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Departments of Molecular Medicine (N.M., A.I., H.N., S.Y.) and International Health and Radiation Research (V.S., S.Y.), Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan; and Transfusion Service (K.N.) and Takashi Nagai Memorial International Hibakusha Medical Center (A.O., S.Y.), Nagasaki University Hospital of Medicine and Dentistry, Nagasaki 852-8501, Japan
Address all correspondence and requests for reprints to: Norisato Mitsutake, M.D., Ph.D., Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: mitsu{at}nagasaki-u.ac.jp.
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Abstract |
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Introduction |
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Identifying CSCs was initially accomplished in the context of acute myeloid leukemia. Several groups demonstrated that the cells capable of initiating leukemia in immunodeficient mice were found in CD34+CD38– fraction (3, 4, 5). Multiple myeloma might also have a CSC population. The majority of the myeloma cells express CD138; however, a minority of CD138– cells preferentially engrafted in immunodeficient mice and also generated CD138+ cells (6).
Recently such CSCs have been also isolated in several types of solid tumors. Al-Hajj et al. (7) demonstrated that breast cancer cells with CD44+CD24–/lowlineage– surface phenotype were tumorigenic in immunodeficient mice. Importantly, as few as 100 tumorigenic cells were able to form tumors in the mice, whereas 105 of CD44+CD24+ cells were not able to form tumors. These tumorigenic cells were serially passaged in the mice, and each time the generated new tumors contained additional CD44+CD24–/lowlineage– tumorigenic cells as well as phenotypically mixed nontumorigenic cancer cells. Further evidence supporting an existence of CSCs in solid tumors came from the studies of brain tumors (8). Singh et al. (9, 10) reported that only CD133+ brain tumor fraction contained cells that were capable of tumor initiation in immunodeficient mouse brains. CD133 is a marker expressed in normal neural stem cells, suggesting that the normal neural stem cell is the target of transforming events that lead to brain tumors. These cells have been identified by the expression of their unique surface markers. However, most tissue-restricted stem cells lack unique and specific expression markers, including those in thyroid.
A small subset of cells, termed side population (SP), has been identified using flow cytometric analysis (11, 12, 13). The SP cells have the ability to exclude the DNA binding dye, Hoechst33342, and are highly enriched for stem cells in many kinds of normal tissues. It has been shown that the SP phenotype is maintained by breast cancer-resistant protein-1 (BCRP1 or ABCG2), one of ATP-binding cassette transporters that are associated with multidrug resistance in many cancers by pumping out the drugs. Because CSCs are thought to be drug resistant, SP cells in cancers might contain CSCs. Indeed, the SP analysis has been recently used in an attempt to isolate CSCs from several types of cancers, and CSCs seem to be enriched in the SP population (14, 15, 16, 17). However, there are some reports demonstrating that SP cells lack stem cell characteristics (18, 19, 20), although primitive stem cells are enriched in the SP in most cases. Little is known about stem cell or CSC in thyroid.
Anaplastic thyroid carcinomas provide less than 8 months of mean survival after diagnosis (21). These tumors are highly resistant to standard therapeutic procedures such as surgical treatment, radiation therapy, and chemotherapy. The identification of CSCs in such thyroid cancers has important therapeutic implications because the elimination of this key population might lead to improved outcome.
In this study, we identified SP cells in several thyroid cancer cell lines and characterized the population in comparison to main non-SP population.
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Materials and Methods |
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Flow cytometry for SP cells
The cells were detached by trypsinization and washed with ice-cold PBS/2% FBS. Cells (1 x 106) were labeled in the growth medium with 5.0 µg/ml Hoechst33342 dye either alone or in combination with 50 µg/ml verapamil at 37 C for 90 min. After washing with PBS/2% FBS, the cells were then incubated with 2 µg/ml propidium iodide to exclude dead cells. SP analysis and sorting were done using a FACSVantage SE (BD Biosciences, San Jose, CA). The Hoechst dye was excited with UV laser and its fluorescence was measured with both 675/20 filter (Hoechst Red) and 424/44 filter (Hoechst Blue).
Cell proliferation assay
The cells were grown in 96-well plate and the relative cell number was determined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, 10 µl of WST-8 solution was added to each well and the plate was incubated for 1 h at 37 C. For quantification, OD was read at 450 nm using a microplate reader ImmunoMini NJ-2300 (System Instruments, Tokyo, Japan).
Nude mouse xenograft model
Animal care and experimental procedures described in this study were done in accordance with the Guidelines for Animal Experiments of Nagasaki University with the approval of the Institutional Animal Care and Use Committee (no. 0202060039). The cells were resuspended in growth medium (
50 µl) and mixed with an equal volume of Matrigel (BD Biosciences). The mixed cell suspension was injected sc into 8-wk-old female BALB/c nu/nu mice (Charles River Japan, Tokyo, Japan).
GeneChip analysis
Total RNA was extracted from approximately 30,000 SP or non-SP cells using an RNeasy micro kit (QIAGEN, Valencia, CA) according to the manufacturer’s instruction. The RNAs were subjected to GeneChip expression array full service with two-cycle target labeling (Biomatrix Research Inc., Chiba, Japan). Briefly, first- and second-strand cDNA was synthesized from total RNA using T7-oligo(dT) primers (first cycle). After first in vitro transcription, second cycle first- and second-strand cDNA was generated. Biotinylated cRNA was then synthesized by second in vitro transcription. Fifteen micrograms of the labeled cRNA were hybridized to a human genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). Array image was scanned and analyzed with GeneChip operating software (Affymetrix). After normalizaion, data mining was done using GeneSpring software (Agilent Technologies, Palo Alto, CA). Probes scored as absent call in both SP and non-SP were excluded from data analysis.
Real-time RT-PCR
Approximately 150 ng of total RNA were reverse transcribed using a SuperScript III first-strand synthesis supermix (Invitrogen, Carlsbad, CA) with random hexamers. The following PCR amplifications were performed using SYBR Premix Ex Taq Perfect real-time kit (TaKaRa Bio, Ohtsu, Japan) in a thermal cycler Dice real-time system (TaKaRa Bio). The cycle threshold value, which was determined using second derivative, was used to calculate the normalized expression of the indicated genes using Q-Gene software (22), using ß-actin as a reference gene. The following primer pairs were used: ß-actin, 5'-ctgaaccccaaggccaaccgcg-3' and 5'-ggcgtacagggatagcacagcc-3'; ABCG2, 5'-ctggataaagtggcagactccaag-3' and 5'-agccagttgtaggctcatccaag-3'; MYC, 5'-tgctctcctcgacggagtcc-3' and 5'-tccacagaaacaacatcgatttcttc-3'; JUN, 5'-aaaaggaagctggagagaatcgc-3' and 5'-tgttccctgagcatgttggc-3'; FZD5, 5'-gggactgtctgctcttctcg-3' and 5'-ccgtccaaagataaactgcttc-3'; HES1, 5'-tctggaaatgacagtgaagcacct-3' and 5'-gttcatgcactcgctgaagcc-3'; JAG1, 5'-ggtcttgcaaactcccaggtg-3' and 5'-actgccagggctcattacagatg-3'.
Statistical analysis
Differences between groups were examined for statistical significance using one-way ANOVA followed by Fisher’s protected least significant difference or unpaired t test as appropriate. P value not exceeding 0.05 was considered statistically significant.
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Results |
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, SP cells were identified in ARO (0.25%), FRO (0.1%), NPA (0.06%), and WRO (0.02%) but not TPC1 cells. In each case, the SP population was markedly diminished by the treatment with verapamil, which is the inhibitor of the pumps responsible for exclusion of the Hoechst dye, indicating that this population was truly SP. To further investigate the function of CSCs in anaplastic cancer, we concentrated on ARO cells, which possessed the highest percentage of SP cells.
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) and cultured in vitro. After 10 d expansion, the cells were restained with Hoechst33342 and reanalyzed by flow cytometry. The SP cells repopulated both SP and non-SP cells (Fig. 2). The ratio of the SP cells to non-SP cells was still much higher than before sorting. The number of cells in the particular area pointed out by the asterisk was also increased, suggesting that those cells are SP cells from G2/M population. At the same time, sorted non-SP cells from G1 main population yielded cells mostly in main population, yet a small number of cells in SP area were also observed (Fig. 2).
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. As shown in Fig. 3A, SP cells were more clonogenic than non-SP cells. There was no difference between non-SP cells and total ARO cells (control). We also investigated the short-term proliferative activity after sorting. Twenty-four hours after sorting, the viability of SP and non-SP cells was identical (data not shown, approximately >90%). The growth of SP cells during additional 72 h was slightly slower than that of non-SP cells (Fig. 3B).
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, the prevalence of SP cells was apparently increased when cultured at low density in all cell lines except TPC1. TPC1 seemed to be a really SP-deficient line.
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) and injected with matrigel into nude mice. Tumor formation was evaluated 4 wk after injection. Unexpectedly, tumors were formed in most cases regardless of cell type (Table 1). Even though two tumors with large size (+++) were from SP cells and two tumor-negative cases were from non-SP cells, there was no statistical significance between any groups.
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). Interestingly, the SP rate did not return to initial level (around 0.2%) but stayed at 5–20% up to 2 months after the last sorting (data not shown). Serial sorting of SP cells seemed to accumulate the cells expressing ABCG2 constitutively (irreversible expression), suggesting the development of mechanism similar to drug resistance.
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Of 54,675 probe sets, 25,583 were scored as marginal or present call (not absent) in either SP or non-SP cells and applied to further analysis. Of the 25,583, 2417 probe sets were up-regulated (fold change > 2.0) and 1009 were down-regulated (fold change < 0.5).
To validate the microarray data, we performed quantitative real-time RT-PCR for six interesting genes (ABCG2, MYC, JUN, FZD5, HES1, and JAG1) which were up-regulated in the microarray data. ABCG2 has been shown to be responsible for SP phenotype. Wnt and Notch signaling pathways play important roles in normal stem cells. MYC, JUN, and FZD5 are related to Wnt signaling, and HES1 and JAG1 are related to Notch. Quantitative real-time RT-PCR revealed that all the six genes were up-regulated in SP cells (Fig. 6). The fold-change of FZD5 in microarray data were 33.63, which was far more than RT-PCR data. A possible explanation for this is that the flag of FZD5 expression was scored as absent in non-SP cells and present in SP cells, and thereby the calculation of normalization was not accurate. In terms of the other five genes, the flags in both SP and non-SP cells were all present.
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Discussion |
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Although the concept of CSCs is not new, it is only recently that advances in stem cell biology have given the impetus to CSC theory (1, 2). Like somatic stem cells, CSCs have the properties of self-renewal, producing heterologous descendent cells and slow cell cycling. In this work, we showed that SP cells were capable of self-renewal and also generated non-SP cells by asymmetric division. This is completely consistent with previous studies showing that SP cells can generate non-SP cells in vitro or in vivo (14, 15, 16, 17). In addition, the cell growth of SP cells was slightly slower than that of non-SP cells during 96 h after sorting. Note that the SP cells probably divided asymmetrically and produced progenitor-like cells, which might be rapidly cycling, implying that the determination of SP cell growth was not exact but rather overestimated.
The clonogenic ability of SP cells was higher than that of non-SP cells. This is consistent with the study by Patrawala et al. (16) using MCF7 cells. The difference is not likely to be a consequence of longer retention of Hoechst dye in non-SP cells because the viability of SP and non-SP cells after sorting was identical and the growth of non-SP cells was even faster. However, we cannot completely rule out the possibility that the difference is due to some effect of the Hoechst dye, which is potentially cytotoxic (23).
This study, for the first time, demonstrates that the SP prevalence depends on cell density in culture. Presumably, the SP cells preferentially survived at very low plating density, which is a severe condition for cells. Kondo et al. (14) reported that MCF7 and Hela cells contained 2.0 and 1.2% SP cells, respectively. On the other hand, Patrawala et al. (16) identified 0.2% SP cells in MCF7 and did not detect SP in Hela cells. The latter group presumed that the difference was due to variation of flow cytometer and staining protocol. Our findings provide a new possible explanation. It is necessary to take into account culture condition when seeking SP population in certain cell lines. If SP cells are not identified in some type of cells that have been cultured at very low density, it is most likely that such cells do not possess SP population, like TPC1 cells.
Comprehensive analysis of gene expression using microarray chip revealed distinct differences between SP and non-SP cells. It is noteworthy that we sorted SP and non-SP cells from G1 population to avoid the effect of cell cycle-specific changes. A few thousand genes were up- or down-regulated in SP cells, indicating that ARO cells are not homogeneous. We next confirmed several interesting gene expressions by quantitative real-time RT-PCR. Wnt signaling is thought to be closely associated with stem cell self-renewal (24). MYC and JUN are important downstream components of Wnt pathway. In addition, Notch signaling maintains survival of stem cells and also inhibits differentiation in certain cell types (25, 26, 27). JAG1 is a ligand and HES1 is a transcription factor regulated by Notch signaling. In SP cells, these genes were up-regulated, suggesting that SP cells retain some stem-like properties. We also compared our microarray data with those by Chiba et al. (17). They isolated SP cells from Huh7 and PLC/PRF/5 cells. In their study, as few as 1 x 103 SP cells were sufficient for tumor formation in nonobese diabetic/severe combined immunodeficiency mice, whereas injection of 1 x 106 non-SP cells did not yield tumors. Sixteen genes were commonly up-regulated in the two studies (Chiba et al. and our study). These genes probably play very important roles in maintenance of CSC properties. To our knowledge, however, none of them have been demonstrated to be involved in thyroid cancer. This can be explained by the fact that SP cells account for only 0.2%, even though they exhibited clearly distinct gene expression patterns from the bulk of cancer cells (main population), which have been used in previous studies for many years.
Previous studies using C6 glioma cells (14), LAPC9 prostate tumor cells (16), MCF7 breast tumor cells (16), U373 glioma cells (16), Huh7 hepatoma cells (17), PLC/PRF/5 hepatoma cells (17), and MOVCAR7 ovarian tumor cells (28) have demonstrated that SP cells are more tumorigenic in vivo using immunodeficient mice. In our xenograft experiment, all injections (eight of eight) with SP cells formed tumors. Regarding non-SP cells, however, six tumors were unexpectedly formed from eight injections. SP cells seemed to be more tumorigenic; however, there was no significant difference. Besides, non-SP cells generated a small number of SP cells (Fig. 2, right panel). Moreover, even though SP cells were more clonogenic than non-SP cells, the clonogenic ability of non-SP cells was not zero (Fig. 3A). These results suggest that tumorigenic cells are probably enriched in SP population, but non-SP main population also contains a small number of tumorigenic cells. Although we found possible SP cells from G2/M population (Fig. 2, middle panel), cancer stem cell is thought to be quiescent and should be in G0/G1 phase. Patrawala et al. (16) proposed the hypothesis in which the ABCG2– population contains primitive CSCs with higher self-renewal and proliferative potentials but slow cycling. These cells then generate ABCG2+ progenitor cells that are more actively proliferating but possess reduced self-renewal and long-term proliferative capacities. The ABCG2+ progenitor cells eventually give rise to ABCG2– fully differentiated tumor cells with limited proliferative potential. Our results are supportive of this hypothesis.
In conclusion, we have identified, for the first time, SP cells in several human thyroid cancer cell lines. Cancer stem-like cells are enriched in the SP population but not exclusively, indicating that CSCs are not identical with SP cells. Our results also suggest that CSCs that have distinct properties might be present even in established thyroid cancer cell lines, which have been cultured in the presence of high concentration of serum for many years. However, it should be noted that there is a possibility that highly clonogenic SP cells might be derived from cells with genomic instability cultured for many years. Further studies are needed to identify thyroid cancer stem cells precisely. The elucidation of key tumorigenic population might contribute to not only clarification of mechanism of thyroid carcinogenesis but also the establishment of a novel therapy for anaplastic thyroid cancer.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online January 18, 2007
Abbreviations: CSC, Cancer stem cell; SP, side population.
Received November 20, 2006.
Accepted for publication January 10, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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