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Endocrinology, doi:10.1210/en.2007-1001
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Endocrinology Vol. 149, No. 3 1314-1322
Copyright © 2008 by The Endocrine Society

Side Population Does Not Define Stem Cell-Like Cancer Cells in the Adrenocortical Carcinoma Cell Line NCI h295R

Urs D. Lichtenauer, Igor Shapiro, Klaus Geiger, Marcus Quinkler, Martin Fassnacht, Roland Nitschke, Klaus-Dieter Rückauer and Felix Beuschlein

Institute of Molecular Medicine and Cell Research (U.D.L., I.S., F.B.), Department of Internal Medicine I (K.G.), Life Imaging Center (R.N.), Centre for Systems Biology, and Department of General and Visceral Surgery (K.-D.R.), Albert-Ludwigs-University Freiburg, D-79085 Freiburg, Germany; Clinical Endocrinology (M.Q.), Department of Internal Medicine, Charite Campus Mitte, Charite University Medicine Berlin, D-10117 Berlin, Germany; Department of Internal Medicine I (M.F.), Endocrine and Diabetes Unit, University of Wuerzburg, D-97074 Wuerzburg, Germany; and Medical Clinic (F.B.), University Hospital Innenstadt, Ludwig Maximilians University, D-80336 Munich, Germany

Address all correspondence and requests for reprints to: Felix Beuschlein, M.D., Division of Endocrine Research, Department of Medicine Innenstadt, University Hospital Munich, Ziemssenstrasse 1, D-80336 Munich, Germany. E-mail: felix.beuschlein{at}med.uni-muenchen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 ± 18.1 vs. 100.0 ± 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN INCREASING body of evidence suggests that a small population of stem cell-like cancer cells define the clinical phenotype of a variety of tumor entities. This concept, which has first been established in hematological malignancies such as acute myeloid leukemia, has only recently been adapted for a growing number of solid tumors including breast cancer (1, 2), neuroblastoma (3), different brain tumors (4, 5), head and neck squamous cell carcinomas (6), colon carcinoma (7), and pancreatic cancer (8). Stem cell-like cancer cells have been proposed to result either from malignant transformation of a normal stem cell or from reacquisition of stem cell characteristics of a differentiated cell, or a progenitor cell, or both (9). Functional characteristics of stem cell-like cancer cells have been defined as the ability to regrow tumors in xenograft in vivo models, to undergo qualitative self-renewal, to recapitulate all cell types within an individual tumor, and to possess extensive proliferative capacity (9, 10, 11). In contrast to this small cell fraction, the vast majority of cells in a given tumor have been hypothesized to represent a mature, nonproliferating population, or at least a population with a more differentiated and less aggressive phenotype. Because therapies specifically targeted to stem cell-like cancer cells could have the potential to eradicate the cellular source of the individual tumor, the pathophysiological concept behind these tumor stem cells has aroused considerable clinical interest.

Tumor cells with stem cell characteristics have been isolated by cell surface markers such as CD24, CD44, and CD133 as well as on the basis of sphere formation after in vitro cultivation (12). An alternative approach, especially in the absence of known cell markers, is the side population (SP) phenomenon, which has been used to identify and isolate enriched stem cell populations from a variety of tissues including bone marrow (13), mammary gland (14, 15), skin (16), liver (17), lung (18), skeletal muscle (19), limb (20), heart (21), and brain (22). This technique is based on Hoechst dye 33342 exclusion, which primarily occurs through the activity of membrane pumps encoded by multidrug-resistance gene 1 (MDR1) (23) and breast cancer-resistance gene 1 (ABCG2) (24). As for the identification and isolation of stem cell-like tumor cells, Hoechst dye exclusion has been successfully applied in neuroblastoma tumors and corresponding cell lines (3). Recent studies have extended these findings for primary cultures, for example of gastrointestinal cancers (25, 26), and ovarian cancer (27), defining the SP characteristics as a valuable marker for cells with stem cell characteristics. Intriguingly, the presence of a specific SP has also been demonstrated in established tumor cell lines of different origin, such as glioma (28), breast (29), thyroid cancer (30), and melanoma (31) cell lines, arguing for the presence of a functionally distinct subpopulation of cells even in a monoclonal cell environment.

Adrenocortical carcinomas are highly malignant tumors that respond poorly to standard chemotherapy treatment regimens (32). The reasons for this clinical phenotype are believed to be based on cellular heterogeneity of the tumor and the presence of multidrug resistance genes, which encode for pumps that actively expel the cytotoxic substances (33). Because the Hoechst efflux capacity of SP cells is also dependent on the presence of membrane pumps (34), we hypothesized that identification of the SP in adrenocortical tumors could potentially represent a suitable isolation method to evaluate stem cell-like tumor characteristics in adrenocortical malignancies. Thus, this study was initiated to investigate whether stem cell-like tumor cells represented by SP cells are present in adrenal tumors and, if so, whether their drug efflux capabilities are responsible for the known chemotherapy resistance in this endocrine malignancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Diagnosis and endocrine work-up of patients with adrenal masses were defined following standard clinical, biochemical, imaging, and histological criteria. All patients gave written informed consent, and the study was approved by the local ethical committee of the University of Freiburg. Ten surgical tissue samples were included from a total of eight patients (one endocrine inactive adenoma, one myolipoma, three hyperplasias, two adrenocortical carcinomas, and one metastasis of an adrenocortical carcinoma as well as adjacent normal adrenal gland tissue from two patients).

Cell line culture conditions and primary cell culture of adrenal surgical samples
NCI h295R cells were grown at 37 C and 5% CO2 in DMEM/F12 medium (Life Technologies, Inc., Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (FCS) (HyClone, South Logan, UT) and 2 mM L-glutamine (BioWhittaker Cambrex, East Rutherford, NJ), whereas murine Y1 and Y6 cells were maintained in DMEM high glucose containing 7.5% horse serum, 2.5% FCS, and 10 µl/ml penicillin/streptomycin (all Life Technologies) as described previously (35, 36).

For primary cultures from human adrenal tumors and adjacent normal adrenal gland tissue, the samples were cleaned of surrounding fat, connective tissue, and blood vessels. Thereafter, tissue samples were minced into pieces smaller than 0.5 mm using a razor blade. Minced samples were transferred into 50-ml Falcon tubes, spun down at 1000 rpm for 5 min, and rinsed twice with fresh PBS. Digestion was performed with 1 mg collagenase II (Biochrom, Berlin, Germany) per ml PBS at 37 C for 50 min in a shaking water bath. Cell suspension was pipetted up and down at least twice during the incubation time. After digestion, pure FCS was added to a minimum concentration of 10% to inactivate the collagenase, followed by a centrifugation step as described above. Cell pellets were resuspended in erythrocyte lysis buffer and incubated for 7 min at room temperature. After another centrifugation step, cells were resuspended in 5–10 ml culture medium depending on the expected cell count (DMEM/F12 with 10% FCS, 3.1 g/liter glucose, 15 mM HEPES, and 10 µl/ml penicillin/streptomycin, all from Life Technologies) and sequentially filtered through a 100- and 70-µm nylon mesh. Cells were counted using a Neubauer counting chamber and further processed for fluorescence-activated cell sorting (FACS) as described below.

Hoechst staining and FACS sorting
NCI h295R and Y1 cells were detached from the cell culture flask with trypsin (Sigma Chemical Co., St. Louis, MO), and viable cells were counted with trypan blue, whereas single-cell suspensions from adrenal surgical material were directly used after preparation as described above. Cells were transferred into DMEM high-glucose medium (Life Technologies) containing 2% FCS and 10 mM HEPES and stained with the fluorescent dye Hoechst 33342 (Sigma) at a concentration of 5 µg/ml at 37 C for 90 min as described (37). After the staining procedure, propidium iodide (2 µg/ml; Sigma) was added to the samples for identification and exclusion of dead cells. Cell analysis and sorting were performed on a triple-laser cell sorter [MoFlo; Dako (formerly Cytomation), Fort Collins, CO]. The Hoechst dye was excited at 350 nm using an argon ion laser. Emission wavelengths were detected at 450 nm using a 450/20 bandpass filter and above 675 nm using a 675LP filter. The SP was defined as described (37), including the verification procedure with verapamil (Sigma), with the only difference that special care was taken to exclusively isolate the tip of the SP cell fraction.

Time course and confocal laser microscopy
For the FACS time-course experiments, 50 million NCI h295R cells were harvested and transferred to the staining medium as described above. A small sample underwent the regular staining procedure as a positive control and to calibrate the FACS settings. The circulating water temperature of the FACS sample chamber was raised to 37 C. Once the sample reached this temperature, the first FACS file was saved; Hoechst dye was then added, and subsequent files were saved every 5 min for a total of 120 min. Recalibration and adjustment of the FACS settings were performed before each set of experiments to ensure optimal technical performance and separation of the cell population. To ensure comparability in experiments for which SP quantification was critical, FACS experiments were performed within the same run with identical experimental conditions.

For confocal laser microscopy cell morphometry, 1000 sorted SP and non-SP cells were transferred to a microslide VI chamber (ibidi, München, Germany), and three-dimensional image stacks were recorded at 405 nm excitation and emission collected with a long-path filter LP420 with a confocal laser scanning microscope (LSM 510 Meta; Zeiss, Jena, Germany). Data were analyzed using the spot detection and analysis functions of the Imaris software (Bitplane AG, Zürich, Switzerland).

For time-course experiments, 450,000 NCI h295R cells were plated on each of two plastic dishes optimized for high-resolution microscopy (µ-dish I; ibidi). After 2 d, the cells reached 80% confluency, at which time the regular growth medium was replaced by the staining medium. One of the wells was then placed in a microscopy heating unit (PeCon, Erbach, Germany) that held the temperature of the optical well at 37 C. Hoechst dye was added as described above. After 90 min, the confocal microscope settings were adjusted for optimal recording conditions for the Hoechst fluorescence (two-photon excitation at 760 nm, emission collected from 400–480 nm). Thereafter, the dish with untreated cells was placed within the microscope, followed by the first photograph and addition of Hoechst dye to the medium. Beginning with this incubation, additional photographs were taken every 90 sec for a total of 120 min.

Tracing experiment
NCI h295R cells were separated into SP and non-SP populations as described above with a final number of 250,000 cells for each population. SP cells were stained with the red fluorescence dye PKH26, whereas non-SP cells were labeled green with PKH67 using the General Cell Membrane Labeling Kit (both Sigma). Images of the cells were taken from both populations with a fluorescence microscope (Axiovert 200; Zeiss) before they were mixed in equal proportions and seeded together in a 12-well plate in regular NCI h295R growth medium as described above. Additional fluorescent images were then taken on d 2, 6, and 14.

Microculture tetrazolium (MTT) assay
A total of 2000 SP and non-SP cells were plated separately in triplicate in 96-well plates and were allowed to grow for 14 d at 37 C in a humidified incubator at 5% CO2. To develop the plate, MTT (Sigma) was dissolved in serum free-medium at a final concentration of 0.5 mg/ml and briefly sonicated. Culture medium was removed from the wells and 100 µl MTT/medium per well was added, followed by an incubation for 2 h at 37 C. Thereafter, the MTT/medium was removed, and 100 µl solubilization (10% SDS, 0.01 M HCl) solution per 100 µl medium was added to stop the coloring reaction and to dissolve the formed formazan crystals. The mixture was kept in the dark and incubated overnight at room temperature. Finally, the plate was shaken at 60 rpm for 5 min to dissolve the precipitate before reading the plate at 555 nm using a microplate reader.

Cell cycle analysis
At least 300,000 SP and non-SP cells were fixed overnight in 4.5 ml of 70% ethanol each and kept at +4 C. Thereafter, cells were centrifuged, the supernatant discarded, and the cell pellet resuspended and washed with PBS. After another centrifugation step, cells were resuspended in 300 µl propidium iodide/Triton X-100 staining solution containing 5 µg propidium iodide/ml PBS, 0.1% Triton X-100, and 100 µg/ml freshly prepared RNase A and were incubated at 37 C for 30 min. For FACS analysis, cells were transferred to FACS tubes and placed on ice. FACS analysis was performed using a FACSCalibur (B&D Biosciences, Heidelberg, Germany) at 488 nm. The data were analyzed using FlowJo version 6.0 and the Watson Pragmatic cell cycle formula.

Real-time PCR
RNA from at least three independent SP and non-SP cell preparations was extracted using the QIAGEN RNA mini kit (QIAGEN, Valencia, CA) following the instructions of the manufacturer. cDNA was transcribed using a RT kit (Promega, Mannheim, Germany) and 1.0 µg total RNA.

Real-time PCR was performed using the FastStart DNA MasterPlus SYBR Green I reaction mix in the LightCycler 1.5 (Roche, Mannheim, Germany). The cycling conditions for real-time PCR included a preincubation step at 95 C for 10 min, followed by an amplification step that consisted of 40–45 cycles at 95 C for 10 sec, annealing (the temperature was primer dependent as given below) for 6 sec, and an extension step at 72 C, for which the time was calculated by the product length in base pairs divided by 25 (Roche, Indianapolis, IN). Primer sequences as well as product lengths and annealing temperatures were as follows: P450scc, 5'-GCAACGTGGAGTCGGTTTAT-3' and 5'-TCCTCGAAGGACATCTTGCT-3' (664 bp; 52 C); steroidogenic acute regulatory enzyme (StAR), 5'-CAGGACAATGGGGACAAAGT-3' and 5'-ATGAGCGTGTGTACCAGTGC-3' (608 bp; 63 C); IGF-II, 5'-CAAATTACCTGCCCATTCGT-3' and 5'-GCGTTAAAGGAGTTGAGTTGAG-3' (357 bp; 58 C); steroidogenic factor 1 (SF-1), 5'-TGCACTGCAGCTGGACCGCCAGGAGTT-3' and 5'-AGGGCTCCTGGATCCCTAATGCAAGGA-3' (390 bp; 58 C); ABCG2, 5'-GAGTGGCTTTCTACCTTGTC-3' and 5'-CATCACAACATCATCTTGTACC-3' (246 bp; 58 C); MDR1, 5'-ATCGTTTGTCTACAGTTCGT-3' and 5'-TATACTTTCATCCAGAGCCT-3' (316 bp; 58 C); claudin-1 (CLDN1), 5'-CCGTTGGCATGAAGTGTATG-3' and 5'-GTTTTGGATAGGGCCTTGGT-3' (309 bp; 60 C); zona occludens-1 (TJP1), 5'-AGCCAAGGAAGGCTTAGAGG-3' and 5'-ACAACACGGAACACCTCTCC-3' (270 bp; 61 C); protooncogene MET, 5'-ACTCCCCCTGAAAACCAAAGCC-3' and 5'-GGCTTACACTTCGGGCACTTAC-3' (536 bp; 60 C); N-cadherin (NCAD), 5'-GACAATGCCCCTCAAGTGTT-3' and 5'-ACCCACAATCCTGTCCACAT-3' (354 bp; 61 C); and vimentin (VIM). 5'-GCAGGCTCAGATTCAGGAAC-3' and 5'-GCTTCAACGGCAAAGTTCTC-3' (330 bp; 59 C).

Melting curve analysis was performed between 65 and 98 C (0.1 C/sec) to determine the melting temperature of the amplified product and to exclude undesired primer dimers. Furthermore, the products were run on a 1% agarose gel to verify the amplified product. Each sample was run at least in triplicate. Quantification was normalized using the β-actin as a reference gene. To facilitate overall comparison, expression levels of the particular genes were set at 100% for non-SP cells.

In vitro treatment with cytotoxic agents
Dose-finding experiments were performed on unsorted NCI h295R cells to assess the therapeutic range of etoposide (Medac, Wedel, Germany), doxorubicin (Medac), streptozotocin (Pfizer, Quebec, Canada), cisplatin (Hexal, Holzkirchen, Germany), and mitotane (Bristol-Myer Squibb, Munich, Germany). In a different set of experiments, NCI h295R cells were Hoechst stained and FACS sorted as described above, and the separated SP and non-SP cells were seeded in 96-well plates at a density of 25,000 cells per well in triplicate. After incubation with the predefined dosage range of the individual cytotoxic agent, cell viability was quantified by MTT assays as described above.

Statistical analysis
All results are expressed as mean ± SEM. If not stated otherwise, all statistical comparisons were analyzed by ANOVA and Fisher’s protective least significant difference test. Statistical significance is defined as P < 0.05 and is indicated in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SP cells are present in a variety of adrenocortical tissues and cell lines
FACS analysis after Hoechst dye labeling consistently revealed the presence of a SP in single-cell suspension from adrenal surgical tissues including an adrenal adenoma (n = 1, SP fraction 0.1%), a myelolipoma (n = 1, SP fraction 0.98%), adrenocortical carcinomas (n = 2, SP fraction 0.03 and 0.09%), adjacent normal adrenal tissue (n = 2, SP fraction 0.00 and 0.03%), a metastasis of an adrenocortical carcinoma (n = 1, SP fraction 0.02%), and adrenal hyperplasia (n = 3, SP fraction 0.03–2.62%). A similar proportion of SP cells was found in normal mouse adrenal glands (data not shown). Moreover, a large SP fraction was detectable in both a human and murine adrenocortical cell line (NCI h295R, 2.79 ± 1.13%; Y1, 0.44 ± 0.37%; Y6, 16.3%). For a general overview, the results are summarized in Table 1Go.


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  		TABLE 1. Presence of the side population (percent) in different adrenal tissues

 
To ensure the specificity of this FACS-derived SP and exclude nonspecific staining effects, coincubation of Hoechst dye with the calcium channel blocker verapamil was performed, which resulted in the expected decrease in the SP cell population (e.g. NCI h295R cells, 2.91% untreated vs. 0.11% after verapamil treatment; Fig. 1AGo). Confocal laser microscopy morphometry proved a significantly lower mean intensity of SP compared with non-SP cells (100 ± 3.11 vs. 81.8 ± 1.82%; P < 0.01) with a comparable mean cell volume (100 ± 2.30 vs. 101.4 ± 1.83%; P = 0.65; Fig. 1BGo). Moreover, FACS time-course experiments and continuous confocal laser microscopy during the Hoechst staining procedure confirmed a gradual dye uptake with a differential nuclear staining and an optimal visual appearance of the Hoechstlow classified SP cells after 90 min (Fig. 1CGo). Prolonged Hoechst incubation over 120 min led to a nearly homogeneous Hoechstpos staining pattern in the confocal microscope (Fig. 1CGo), so the duration of Hoechst incubation was set to 90 min for all additional experiments.


Figure 1
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  		FIG. 1. A, Characteristic Hoechst 33342 dye staining profile for the human NCI h295R adrenocortical cell lines, demonstrating the existence of a SP, which can successfully be blocked by simultaneous incubation with the Ca2+ channel antagonist verapamil; B, confocal three-dimensional cell reconstruction exemplary shown for SP cells (left), and the resulting quantification diagram for the mean cell Hoechst 33342 intensity per pixel and the cell volume in cubic micormeters in SP and non-SP cells (right); C, Hoechst 33342 staining time-course experiment with NCI h295R cells using reapplied FACS analysis (upper panel) and continuous confocal microscopy (lower panel) demonstrating differential Hoechst dye uptake and formation of the typical FACS pattern at 90 min incubation time. *, Statistically significant differences (P < 0.05) between SP and non-SP cells.

 
SP cells from the NCI h295R cell line express lower levels of steroidogenic enzymes and higher levels of multidrug resistance genes
As would be expected from a cell population with stem cell characteristics, semiquantitative RT-PCR analysis revealed lower expression of marker genes specific for differentiated adrenocortical cells including SF-1, P450 side chain cleavage enzyme (SCC), and StAR in SP cells relative to non-SP cells (Fig. 2AGo). This finding was further substantiated by real-time PCR with lower expression levels found in SP cells (SCC, 59.3 ± 3.1%; P < 0.01, and StAR, 59.0 ± 1.1%; P < 0.01) compared with non-SP cells (SCC, 100 ± 5.9%, and StAR, 100 ± 4.8%; Fig. 2BGo). In contrast, the expression of IGF-II, a major regulator of adrenocortical development and function, was not different between the two groups (Fig. 2BGo).


Figure 2
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  		FIG. 2. A and B, Expression pattern of adrenocortical marker genes by means of semiquantitative RT-PCR (A) and real-time analysis (B) indicating lower levels of steroidogenic enzyme expression and similar levels of IGF-II expression as a marker of the fetal adrenal in SP compared with non-SP cells; C and D, in addition, real-time analysis of ABCG2 (C) and MDR1 (D) reveals profoundly elevated expression levels in SP cells. *, Statistically significant differences (P < 0.05) between SP and non-SP cells.

 
Furthermore, expression levels of the multidrug resistance genes ABCG2 and MDR1, which are considered to be mainly responsible for the Hoechst drug efflux effect, were measured in triplicate in independent samples and were found to be substantially increased in SP (ABCG2, 27089 ± 3283%, P < 0.04, and MDR1, 176.0 ± 23.7%, P < 0.03) compared with non-SP cells (ABCG2, 100 ± 47.1%, and MDR1, 100 ± 0.5%; Fig. 2Go, C and D).

NCI h295R SP cells display similar growth potential and self-renewal capacity in comparison with non-SP cells
In contrast to the assumed phenotypic properties of a stem cell-like tumor cell, after FACS-based cell sorting followed by separate cultivation and quantification of cellular viability, NCI h295R SP cells displayed a proliferative potential comparable to that of non-SP cells (105.6 ± 18.1 vs. 100.0 ± 3.5%, P = 0.7; Fig. 3AGo). To further verify this finding, both populations of cells were labeled with different live-cell stains, which allowed detection of the original cell as well as several daughter cell generations. The resulting red-stained SP cells and green-stained non-SP cells were repooled with equal cell numbers (2500 cells per population per well; Fig. 3BGo) and separately quantified over the course of the next 14 d. Repeated cell counting revealed a stable and equal ratio of the original cell populations (SP fraction: d 2, 49.9 ± 2.2%; d 8, 46.1 ± 1.8%; d 14, 46.5 ± 2.8%; Fig. 3BGo) indicating the inability of the SP cells to outgrow the non-SP population.


Figure 3
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  		FIG. 3. A, FACS-based cell sorting and in vitro cultivation demonstrating similar growth potential between the SP and non-SP of NCI h295R cells after 6 d in culture; B, tracing experiments with life dye colors (green, non-SP cells; red, SP cells) indicating indistinguishable growth kinetics with equal distribution of both cell populations after coculturing.

 
Based on stem cell physiology, SP cells should give rise to SP and non-SP cells by means of asymmetric cell division, whereas non-SP cells should not have this potential. To further analyze the SP and non-SP distribution over several generations, NCI h295R cells were repeatedly stained, sorted, restained and re-sorted over four passages. Although the comparable numbers within a given passage consistently demonstrated an enrichment of SP cells after original SP sorting, isolated non-SP cells regularly gave rise to a significant proportion of SP cells (Fig. 4Go).


Figure 4
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  		FIG. 4. Iterative sorting and separate culturing of SP and non-SP NCI h295R cells over four passages, demonstrating persistence of similar distributions for both cell populations within each passage.

 
SP phenotype in NCI h295R SP is not dependent on cell cycle or epithelial-mesenchymal transition
Because the results from tracing and re-sorting experiments suggested a similar growth potential and self-renewal capacity between SP and non-SP NCI h295R cells, we hypothesized that phenotypic changes that were associated with Hoechst staining properties could be dependent on the cell cycle of the individual cell. However, FACS analysis of propidium iodine-stained cells revealed no differences in cell cycle distribution (non-SP: G1 phase, 66.9%; S phase, 23.1%; G2 phase, 9.47% vs. SP: G1 phase, 66.8%; S phase, 24.8%; G2 phase, 6.7%; Fig. 5Go) between SP and non-SP cells.


Figure 5
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  		FIG. 5. A, Demonstration of similar cell cycle distribution of SP and non-SP of NCI h295R cells arguing against a cell cycle-dependent association of the Hoechst dye exclusion phenomenon in NCI h295R cells; B, expression analysis of NCI h295R SP and non-SP cells indicating similar expression pattern for epithelial (hCLDN1. hTJP, and hMET) and mesenchymal (hNCAD and hVIM) marker genes. *, Statistically significant differences (P < 0.05) between SP and non-SP cells.

 
Another explanation for the free conversion of the presumably more undifferentiated SP cells with a rather mesenchymal phenotype into the more differentiated non-SP cells, which possess a more epithelial phenotype, has been proposed to occur by epithelial-mesenchymal transition. However, real-time PCR analysis did not reveal consistent differences between SP and non-SP cells for the expression levels of the epithelial markers CLDN1, TJP1, and MET (non-SP vs. SP, 100.0 ± 1.3 vs. 90.3 ± 1.7%, P < 0.01; 100.0 ± 5.9 vs. 100.4 ± 11.1%, P = 0.97; 100.0 ± 2.7 vs. 101.1.3 ± 10.5%, P = 0.92, respectively), and the mesenchymal markers VIM and NCAD (100.0 ± 0.6 vs. 93.7 ± 3.4%, P < 0.01; 100.0 ± 1.1 vs. 81.2 ± 1.6%, P = 0.03, respectively; Fig. 5Go B). Thus, a close association between the SP and non-SP phenotype with a particular mesenchymal or epithelial state in NCI h295R cells seems unlikely.

SP cells in the NCI h295R cell line do not display resistance to cytotoxic agents in comparison with non-SP cells
Chemotherapy resistance represents the key survival benefit for stem cell-like tumor cells. However, similar to the finding for cell proliferation under baseline conditions, incubation with a variety of chemotherapeutic agents commonly used in the treatment of adrenocortical cancer (32) did not demonstrate a significantly higher survival of SP cells in comparison with non-SP cells (SP vs. non-SP: etoposide, 100 µg/ml, 30.1 ± 7.6 vs. 36.8 ± 4.1%, P = 0.07; doxorubicin, 1.0 mg/ml, 84.6 ± 13.1 vs. 77.1 ± 19.7%, P = 0.86; streptozotocin, 3 mg/ml, 80.0 ± 6.0 vs. 63.2 ± 12.2%, P = 0.14; cisplatin, 3 µg/ml, 36.0 ± 21.1 vs. 34.8 ± 15.4%, P = 0.91; mitotane, 25 µg/ml, 13.1 ± 1.3 vs. 13.2 ± 3.4%, P = 0.84; Fig. 6Go). Taken together, these findings indicate an indistinguishable response of SP and non-SP cells to cytotoxic actions of the applied pharmacological agents.


Figure 6
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  		FIG. 6. Cell viability after incubation with increasing doses of different chemotherapeutic (cisplatin, streptozotocin, doxorubicin, and etoposide) and adrenolytic (mitotane) agents assessed by MTT assay, indicating no survival benefit for SP vs. non-SP NCI h295R cells. *, Statistically significant differences (P < 0.05) between SP and non-SP cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of their potential impact in general tumor pathophysiology, stem cell-like tumor cells have gained much attention over the last 2 yr (9, 11). Sparked by the initial publication from Al Hajj and colleagues (1), who first isolated cells with stem cell properties in breast cancers, a number of groups have identified cancer stem cells derived from other tumors in rapid succession, including ovarian (27) and lung cancers (38), brain tumors (4), and colon carcinomas (7). The prerequisite for the successful isolation of these cell populations was the identification of surface markers that could be used for cell sorting-based approaches.

Cell surface markers in adrenocortical tumor entities have not been identified so far. In a candidate gene approach, we unsuccessfully analyzed the human adrenocortical cancer cell line NCI h295R for known tumor stem cell markers such as prominin (CD133), SCA1 (LY6), and CD24 (data not shown) until we detected a distinct SP in a variety of adrenal tumors. Until 2005, the Hoechst efflux phenomenon of the SP was used solely to isolate a stem cell-enriched population from normal bone marrow, liver, and brain tissue (37). Recently, however, Hirschmann-Jax and colleagues (3) were the first to successfully adapt this technique to isolate stem cell-like tumor cells from neuroblastoma cell lines and primary cultures. Similar to the findings achieved with stem cell-like tumor cells that had been isolated on the basis of surface stem cell markers, SP cells were shown to proliferate and possess self-renewal capacity and chemotherapy resistance beyond that of non-SP cells. Since then, SP cells from ovarian (27), brain (39), and thyroid (30) cancers have been demonstrated to exhibit similar stem cell-like functional properties.

Herein we demonstrate the presence and successful isolation of SP cells from a variety of adrenocortical tumor specimens, including primary cultures of human adrenocortical tumors with different endocrine activities as well as established human and murine adrenocortical cancer cell lines. Because sources of surgical tumor specimens are limited and the presence of functionally distinct SP in permanent cell lines have been demonstrated for other tumor entities, we chose to further analyze SP cells isolated from the well described human cell line NCI h295R (40), which has been used extensively as an adrenocortical model system in the past (41).

To ensure the specificity of the isolation procedure, the following precautions were implemented. First, we show the expected pharmacological blockage of the Hoechst dye efflux capacity of SP cells using the calcium channel blocker verapamil. Second, we demonstrate the appearance of a distinct SP within a detailed time-course experiment based on both FACS analysis as well as visualization by confocal microscopy. Findings from these time-course experiments were used to specify incubation times for the further experimental settings. Third, special care was taken to exclusively isolate cells from the tip of the SP fraction, because it has been proposed that these cells contain the highest proportion of undifferentiated cells (42, 43). The expected lower Hoechst intensity of the isolated SP cells compared with the non-SP cells was quantified by confocal laser microscope morphometry. Fourth, we show that SP cells derived from NCI h295R cells have a distinct expression profile in comparison with cells from the non-SP cells; ABCG2 and MDR1, which are believed to encode for the membrane-associated pumps mainly responsible for the Hoechst 33342 efflux, are substantially more highly expressed in SP cells. Also, because expression of steroidogenic enzymes can be regarded as a marker for differentiated adrenocortical cells, the lower expression levels detected are in line with the expected phenotype of the isolated SP, which would be that of less differentiated cells. Overall, these experiments provide ample evidence for the presence of a distinct SP of cells within adrenocortical tumors specimen and cell lines.

Stem cell-like cancer cells have been characterized on the basis of functional properties that include higher proliferative potential with the ability to outgrow other cell populations, self-renewal capacity that is not present in the remaining tumor, and the ability to withstand higher levels of cytotoxic agents included in standard chemotherapeutic regimens (44, 45, 46). In line with this notion, we performed a number of experiments to assess similar functional characteristics of the isolated NCI h295R SP cells and define them as potential stem cell-like cancer cells.

In contrast to what has been reported for other tumor cell lines, however, FACS-based cell sorting and separate culturing of NCI h295R cells from the SP and non-SP revealed similar growth rates under baseline conditions. These studies were further validated by tracking experiments in which separated cells from the SP and non-SP were marked by means of different dyes that constantly tag the original cell as well as several generations of daughter cells. When the two cell populations were cocultured in an equal distribution, this proportion was maintained over the observation period, indicating parallel and indistinguishable growth kinetics of the two different populations of cells. To further highlight this finding, iterative re-sorting experiments were carried out. If SP cells would possess significantly higher stem cell capacities than non-SP cells, one would expect not only an enrichment of the SP cells after recurrent re-sorting for the SP phenotype but also the loss of SP cells after repeated re-sorting for the non-SP phenotype. As expected, a slightly higher proportion of SP cells was evident in subsequent FACS analyses performed on separated and cultured SP cells in comparison with that of separated and cultured non-SP cells. However, recurrent sorting and culturing of non-SP cells consistently resulted in a cell population that contained an easily detectable proportion of SP cells.

A potential explanation for the findings of the re-sorting and color-tracking experiments could be that a free and undirected transition of an individual cell between the SP and non-SP phenotype occurs. One possibility would be a continuous cell cycle-dependent change in membrane pump expression or activity, responsible for the SP characteristics. Alternatively, transition of a cell from a more differentiated epithelial-like to a less differentiated mesenchymal-like phenotype could go in parallel with the observed Hoechst efflux capacities. This epithelial-mesenchymal transition has been considered a prerequisite for tumor infiltration and metastasis. Although the original model proposes a unidirectional change toward the less differentiated mesenchymal phenotype, recent evidence has suggested the presence of incomplete epithelial-mesenchymal transition as well as a reversion of this transition (47). To further explore these possibilities for NCI h295R cells, cell cycle-dependent phenotypic changes and the expression patterns of proposed epithelial and mesenchymal markers were investigated. However, cell cycle analysis did not reveal differences between SP and non-SP cells, and there was no evidence for a significant association between Hoechst dye exclusion properties and a specific stage in the cell cycle. Similarly, real-time analysis of a number of epithelial and mesenchymal markers did not indicate a common epithelial or mesenchymal pattern for SP or non-SP cells.

Probably the most relevant aspect of the tumor stem cell concept from the clinical perspective is the ability for stem cell-like tumor cells to possess an additional survival benefit in the context of various chemotherapeutic agents. Adrenocortical carcinomas are characterized by their overall resistance to several chemotherapies (32). As such, the targeted therapy of a subpopulation of cells within an adrenocortical carcinoma responsible for this deleterious clinical phenotype would be of major clinical interest. However, although SP cells derived from NCI h295R cells obviously express the postulated pumps responsible for Hoechst efflux, this equipment seems to be insufficient to ensure chemotherapy resistance associated with a survival benefit over non-SP cells. Hence, the SP of NCI h295R cells seems not to represent a relevant experimental target to explore future stem cell-directed therapies.

Taken together, we demonstrate the presence of a distinct SP of cells in adrenocortical tumors and tumor cell lines. Although these findings are in line with those reported for a number of other tumor entities, we provide evidence that SP cells isolated from the NCI h295R adrenocortical cancer cell line are not distinguishable from non-SP cells based on their proliferative potential, self-renewal capacity, or resistance to chemotherapeutic agents. As such, the SP characteristic is unlikely to determine the malignant phenotype within the NCI h295R cell line. Whether this phenomenon is restricted to this particular cell line or poses a general concept for adrenocortical tumorigenesis needs to be addressed in future studies. However, as Hoechst exclusion is increasingly used for the isolation of stem cell-like tumor cells, it has to be stressed that the mere demonstration of a SP cannot be equated with the presence of a tumor-like stem cell population in a given sample. In fact, the finding of SP cells has to be scrutinized by detailed functional studies to prove or disprove the stem cell capacity of the SP in each tumor entity.


   Acknowledgments
 
We are indebted to Dirk Engelbert for assistance with the cell cycle analysis and Lilia Spady for excellent technical assistance. Cell sorting was carried out in the Core Facility, Department of Internal Medicine I, Universitaetsklinikum, Freiburg, and confocal microscopy at the Life Imaging Center, University of Freiburg.


   Footnotes
 
This work was supported by Landesstiftung Baden-Württemberg Grant P-LS-ASN/5 (to F.B.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 6, 2007

Abbreviations: FACS, Fluorescence-activated cell sorting; FCS, fetal calf serum; MTT, microculture tetrazolium; SP, side population.

Received July 23, 2007.

Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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