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. 2014 Mar 19;9(3):e91983.
doi: 10.1371/journal.pone.0091983. eCollection 2014.

The Notch pathway is important in maintaining the cancer stem cell population in pancreatic cancer

Ethan V Abel  1 , Edward J Kim  2 , Jingjiang Wu  1 , Mark Hynes  1 , Filip Bednar  1 , Erica Proctor  1 , Lidong Wang  1 , Michele L Dziubinski  1 , Diane M Simeone  3
Affiliations

The Notch pathway is important in maintaining the cancer stem cell population in pancreatic cancer

Ethan V Abel et al. PLoS One. .

Abstract

Background: Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.

Methods: Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.

Results: Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.

Conclusion: The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression profile of Notch pathway components in cancer stem cells (CSC).
A. Flow cytometry analysis. Dissociated low-passage human pancreatic cancer xenograft cells were stained with DAPI and antibodies to H2K, ESA, CD44 and CD24. DAPI positive dead cells and H2K positive mouse cells were both eliminated from the analysis. The CSC population (with surface marker ESA+/CD44+/CD24+) was gated by both P5 and P7, and the non-CSC population from P6. B. Transcript levels of Notch pathway components in CSC compared to in non-CSC obtained by qPCR, normalized to GAPDH. The mean fold change between tumors is represented by a horizontal bar.
Figure 2
Figure 2. GSI treatment of pancreatic cancer cells.
A. Primary pancreatic cancer cell tumorspheres (derived from patient 5) were cultured in sphere medium containing different concentration of GSI as indicated for 48 hours before Western blot analysis with an antibody to Hes1 or β-actin. B. Quantitation of Hes1 mRNA levels following GSI treatment for 48 hours (*p<.001 vs. control). C. Pancreatic cancer cells in sphere medium were treated with control vehicle (DMSO) or 8 μM GSI for 48 hours. FACS analysis of control DMSO treated cells (upper panel) and GSI treated cells (lower panel). CSC population with surface marker of ESA+/CD44+/CD24+ was identified by those cells in both gates P5 and P7. D. Quantitation of ESA+/CD44+/CD24+ CSC population following DMSO and GSI treatment (*p = 0.013 vs. DMSO).
Figure 3
Figure 3. GSI treatment effect on pancreatic tumorspheres.
A. Pancreatic cancer cell tumorspheres cultured for 5 days in sphere medium containing indicated concentrations of GSI. Representative images of primary tumorspheres that developed from 1000 cells per well cultured with increasing concentrations of GSI. Quantitation of number of primary tumorspheres (black bars) formed at each concentration with 1000 cells seeded per well (*p<.01 vs. control). Secondary tumorsphere formation (gray bars) from tumorspheres treated with GSI were dissociated into single cells, washed, and cultured for 5 days in normal sphere medium without GSI (**p<.01 vs. control). B. Tumorspheres treated with GSI at increasing concentrations stained with propidium iodide (PI) and FITC-Annexin V (AnV) and analyzed by flow cytometry to evaluate the extent of apoptosis.(*p<.05 8 μM vs. control, **p<.01 16 μM vs. control). C. Cell cycle analysis of pancreatic tumorspheres treated with DMSO control or GSI at 0, 24, 48, and 72 hrs. (*p<.01 vs. control). D. Pancreatic cancer cells cultured with either 8 μM GSI or vehicle control (DMSO) for 48 hours injected subcutaneously into NOD/SCID mice. Representative images of mice implanted with cells from the indicated treatment groups taken 21 days after injection. Arrows indicate location of the tumors. Serial tumor sizes measurements from mice treated with DMSO control or GSI at indicated time points. N = 5 (*p<.01 vs. control).
Figure 4
Figure 4. Hes1 shRNA treatment effect on pancreatic tumorspheres.
A. Pancreatic cancer cells transduced with either control or Hes1 shRNA harvested after culturing for 4 days. Hes1 transcript levels quantitated by qRT-PCR, normalized to GAPDH (*p<.001 vs. control). B. Cell lysates were Western blotted for Hes1, Notch1, cleaved Notch1, or β-actin. C. Representative images of tumorspheres generated from pancreatic cancer cells transduced with either control scramble sequence or shRNA to Hes1 cultured for 5 days. D. Quantitation of number of tumorspheres generated per 1000 cells plated (*p<.001 vs. control).
Figure 5
Figure 5. Notch pathway activation with DSL peptide stimulates tumorsphere formation and increased CSC population.
A. Pancreatic cancer cell tumorspheres were cultured in sphere medium containing increasing concentration of DSL peptide as indicated for 5 days. mRNA levels of Hes1 from treated tumorspheres were analyzed by qRT-PCR and normalized to internal control GAPDH (*p<.001 vs. control). B. Tumorspheres cultured as in A. were Western blotted for cleaved-Notch1, Notch1, Hes1 or β-actin. C. Pancreatic cancer cells cultured in sphere medium containing increasing concentration of DSL analyzed for percentage of CSC by FACS analysis of cells stained with DAPI and antibodies to ESA, CD44, and CD24. Percentage of CSC represents ESA+/CD44+/CD24+ cells as a percentage of live DAPI-negative cells (*p<.02 vs. control). D+E. Tumorsphere formation assay performed on pancreatic cancer cells cultured in increasing concentration of DSL peptide. Representative images shown in D. with quantitation of number of tumorspheres per 1000 cells plated shown in E. (*p<.01 vs. control).
Figure 6
Figure 6. GSI treatment slowed tumor growth in an orthotopic model of primary pancreatic cancer.
A. Representative images of tumors monitored using bioluminescent imaging. Fold change in bioluminescence of tumors over time from day of implantation. B. Images of tumors excised at the completion of 4 weeks of treatment for each treatment group. Final tumor weights (in grams) of the excised tumors (*p<.01 vs. control). C. (upper panel) mRNA harvested from tumors excised at completion of 4 weeks of treatment was analyzed by qRT-PCR for Hes1, shown normalized to GAPDH (*p<.02 vs. control). (Lower panel) Single cells were isolated from tumors excised at completion of treatment were analyzed for percentage of CSC by FACS analysis of cells stained with DAPI and antibodies to H2k, ESA, CD44, and CD24. Percentage of CSC represents ESA+/CD44+/CD24+ cells as a percentage of live DAPI-negative and human H2k-negative cells (*p<.01 vs. control). D. H+E stained sections from tumors treated with GSI, gemcitabine, or combination of GSI and gemcitabine. Proliferative index calculated as number of Ki67+ stained cells from formalin-fixed sections of tumor treated with drug. Graph representing mean number of Ki67+ cells per 40x field from 5 random sections from each of 3 tumors per treatment group (*p<.01 vs. control). TUNEL assay performed on sections of tumor treated in vivo and tabulated mean number of TUNEL+ apoptotic cells (*p<.001 vs. control).
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