 2
Figure 2. ALDH+ cells detected in lung cancer
A. Flow cytometry analysis of a lung cancer samples using the Aldefluor assay. Baseline fluorescence was established by inhibiting ALDH activity with DEAB (left panel) and used to generate a gate that will identify ALDH+ cells in lung cancer cells that have not been incubated with DEAB (right panel). B. Example Aldefluor analysis of a NSCLC cell line, HCC461 and a SCLC cell line, H69. C. ALDH+ cells can be observed by microscopy through accumulation of BAA (green, arrows) with nuclei identified by DAPI (blue). D. Ink4a/ARF−/− Kras-induced mouse lung tumors were analyzed for the presence of ALDH. Gating based on the DEAB control (left panel) was applied to tumor cells without the ALDH inhibitor (center panel), revealing a sub-population (~2%) of ALDH+ tumor cells. Cells co-stained with Sca-1 and gated for elevated ALDH were observed to be enriched in Sca-1+ tumor cells compared to ALDH− tumor cells (right panels).
Figure 3
Figure 3. ALDH+ cells are more tumorigenic than ALDH− lung cancer cells
A. Tumor growth curves were generated from 106 isolated ALDH+ and ALDH− H1299 cells. B. Similarly, sorted H358 cells were injected subcutaneously into NOD/SCID mice and tumor growth was monitored over time (n = 5). C. BLI imaging of NOD/SCID mice injected with 104 sorted ALDH+ and ALDH− H358-luc cells. D. ALDH activity was assayed in tumors derived from ALDH+ and ALDH− cells, with DEAB treated cells serving as a negative control, revealed ALDH+ cell derived tumors possessed a greater proportion of ALDH+ cells than ALDH− derived tumors. E. Isolated EpCam+ALDH+ cells from LT7 generated xenografts with a similar distribution of ALDH+ cells as the parental patient tumor.
Figure 4
Figure 4. ALDH+ cells are more clonogenic than ALDH− lung cancer cells
A. Colonies derived from sorted ALDH+ and ALDH− cells in soft agar were counted after three weeks in culture (n = 4, *P < 0.05, **P < 0.01). B. Colony formation efficiencies of sorted H358 (25 ± 3.6% vs. 9 ± 2.9%) and H1299 (40 ± 3.5 vs. 11 ± 0.6%) cells were determined in liquid culture after two weeks of growth in limiting dilutions. C. Microscopy of ALDH+ and ALDH− cell derived colonies stained with methylene blue. ALDH+ cells produced dense “holoclone” colonies (left panel) whereas ALDH− cells generated primarily diffuse “meroclone” colonies (right panel). D. ALDH+ and ALDH− cells were isolated by FACS from parental H358 cells (left panel), grown in culture for two weeks and reanalyzed for proportion of ALDH+ cells by flow cytometry.
Figure 5
Figure 5. Notch signaling maintains ALDH+ lung cancer cells and sensitized ALDH+ cells to Notch signaling inhibition
A. Expression of Notch signaling transcript (shown as an expression ration in ALDH+/ALDH− cells) is elevated in ALDH+ lung cancer cells. B. qRT-PCR analysis revealed a decrease in Notch signaling transcription factors in unsorted lung cancer cells treated with 10 µM DAPT for 24hrs. C. Cell lines treated with 10 µM DAPT for five days retained significantly smaller ALDH+ populations compared to DMSO treated cells (n = 3, *P < 0.05). D. Sorted H358 ALDH+ and ALDH− cells were treated with DMSO and DAPT and cell cycle analysis was performed revealing a greater accumulation of cells in G1/G0-phase as well as a decrease of cells in S-phase in DAPT treated ALDH+ cells compared to DAPT treated ALDH− cells.
Figure 6
Figure 6. shRNA mediated knockdown of NOTCH3 reduces clonogenic and ALDH+ lung tumor cell population
A. qRT-PCR analysis reveals an approximately 2 fold reduction of NOTCH3 expression, as well as a reduction in HEY1 and HEY2 expression in H358 and H2009-shNOTCH3 cells compared to control shGFP cells. B. Colony formation analysis of H358 and H2009 reveal shNOTCH3 expressing cells exhibit a reduction in clonogenicity (***P < 0.005). C. Aldefluor analysis of H358 and H2009 cells expressing shGFP and shNOTCH3 indicate a reduction of ALDH+ cells in shNOTCH3 expressing cell lines.
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