Identification of Pancreatic Cancer Stem Cells and Selective Toxicity of Chemotherapeutic Agents

doi:10.1053/j.gastro.2012.03.054

Gastroenterology

Volume 143, Issue 1, July 2012, Pages 234-245.e7

https://doi.org/10.1053/j.gastro.2012.03.054 Get rights and content

Background & Aims

Identification and purification of cancer stem cells (CSCs) could lead to new therapeutic targets, but their heterogeneous expansion is an obstacle to their study. We investigated whether it is possible to monitor pancreatic CSCs in real time, based on their intrinsic low level of proteasome activity.

Methods

We engineered human pancreatic adenocarcinoma cells (PANC1, MIAPaCa2, BxPC3, and KLM1) to express a green fluorescent molecule fused to the degron of ornithine decarboxylase (Gdeg) from a retroviral vector; the fluorescent Gdeg accumulates in CSCs as a result of low activity of the 26S proteasome. Cells with high and low levels of fluorescence (Gdeg^high and Gdeg^low) were isolated by flow cytometry; tumor growth was analyzed in immunocompromised mice. We performed a screen for agents that were specifically toxic to pancreatic CSCs, in a synthetic lethal manner.

Results

Gdeg^high cells, but not Gdeg^low cells, formed spheres and underwent asymmetric division—features of CSCs. Injection of as few as 10 Gdeg^high cells led to tumor formation in mice. Gemcitabine was toxic to cultured Gdeg^low cells, whereas Gdeg^high cells were resistant. We observed that quercetin was toxic to Gdeg^high cells in culture and in pre-established tumors grown from these cells in mice. Nuclear accumulation of β-catenin was detected in Gdeg^high, but not Gdeg^low, and lost after exposure to quercetin.

Conclusions

We used a fluorescence marker system for level of proteasome activity to identify pancreatic cancer cells with features of cancer stem cells. We identified quercetin as a compound that is specifically toxic to pancreatic CSCs.

Section snippets

Cell Culture

Human pancreatic adenocarcinoma cell lines (PANC1, MIAPaCa2, and BxPC3) were purchased from the American Type Culture Collection (Manassas, VA). In addition, we used KLM1 cells derived from a Japanese patient with a pancreatic adenocarcinoma.²³ PANC1, KLM1, and BxPC3 cells were cultured in log-growth phase in 1640 RPMI (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO) and PenStrep (Sigma) as antibiotics, and were grown in an incubator in 5% CO₂ at 37°C.

Real-Time Imaging of CSCs in Human Pancreatic Cancer Cells

The stable transfection of the Gdeg reporter into PANC1 human pancreatic cancer cells revealed that a Gdeg^high population did exist at approximately 0.5% of the total cell number (Supplementary Figure 1), but there was no Gdeg^high population in normal pancreatic epithelial cells (data not shown). As shown by time-lapse microscopy, asymmetric divisions of Gdeg^high cells into Gdeg^low and Gdeg^high cells were clearly recognized, and Gdeg^low cells never divided into Gdeg^high cells (Figure 1A,

Discussion

CSCs consist of a small subpopulation of cancer cells that selectively possess tumor initiation, a self-renewal capacity, and the ability to give rise to bulk populations of nontumorigenic progeny through differentiation.³ In our present report, asymmetric cell division of human pancreatic CSCs clearly was shown by the use of the Gdeg real-time imaging system with low proteasome activity (Figure 1A, Supplementary Video 1). Kirshner et al³³ reported that a subpopulation of human multiple myeloma

Acknowledgments

The authors thank Dr Frank Pajonk and Dr Erina Vlashi from the University of California for providing the pQCXIN-ZsGreen-cODC plasmid and Dr Ming-Sound Tsao from the University Health Network, Toronto, for human pancreatic duct epithelial cells. The authors also thank Ms Ayumi Shioya, Dr Hiroyuki Kagechika, and Dr Mari Yuasa (Chemical Biology Screening Center, Tokyo Medical and Dental University) for their technical assistance.

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Citation Excerpt :
The latter targets ZsGreen to ubiquitin-independent degradation by the 26S proteasome, thus reporting lack of proteasome function through accumulation of ZsGreen-cODC. We previously reported that cancer cell populations lacking proteasome activity are enriched for tumor-initiating cells in glioblastoma, breast cancer, and cancer of the head and neck region [12–15] and others have confirmed these findings independently in tumors of the liver, lung, cervix, pancreas, osteosarcoma, and colon [16–21]. After infection with the lentivirus, cells expressing the ZsGreen-cODC fusion protein were further selected with G418 for 5 d. Successful infection was verified using the proteasome inhibitor MG132 (Sigma, MO).
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: Conflicts of interest The authors disclose no conflicts.

: Funding This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Research (A) from the Ministry of Education, Culture, Sports, Science & Technology of Japan, and a Health & Labour Sciences Research Grant from the Ministry of Health Labour & Welfare of Japan.

View full text

Original ResearchBasic and Translational—PancreasIdentification of Pancreatic Cancer Stem Cells and Selective Toxicity of Chemotherapeutic Agents

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell Culture

Real-Time Imaging of CSCs in Human Pancreatic Cancer Cells

Discussion

Acknowledgments

Cell

Cancer Cell

Cell Stem Cell

Cell

Curr Biol

J Biol Chem

Am J Pathol

Biochem Biophys Res Commun

Blood

Blood

Immunity

Cancer Cell

Cell

Gastroenterology

Life Sci

Lancet

J Nutr

Cells of origin in cancer

Nature

Stem cells, cancer, and cancer stem cells

Nature

Prospective identification of tumorigenic breast cancer cells

Proc Natl Acad Sci U S A

Identification of human brain tumour initiating cells

Nature

Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer

Genes Dev

CD13 is a therapeutic target in human liver cancer stem cells

J Clin Invest

Identification and expansion of human colon-cancer-initiating cells

Nature

Tumour-initiating cells: challenges and opportunities for anticancer drug discovery

Nat Rev Drug Discov

In vivo imaging, tracking, and targeting of cancer stem cells

J Natl Cancer Inst

Elimination of damaged proteins during differentiation of embryonic stem cells

Proc Natl Acad Sci U S A

Stem cell quiescence

Clin Cancer Res

Original Research
Basic and Translational—Pancreas
Identification of Pancreatic Cancer Stem Cells and Selective Toxicity of Chemotherapeutic Agents