Characterization of a Side Population of Cancer Cells from Human Gastrointestina - 《stemcells》干细胞学杂志干细胞之家



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作者：Naotsugu Haraguchia, Tohru Utsunomiyaa, Hiroshi Inouea, Fumiaki Tanakaa, Koshi Mimoria, Graham F. Barnardb, Masaki Moria作者单位：a Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan;b Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA 2 \' N2 |% j& A6 _- C
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      【摘要】/ Z8 T* x5 J4 N9 r
   A subset of stem cells, termed "side population" (SP) cells, has been identified and characterized in several mammalian tissues and cell lines. However, SP cells have never been identified or isolated from gastrointestinal cancers. We used flow cytometry and the DNA-binding dye Hoechst 33342 to isolate SP cells from various human gastrointestinal system cancer cell lines. Fifteen of sixteen cancer cell lines from the gastrointestinal system contained 0.3%¨C2.2% SP cells. Next, we used an oligonucleotide microarray to analyze differentially expressed genes between SP and non-SP cells of hepatoma HuH7. The expression of GATA6, which is associated with embryonic development and hepatocytic differentiation, was significantly upregulated in HuH7 SP cells. The expression of ABCG2, ABCB1, and CEACAM6, which are associated with chemoresistance, was also significantly increased in SP cells. In addition, some epithelial markers and mesenchymal markers were overexpressed in SP cells. Reverse transcription-polymerase chain reaction and immunocytochemical staining validated these results and suggested a multilineage potential for HuH7 SP cells. In hepatoma HuH7 and colorectal SW480 cell lines, SP cells showed evidence for self-renewal, generating both SP and non-SP cells. Finally, chemoresistance to anticancer agents, including doxorubicin, 5-fluorouracil, and gemcitabine, were compared between HuH7 SP and non-SP cells using an ATP bioluminescence assay. The HuH7 SP cells expressed a higher resistance to doxorubicin, 5-fluorouracil, and gemcitabine compared with non-SP cells. These findings demonstrate that cancers of the gastrointestinal system do contain SP cells that show some characteristics of so-called stem cells.
      【关键词】 Liver cancer MDR BCRP Microarray Chemosensitivity assay Invasion assay
   INTRODUCTION

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells that give rise to phenotypically diverse cancer cells .

Cancers of the gastrointestinal system are a leading cause of death and, worldwide, are much more prevalent compared with breast cancer and brain tumors. In Japan, cancers of seven gastrointestinal system sites, including the esophagus, stomach, colorectum, liver, and pancreas, are listed among the top 10 causes of cancer death. In this study, we tried to identify cancer SP cells in human gastrointestinal system cancer cell lines. Such SP cells were isolated in 15 of 16 examined cancer cell lines. Moreover, we evaluated the capacity for self-renewal, multilineage potential, and resistance to chemotherapeutic agents in the SP cells of representative hepatoma and colorectal cell lines, HuH7 and SW480., c: x+ z/ d: N& V' s5 M$ r

MATERIALS AND METHODS. [9 X* k( x( ?& M' p

Cell Culture
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Human gastrointestinal system cancer cell lines included esophageal (TE1, TE2 and TE13), gastric (NUGC3, MKN1, MKN7, and MKN28), colorectal (WiDr, SW480, HSC15, and CCK81), pancreas (PK9 and PK45H), and liver (HuH7, Hep3B and HepG2) cell lines. CCK81 was cultured in minimal essential medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), Hep3B was maintained in GIT (Nihon Pharmaceutical, Tokyo, http://www.nihon-pharm.co.jp), and the remaining cell lines were cultured in RPMI 1640 (Invitrogen). Each medium was supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, http://equitech-bio.com), 100 units per ml penicillin G, and 100 µg/ml streptomycin (Gibco, Grand Island, NY, http://www.invitrogen.com). The cells were cultured at 37¡ãC in a humidified atmosphere containing 5% CO2. NUGC3, MKN1, MKN7, MKN28, WiDr, SW480, HSC15, and CCK81 were obtained from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, http://cellbank.nibio.go.jp), and the remaining cell lines were from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan).2 D" A9 }5 A+ z2 w4 [
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Flow Cytometry
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To identify and isolate SP and non-SP fractions, cells were removed from the culture dish with trypsin and EDTA, pelleted by centrifugation, washed with phosphate-buffered saline (PBS), and resuspended at 37¡ãC in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) containing 2% FBS and 1 mM HEPES. The cells were then labeled with Hoechst 33342 (Invitrogen) at a concentration of 5 µg/ml for TE2, TE13, MKN7, MKN28, PK9, and PK45H and 10 µg/ml for TE1, NUGC3, MKN1, WiDr, CCK81, HuH7, Hep3B, and HepG2. The labeled cells were incubated for 70¨C90 minutes at 37¡ãC, either alone or with 50 µM verapamil (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). After staining, the cells were suspended in Hanks¡¯ balanced saline solution (HBSS; Invitrogen) containing 2% FBS and 1 mM HEPES, passed a through 40 µm mesh filter, and maintained at 4¡ãC until flow cytometry analysis.2 c- s* g8 c$ t! m* t

Cells were counterstained with 1 µg/ml propidium iodide to label dead cells. Then, 1 x 106 viable cells were analyzed and sorted by an EPICS ALTRA (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The Hoechst dye was excited at 350 nm, and its fluorescence was measured at two wavelengths using a 450 DF10 (450/20 nm band-pass filter) and a 675LP (675 nm long-pass edge filter) optical filter. The gating on forward and side scatter was not stringent, and only debris was excluded .

RNA Extraction and Oligonucleotide Microarray; i8 l! }4 `) m+ f  G& O0 J

We collected both SP and non-SP cells of HuH7, 1 x 104 cells each, in a microcentrifuge tube with 350 µl RLT buffer containing 1% 2-mercaptoethanol. We extracted total RNA from these cells using an RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) according to the manufacturer¡¯s protocol. The purity and concentration of RNA were determined by a Nano Drop (NanoDrop Technologies, Wilmington, DE, http://www.nanodrop.com) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, http://www.agilent.com) as described previously .

Identification of Differentially Expressed Genes in HuH7 SP Cells5 L7 t6 _4 M# V; t; A
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After subtracting local and global background signals, expression values were calculated as the log ratio of the dye-normalized red (Cy5) and green (Cy3) channel signals. The data flagged as being of poor quality according to the Agilent data extraction software were removed from the analysis. All intensity data were imported to the Rosetta Luminator system version 2.0. (Rosetta Biosoftware, Seattle, WA, http://www.rosettabio.com) . Sequences that were twofold upregulated or down-regulated in SP cells compared with non-SP cells and that had a p 6 `, B! X. L3 U: d% P

RT-PCR Assay( l2 z( f* C" }7 K6 o* e8 u

From the isolated SP and non-SP cells, total RNA was extracted, and T7 linear amplification was applied as described above. Each 200 ng aRNA (amplified RNA) was reverse-transcribed into cDNA using 5x first-strand buffer (Invitrogen), random primers (TaKaRa, Shiga, Japan, http://www.takara-bio.co.jp), dNTP (TaKaRa), DTT (Invitrogen), RNase Inhibitor (Promega, Madison, WI, http://www.promega.com), and M-MLVRT (Invitrogen). To validate the oligomicroarray results, we used the aRNA samples for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The PCR primers used for amplification were as follows: BCRP1, 5'-GGAGGCCTTGGGATACTTT-GAA-3' and 5'-GAGCTATAGAGGCCTGGGGATTAC-3' for a 380-bp fragment; multidrug resistance 1 (MDR1), 5'-GCCTG-GCAGCTGGAAGACAAATAC-3' and 5'-ATGGCCAAAAT-CACAAGGGTTAGC-3' for a 253-bp fragment; Keratin 19, 5'-TCCCGCGACTACAGCCACTACTAC-3' and 5'-TTGGCTT-CGCATGTCACTCAGGAT-3' for a 396-bp fragment; Albumin, 5'-CGGCTTATTCCAGGGGTGTG-3' and 5'-GGGGGAGGTT-TGGGTTGTC-3' for a 369-bp fragment; CEACAM6, 5'-GAAATACAGAACCCAGCGAGTGC-3' and 5'-CAGTGAT-GTTGGGGATAAAGAGC-3' for a 226-bp fragment; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-TTGG-TATCGTGGAAGGACTCA-3' and 5'-TGTCATCATATTTG-GCAGGTTT-3' for a 249-bp fragment. The PCR products were separated by electrophoresis in 2% agarose gel.

Immunocytochemical Staining3 L( Y3 e  s( [: v5 [  h

To examine the cellular characteristics of HuH7 SP cells, we originally established the appropriate cell culture conditions. We first maintained HuH7 SP and non-SP cells in DMEM/Ham¡¯s F-12 (Invitrogen) medium, supplemented with 1 µg/ml insulin (Wako, Osaka, Japan, http://www.wako-chem.co.jp), 1 x 10¨C7 M dexamethasone (Wako), 10 mM nicotinamide (Wako), 2 mM L-glutamine (Invitrogen), 50 µM 2-mercaptoethanol (Wako), 5 mM HEPES, 10% FBS, 100 units per ml penicillin G, and 100 µg/ml streptomycin. We adopted this medium here because it has been used to maintain mouse hepatic progenitor cells . Although the HuH7 SP cells survived and proliferated in this culture medium, few SP cells could be detected after several weeks. We thus examined which factors could adequately maintain HuH7 SP cells in a serum-free culture media. We tested stem cell factor (Chemicon, Temecula, CA, http://www.chemicon.com), platelet-derived growth factor (Pepro Tech, Rocky Hill, NJ, http://www.peprotech.com), basic fibroblast growth factor (R&D Systems, Minneapolis, http://www.rndsystems.com), and leukemia inhibitory factor (LIF; Chemicon) as candidate factors. We subsequently determined that the above culture medium, supplemented with 20 ng/ml recombinant human LIF, could effectively expand the HuH7 SP cells.7 f/ O2 M; x, x7 ~
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To study the expression of lineage markers, including a hepatocyte marker (albumin: ALBU), a cholangiocyte marker (cytokeratin 19: KRT 19), liver stem cell marker (cytokeratin 14: KRT 14), and hematopoietic and neural stem cell marker (prominin 1: CD133), in the HuH7 SP and non-SP cells, we carried out immunocytochemical staining. The cells were maintained for 24 hours in collagen type 1 precoated chamber slides (Bio-coat; Beckton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Cells were fixed with 2% paraformaldehyde/PBS for 30 minutes at 4¡ãC for KRT19, KRT14, and ALBU and 4% paraformaldehyde/PBS for 20 minutes at room temperature for CD133. Then they were washed three times with cold PBS and with cold 0.2% TritonX/PBS for 10 minutes at 4¡ãC and stained with the following antibodies overnight at 4¡ãC: anti-KRT19 (mouse monoclonal immunoglobulin G (IgG) 2b; 1:10; Progen Biotechnik GmbH, Heidelberg, Germany, http://www.progen.de), anti-KRT14 (mouse monoclonal IgG3; 1:100; Chemicon), anti-serum albumin (mouse monoclonal IgG1; 1:100; Zymed Laboratories, San Francisco, http://www.invitrogen.com) and anti-CD133 (mouse monoclonal IgG; 5 µg/ml; Genzyme-Techne, Minneapolis, http://www.rndsystems.com). The chamber slide was washed with cold 0.2% Triton-X/PBS for 5 minutes at 4¡ãC three times and with cold 0.2% Triton-X/PBS containing 3% bovine serum albumin for 5 minutes at 4¡ãC. The primary antibodies were detected with fluorescein isothiocyanate-conjugated anti-mouse IgG1. The cells were counterstained with mounting medium containing 4', 6-diamidino-2-phenylindole (Molecular Probes, Invitrogen) to identify all nuclei.
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Survival Studies for Doxorubicin, Gemcitabine, and 5-Fluorouracil7 o5 S+ t( G, t, s2 x" F; [0 t' v( I

We isolated the HuH7 SP and non-SP cells and seeded them into 96-well culture plates at 5 x 102 cells per well for each population of cells. We incubated them in culture medium as described above at 37¡ãC in an atmosphere containing 5% CO2 for 24 hours. The cells in both populations were treated with doxorubicin (0.01 and 0.1 µg/ml) or 5-fluorouracil (5-FU; 0.1 and 10 µg/ml) or gemcitabine (0.1 and 100 µg/ml). After 72 hours of exposure to the chemotherapeutic agents, viability of the cells was determined using an ATP bioluminescence assay (CellTiter-Glo Luminescent Cell Viability Assay; Promega), and the luminescence signal was detected by a luminometer (ARVO MX; PerkinElmer, Boston, http://las.perkinelmer.com) according to the manufacturer¡¯s protocol .( P5 \: F0 I( c. I: O
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Invasion Assay
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The invasiveness of SP and non-SP cells was evaluated by a Matrigel assay. Each cell population was added to 8.0-µm pore FluoroBlok (BD Biosciences, San Diego, http://www.bdbiosciences.com) inserts at a density of 5 x 104 per insert. The culture medium (described above) with 20 ng/ml LIF and 10% FBS was used as chemoattractant in the lower wells and cultured for 72 hours. The cells were then labeled with 4 µg/ml Calcein-AM (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com) in HBSS and incubated at 37¡ãC in a humidified atmosphere containing 5% CO2. Fluorescence of cells that had invaded through the FluoroBlok inserts was measured on a plate reader (ARVO MX; PerkinElmer).
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RESULTS" W4 t0 i/ k  e

Existence of SP Cells in Gastrointestinal Cancer Cell Lines2 ]. F- u0 p, u4 o
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To determine whether a selection of cancer cell lines contains SP cells, we stained them with Hoechst 33342, which is actively extruded by verapamil-sensitive ABC transporters. Representative results analyzed by flow cytometry (Fig. 1) are shown. Cancer cell lines from the esophagus contained 0.3¨C1.4% SP cells; stomach 0.6¨C2.2% SP cells; colorectal 0.3¨C0.5% SP cells; liver 0¨C1.8% SP cells; and pancreas 0.3¨C1.3% SP cells (Table 1). In each case, by definition, the number of SP cells was decreased greatly by treatment with verapamil (Fig. 1). We could not detect SP cells in just a single liver cancer cell line HepG2. Thus, most of the gastrointestinal system cancer cell lines do contain a small component of cancer SP cells. Because the existence of bipotent human liver progenitor cells with both hepatocyte and cholangiocyte lineages has recently been suggested , further experiments were focused on one of the human liver cancer cell lines, HuH7.8 n. V3 b$ C3 r0 s5 I7 M
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Figure 1. Analysis of side population (SP) cells in gastrointestinal cancers. Representative SP cell isolation from various human gastrointestinal system cancer cell lines. Cell lines of esophageal (TE13), gastric (MKN28), colon (CCK81), liver (Hep3B), and pancreas cancers (PK9) were labeled with Hoechst 33342 and analyzed by flow cytometry. Results when cells are treated with 50 µM verapamil during the labeling procedure. The SP cells, which disappear in the presence of verapamil (bottom panel), are outlined and shown as a percentage of the total cell population.. ]1 j  C0 b  V5 E
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Table 1. Percentages of SP fractions in various human cancer cell lines of gastrointestinal organ system5 i) K; p! I" D; `  U
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Identification of Differentially Regulated Genes in HuH7 SP Cells* z" l3 E- n5 Y. s
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Using a Human Whole Genome Oligo Microarray, we determined differentially expressed genes between SP and non-SP lineages of HuH7. We identified 268 upregulated and 170 downregulated genes in the HuH7 SP cells compared with the non-SP cells (supplemental online Table 1). We categorized the specifically upregulated genes in HuH7 SP cells, according to their biologic functions, as summarized in Figure 2A. First, we succeeded in confirming that the expression of ABC transporters, such as BCRP1 (ABCG2, 2.0-fold, p = .032) and multidrug resistance 1: MDR1 (ABCB1, 2.3-fold, p = .011), was upregulated in the HuH7 SP cells. Including these ABC transporters, genes associated with a "transporter" (13%) were the most frequently upregulated (Fig. 2A). The expression of genes related to signaling pathways of normal stem cells, such as BMP2 (2.6-fold, p = .011) and JAG1 (2.5-fold, p = .072), was also increased. The most pronounced (9.8-fold, p = .017) upregulated element was carcinoembryonic antigen-related cell adhesion molecule (CEACAM6), which may serve as a determinant of gemcitabine chemoresistance .
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Figure 2. Identification of differentially expressed genes in HuH7 side population cells. (A): Biologic functions of the upregulated genes determined by the Human Whole Genome Oligo Microarray; 15 of the 268 upregulated genes were spotted on this microarray slide in duplicate¡ªwe divided the 253 unique genes into 12 categories, including genes whose functions are unknown (73 genes, 29%). Detailed information on the biological functions of the remaining genes (180 genes, 71%) is available in supplemental online Table 1. (B): Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The oligomicroarray data of five genes were successfully validated by RT-PCR. GAPDH was used as a control. Abbreviation: GAPDH, glyceral-dehyde-3-phosphate dehydrogenase.8 i( [; W5 O) \0 X0 [; B

Verification of Microarray Data by RT-PCR Analysis

We carried out semiquantitative RT-PCR amplification to verify the data of several differentially expressed genes identified by the oligomicroarray. Sufficient expression of ALBU in both SP and non-SP cells was determined in our microarray analysis (data not shown). The RT-PCR results corresponded very well to the microarray data for all five genes tested (Fig. 2B).8 c- B; S! X$ v' M4 d+ P

Repopulation of Both SP and Non-SP Fractions by HuH7 SP Cells# ]8 W& N: D: g

To further examine the cellular characteristics of HuH7 SP cells, we examined which factors adequately maintained HuH7 SP cells. We determined that culture medium supplemented with LIF could effectively expand the HuH7 SP cells. To compare the self-renewal ability of SP cells, we maintained them in this supplemented medium for 2 weeks and then sorted them again into SP and non-SP cells by flow cytometry. We found that the cultures initiated with SP cells contained both SP and non-SP cells, whereas those with non-SP cells generated only non-SP cells (Fig. 3A). The percentage of SP cells was increased when analyzed after 2 weeks of culture of the SP cells compared with that of the pre-isolated HuH7 cell line (pre-isolation vs. first sort and 2 weeks of culture: 0.9 vs. 9.1%). In addition, when the sorting and 2-week culture was repeated four times, the percentage of SP cells was greatly increased compared with that of the first sort and 2-week culture (first sort and culture vs. four repeats of sorting and culture: 9.1 vs. 31.6%) (Fig. 3A). Even after a fourth repeat of sorting and culture, when these were subcultured without another repeated sorting step, the percentage of SP cells was decreased and almost equaled that of the untreated HuH7 cell line (data not shown). Comparable results were obtained for colorectal cancer cell line SW480, confirming the pattern seen with HuH7 (Fig. 3B). We performed our analyses in triplicate with similar results in each case.* ]! E, a& R& w( I  \3 S

Figure 3. Side population (SP) cells display a capacity for self-renewal. (A): Self-renewal capacity of HuH7 SP cells. The HuH7 cell line contains 0.9% SP cells (left). After the isolation of both the HuH7 SP and non-SP fractions, we cultured them in media supplemented with leukemia inhibitory factor for 2 weeks and then reanalyzed them by flow cytometry. In these culture conditions, the SP cells expanded sufficiently and maintained the SP subpopulation (9.1%), whereas no SP cells were found in the fraction initiated from non-SP cells (middle). When this sorting and culture were repeated four times, the percentage of SP cells was greatly increased (31.6%). The experiments were repeated in triplicate with similar results. (B): The SW480 (colorectal cancer) cell line showed results similar to HuH7 (liver cancer).
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Immunocytochemical Analysis

We confirmed the expression of ALBU, KRT19, KRT14, and CD133 immunocytochemically in both SP and non-SP cells of HuH7 (Fig. 4). Corresponding to the observed increased gene expression in the microarray analysis, staining for KRT19 (cholangiocyte marker; 2.5-fold, p = .06) and KRT14 (thought to be a liver stem cell marker; 2.6-fold, p = .03) was strongly positive in SP cells and negative in non-SP cells. Conversely, ALBU (mature hepatocyte marker) expression was very weak in SP cells and strong in non-SP cells. The staining for CD133 (hematopoietic and neural stem cell marker) was strongly positive in SP cells and negative in non-SP cells. In addition, SP cells were morphologically quite distinguishable (small and round in shape) from non-SP cells (fibroblast-like).

Figure 4. Side population (SP) cells express multilineage markers. Immunocytochemical staining to examine the multilineage potential of HuH7 SP cells. Both SP and non-SP cells were cultured as described for 2 weeks and then stained with a hepatocyte marker (ALBU), a cholangiocyte marker (KRT19), a liver stem cell marker (KRT14), and a hematopoietic stem cell marker (CD133). The shape of the cells was observed under differential interference contrast (DIC). Scale bar = 20 µm.

Chemoresistance of HuH7 SP Cells to Anticancer Agents

The chemoresistant ability of SP cells has been reported to depend mainly on ABC transporters . To determine whether SP cells resist ABC transporter-independent anticancer drugs more than do non-SP cells, we tested 5-FU and gemcitabine because they are usually used for the treatment of hepatobiliary tracts neoplasms. In the case of 5-FU treatment, viability of SP cells was again significantly higher than that of non-SP cells (p =.02 for 0.1 µg/ml, p # o  g" R) r3 v. h

Figure 5. Side population (SP) cells show a high resistance to anti-cancer drugs. Chemoresistance of HuH7 SP cells to anticancer drugs. We maintained cells in medium supplemented with leukemia inhibitory factor for 24 hours and incubated them with doxorubicin (left), 5-fluorouracil (middle), and gemcitabine (right) for 72 hours, and then cell viability was determined by an ATP bioluminescence assay (black bar, SP cells; white bar, non-SP cells). The experiments were repeated in triplicate with similar results.# a! E" J7 o' H! K; p( y

Invasion Assay

To evaluate any differences in biological behavior between SP and non-SP cells, we performed invasion assays (Fig. 6). Invasiveness of non-SP cells was higher than that of SP cells (2.45-fold, p = .01). We performed these analyses in triplicate.

Figure 6. Non-SP cells show more invasiveness than do SP cells. Invasion assay of HuH7 SP and non-SP cells (black bar, SP cells; white bar, non-SP cells). The invasiveness of non-SP cells was higher than that of the SP cells (2.45-fold, p = .01). Abbreviation: SP, side population.

DISCUSSION9 {7 n3 J" `* Z% T+ t
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In the current study, we were able to identify a small SP component (0.3%¨C2.2%) of cancer cells from various human gastrointestinal organ cancer cell lines. To the best of our knowledge, this is the first report demonstrating the existence of cancer SP cells in gastrointestinal cancers. The percentages of SP cells detected are similar to most previous reports: 2% in human breast cancer cell line MCF7, 0.4% in rat C6 glioma, and 1.2% in human Hela carcinoma .
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Stem cells identified from solid tumors usually express organ-specific markers. In the case of breast cancers, a cell surface marker profile CD44  CD24¨C/low Lin¨C was reported . Therefore, these molecules could be distinctive markers for liver cancer SP cells. Downregulated genes in SP cells could equally be very important but are not further discussed here.
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Stem cells show properties of self-renewal and pluripotential differentiation. The CSC shares many properties with normal stem cells. In central nervous system tumors, stem cells with a capacity for self-renewal and pluripotential development have been isolated . We have not screened the other cell lines in the panel, and the experiments are ongoing. In addition, it will also be important to confirm the existence of SP cells, which are observed in in vitro assays, in primary clinical tumor cells.1 i/ T5 w; h1 N
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Intriguingly, we noticed a greater expression of KRT19 mRNA in HuH7 SP cells in both oligonucleotide microarray and RT-PCR analysis. We thus evaluated the expression of KRT19 protein immunocytochemically and found its distinct expression only in HuH7 SP cells. ALUB expression was observed in both SP and non-SP cells but was very weak or almost negative in SP cells. Because HuH7 SP cells expressed cell lineage markers for both cholangiocytes (KRT19) and hepatocytes (ALBU), as reported in human liver progenitor cells , the liver cancer SP cells may have a capacity for bipotential differentiation. In addition to these findings, liver stem cell marker (KRT14) and hematopoietic and neural stem cell marker (CD133) were strongly positive in SP cells only.
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The association of increased expression of ABC transporters in cancer SP cells with resistance to chemotherapeutic agents has been reported . We therefore examined the sensitivity of HuH7 SP cells to doxorubicin, a commonly used agent in the treatment of hepatocellular carcinoma, using an ATP bioluminescence assay. After 72 hours of exposure to doxorubicin, the viability of the SP cells was markedly higher than that of the non-SP cells. The results of the assay also demonstrated the apparent chemoresistance of HuH7 SP cells to the ABC transporter-independent anticancer drugs, 5-FU and gemcitabine. Data from a whole genome DNA microarray analysis provide insight into the existence of key molecules, which may be associated with chemoresistant properties of CSCs not only through ABC transporters but also through other mechanisms, possibly associated with the increased expression of genes such as CEACAM6, AREG, and ABCC2/GSTA1.  v" K" r6 [* ^+ E9 Q  u4 ?7 b

In our invasion assay results, non-SP cells showed more invasiveness than did SP cells. It is generally accepted that normal stem cells show properties that provide for a long lifespan, such as relative quiescence, resistance to drugs through the expression of ABC transporters, and anti-apoptosis. These cancer SP cells may also be relatively quiescent compared with non-SP cells, while still playing important roles in maintaining cancer foci (that is, to differentiate and proliferate after chemotherapy and radiotherapy using their self-renewal and chemoresistance capacities). It is difficult to identify CSCs, because they are usually present in very small numbers and specific surface markers are still unknown. A whole genome DNA microarray analysis may be useful for identifying potential candidates for specific CSC markers or even for defining a definitive CSC pattern. If CSC specific surface markers are discovered, that may permit isolation of this important population of cells and cancer therapy and science will be revolutionized.$ W" \/ w7 E8 X' M

DISCLOSURES

The authors indicate no potential conflicts of interest.  n4 a- M0 s$ S7 O/ X
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ACKNOWLEDGMENTS3 d. r+ F! M" |( `) M1 S

We thank T. Shimooka, H. Yasunami, M. Oda, and M. Kasagi for excellent technical assistance. This study was supported by the Core Research for Evolutional Science and Technology of Japan, Science and Technology Agency; a grant-in-aid for scientific research (S) (17109013) from the Japan Society for the Promotion of Science; a grant-in-aid for scientific research on priority areas (17015032) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a grant from the Third-Term Comprehensive Ten-Year Strategy for Cancer Control; and a grant for cancer research from the Ministry of Health, Labor and Welfare of Japan.
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