Original Article  /  Pancreas

Detection of tumor stem cell markers in pancreatic carcinoma cell lines Monika Olempska, Patricia Alice Eisenach, Ole Ammerpohl, Hendrik Ungefroren, Fred Fandrich and Holger Kalthoff Kiel, Germany

BACKGROUND: Cancer of the pancreas is the fourth leading cause of cancer death in industrialized countries. In malignancy, actively proliferating cells may be effectively targeted and killed by anti-cancer therapies, but stem cells may survive and support re-growth of the tumor. Thus, new strategies for the treatment of cancer clearly will also have to target cancer stem cells. The goal of the present study was to determine whether pancreatic carcinoma cell growth may be driven by a subpopulation of cancer stem cells. Because previous data implicated ABCG2 and CD133 as stem cell markers in hematopoietic and neural stem/progenitor cells, we analyzed the expression of these two proteins in pancreatic carcinoma cell lines. METHODS:  Five established pancreatic adenocarcinoma cell lines were analyzed. Total RNA was isolated and realtime RT-PCR was performed to determine the expression of ABCG2 and CD133. Surface expression of ABCG2 and CD133 was analyzed by flow cytometric analysis. RESULTS:  All pancreatic carcinoma cell lines tested expressed significantly higher levels of ABCG2 than non-malignant fibroblasts or two other malignant nonpancreatic cell lines, i.e., SaOS2 osteosarcoma and SKOV3 ovarian cancer. Elevated CD133 expression was found in two out of five pancreatic carcinoma cell lines tested. Using flow cytometric analysis we confirmed surface expression of ABCG2 in all five lines. Yet, CD133 surface expression was detectable in the two cell lines, A818-6 and PancTu1, which exhibited higher mRNA levels.

Author Affiliations: Department of General and Thoracic Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Arnold Heller Str. 7, 24 105 Kiel, Germany (Olempska M, Eisenach PA, Ammerpohl O, Ungefroren H, Fandrich F and Kalthoff H) Corresponding Author: Holger Kalthoff, PhD, Department of General and Thoracic Surgery, Division of Molecular Oncology, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller Str. 7, 24105 Kiel, Germany (Tel: +49-431-5971937, Fax: +49-431-5971939, Email: [email protected]) © 2007, Hepatobiliary Pancreat Dis Int. All rights reserved.

CONCLUSIONS: Two stem cell markers, ABCG2 and CD133 are expressed in pancreatic carcinoma cell lines. ABCG2 and/or CD133 positive cells may represent subpopulation of putative cancer stem cells also in this malignancy. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, they may be a very promising target for new drug developments. (Hepatobiliary Pancreat Dis Int 2007; 6: 92-97) KEY WORDS: pancreatic adenocarcinoma; cancer stem cells; stem cell markers; ABCG2; CD133

Introduction

T

he cell population of most tumors is heterogeneous with regard to its proliferation capacity, apoptosis-resistance mechanisms, and ability to reconstitute the tumor upon xeno-transplantation. This phenomenon arises as the result of accumulation of multiple genetic and epigenetic changes. Current evidence suggests that only a few cells within the tumor, the cancer stem cells, possess unlimited proliferative capacity and give rise to tumors that phenotypically resemble their initializing cells.[1-3] The postulated significance of stem cells in cancer development is based on two fundamental characteristics. First, they are the only long-lived cells within tissues and are capable of accumulating multiple transforming mutations. Second, they can self-renew into new stem cells with identical proliferation and differentiation potential. Cancer stem cells are therefore defined as cancer cells that have the ability to divide into a new malignant stem cell and a cell that gives rise to a heterogeneous tumor cell population. The existence of cancer stem cells was first documented in haematological malignances[4,  5] and was subsequently discovered in several solid tumors,

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Detection of tumor stem cell markers in pancreatic carcinoma cell lines including breast,[6] brain,[7] prostate,[8] liver,[9, 10] lung,[11] melanoma,[12] and colon[13, 14] tumors. It was shown that only a small subset of human breast cancer cells, with the phenotype CD44+CD24- /low, gives rise to new tumors in mice.[6] These breast cancerinitiating cells can be isolated and propagated in vitro as extensively proliferating, clonal, nonadherent spherical clusters with the ability to differentiate along different mammary epithelial lineages.[15] Singh et al[16] detected a minor population of human brain cancer cells based on the expression of cell-surface marker CD133, showing that this population is necessary for the proliferation and self-renewal of the tumor in culture as well as initiation of tumor growth in SCID mice. The presence of a small subpopulation of cancer stem cell-like cells was also confirmed in the C6 glioma and other cell lines, indicating that established malignant cell lines that have been maintained for many years in culture retain a subpopulation of cancer stem cells.[17, 18] Moreover, a cancer stem cell population with the CD44+/integrinα2β1hi /CD133+ phenotype and the capacity for extensive proliferation, self-renewal, differentiation, and invasion was identified in prostate cancer.[8] Recently, it was shown that colon cancer CD133+ cells were able to maintain themselves as well as differentiate and re-establish tumor heterogeneity upon serial transplantation.[13, 14] Adult stem cells possess the ability to efflux fluorescent dyes such as rhodamine 123 or Hoechst 33342. This 'side population' of stem cells can be identified and isolated as cells displaying low levels of Hoechst fluorescence by flow cytometric analysis.[19] Although the molecular mechanisms that provide a side population phenotype are still unclear, one likely important contributor is a member of the ATPbinding cassette transporter ABCG2 family, also known as ABCP/MXR/BCRP.[20] ABCG2 functions as a high capacity drug transporter with wide substrate specificity.[21] It is expressed in a variety of tissues at high levels in the placenta, liver, and small intestine, and at lower levels in the kidney, heart, and brain.[21,  22] Elevated expression of ABCG2 is observed not only in adult stem cells but also in primary cancer cells and cell lines, conferring a multidrug resistant phenotype.[23, 24] So far, the presence of cancer stem cells in pancreatic carcinoma has not been demonstrated. The aim of the present study was to identify putative cancer stem cells within established pancreatic carcinoma cell lines. As markers, ABCG2 and CD133 were selected. The ABCG2 transporter is responsible for the side population phenotype in a number of

unrelated human cancers and the corresponding nonmalignant tissues and is widely used to detect and isolate somatic stem/progenitor cells. The second marker, CD133, is expressed in a subset of stem/ progenitor cells in the hematopoietic system as well as in a few solid tumors of the brain, prostate, colon, and liver and is a commonly used stem cell marker. Based on our findings on surface expression of ABCG2 and CD133, we identified a subpopulation(s) that likely represents cancer stem cells.

Methods Cell lines and culture conditions Pancreatic adenocarcinoma (Panc1, Panc89, Colo357, PancTu1, and A818-6), breast cancer (MCF7), ovarian cancer (SKOV3), and osteosarcoma (SaOS2) cell lines as well as human diploid fibroblasts (Kif5) were cultured in RPM1 1610 medium containing 10% FBS under standard culture conditions. The pancreatic adenocarcinoma cell lines represent two grades of differentiation, i.e., moderately differentiated Panc89, Colo357, and A188-6, and poorly differentiated Panc1 and PancTu1.[25] Real-time RT-PCR analysis Total RNA was isolated using RNeasy mini kit (Qiagen). The PCR primers included ABCG2 (sense, 5'-GGG TTC TCT TCT TCC TGA CGA CC-3'; antisense, 5'-TGG TTG TGA GAT TGA CCA ACA GAC C-3'), CD133 (sense, 5'-TCT CTA TGT GGT ACA GCC G-3'; antisense, 5'-TGA TCC GGG TTC TTA CCT G-3'), and G6PD (sense, 5'-ACG TGA TGC AGA ACC ACT ACT G-3'; antisense, 5'-ACG ACG GCT GCA AAA GTG GCG-3'). Thermal cycling was performed for 40 cycles of 95˚/40 s, 61˚/40 s, and 72˚/1 min. All PCR reactions were carried out using a BioRad MiniOpticon Cycler with SYBR Green Supermix (BioRad) and analyzed by MJ Opticon Monitor software. ABCG2 and CD133 mRNA levels in cancer cells and fibroblasts were measured and were normalized to glucose-6-phosphate dehydrogenase (G6PD). Flow cytometry analysis 106 live cells were stained in the staining solution containing 1% BSA, 2 mmol EDTA, and monoclonal fluorescein (FITC)-conjugated BCRP1 antibody (Chemicon Int., Hampshire, UK), or monoclonal allophycocyanin (APC)-conjugated CD133 antibody (Miltenyi Biotech, Bergisch Gladbach, Germany) for

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Hepatobiliary & Pancreatic Diseases International

Fig. 1. Relative expression of (A) ABCG and (B) CD133 genes in five pancreatic and three non-pancreatic carcinoma cell lines shown by quantitative real-time RT-PCR analysis. ABCG2 and CD133 mRNA levels in cancer cells were related to their respective levels in fibroblasts and were normalized to G6PD. (C) PCR amplification products were separated on 2% agarose gel. The results in A and B are expressed mean±SD and are representative of three independent experiments.

Fig. 2. Surface expression of (A) ABCG2 and (B) CD133 in five pancreatic and three nonpancreatic carcinoma cell lines shown by flow cytometric analysis. The cells were incubated with FITC-ABCG2 or APC-CD133 antibody, or corresponding isotype controls, followed by flow cytometric analysis. Dot plots of CD133-negative cells are not shown. The results are expressed by mean±SD and are representative of three independent experiments.

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Detection of tumor stem cell markers in pancreatic carcinoma cell lines

20 minutes at 4 ℃. Mouse FITC-IgG2b (Chemicon Int., Hampshire, UK) or APC-IgG1 (Miltenyi Biotech) were used as controls, respectively. Flow cytometric analysis was carried out with a FACSCalibur, and data were analyzed with the Cell Quest software (Becton Dickinson, Heidelberg, Germany). A minimum of 500 000 viable cells were measured per sample. Using forward and side scatter profile, debris and dead cells were gated out.

Results Expression of ABCG2 and CD133 in pancreatic carcinoma cell lines The relative expression of ABCG2 and CD133 in five pancreatic carcinoma cell lines was determined by real-time RT-PCR. Recent evidence suggests that the side population phenotype is associated with highlevel expression of the ABCG2 transporter.[20] It was previously shown that MCF7 and SKOV3 cell lines contain a side population of stem cells, whereas SaOS2 cells lack it.[17, 18] Therefore, MCF7 and SKOV3 cells served as positive, whereas SaOS2 cells as a negative control for real-time RT-PCR. As shown in Fig. 1, all five pancreatic carcinoma cell lines tested expressed significantly higher levels of ABCG2 than non-malignant fibroblasts. The highest expression was detected in Panc89, PancTu1, and A818-6 cells. The expression of CD133 was strongly elevated in two cell lines tested, PancTu1 and A818-6, whereas it was only slightly increased in Panc1 cells. In addition, SaOS2 cells showed a higher level of CD133 expression, whereas MCF7 and SKOV3 cells lacked detectable CD133 expression. Presence of ABCG2 and CD133 on the cell surface in pancreatic carcinoma cell lines To determine the presence of ABCG2 and CD133 on the cell surface in pancreatic carcinoma cell lines, flow cytometric analysis was made (Fig. 2). ABCG2 expression was confirmed in all pancreatic carcinoma cell lines although surface protein levels did not correlate very well with mRNA expression levels (Fig. 2A). Being consistent with mRNA expression data, both PancTu1 and A818-6 as well as SaOS2 displayed CD133 surface expression (Fig. 2B).

Discussion The existence of cancer stem cells in several solid and hematopoietic tumors has recently been

proven.[4-9,  11,  12] Moreover, it was demonstrated that cancer stem cells may be present also in established cancer cell lines.[17, 18] It is widely accepted that these cells are responsible for tumor recurrence after chemo- or irradiation therapy. Although it is still not clear whether the cancer stem cells are derived from original tissue-derived stem cells, bone marrow stem cells or mature cells that have undergone a dedifferentiation process, it has been suggested that novel strategies for successful cancer therapy should focus on the elimination of cancer stem cells. Because pancreatic tumors are highly apoptosis-resistant,[26] we investigated whether pancreatic carcinoma cell lines contain a subpopulation of putative stem cells that could be targeted for anti-cancer therapy. It is possible that cell culture conditions may contribute to phenotypic differences between cancer stem cells and non-cancer stem cells as well as between cancer stem cells within established cell lines and cancer stem cells in the original tumors. On the other hand, growing evidence suggests that both primary tumors and cell lines derived from them represent the same surface immunophenotypes.[17, 18] It is believed that the research limitations that result from the small number of cancer stem cells isolated from clinical tumors may be overcome by in vitro propagation to obtain a sufficient number of cells to develop new anti-cancer therapies. In the current study, all pancreatic carcinoma cell lines tested over-expressed ABCG2 compared with non-malignant cells. The presence of ABCG2 at the cell surface was confirmed by flow cytometric analysis. Relative surface protein levels, however, did not correlate very well with mRNA levels (compare Fig. 1A and Fig. 2A). It is known that protein regulatory mechanisms such as post-translational modifications, proteolysis or sequestration in cell compartments, affect proteins but not transcripts. In addition, a small decrease/increase in mRNA level may not necessary lead to a significant decrease/increase in protein level. ABCG2 has been identified as a transporter responsible for the Hoechst 33342 dye efflux pattern within the side population of stem cells by flow cytometric analysis.[20] It is accepted that the side population approach is a valid marker-independent method to identify stem cells. On the other hand, it is possible that the side population of some cancers is too small (i.e.,