Replacing Hoechst33342 with Rhodamine123 in isolation of cancer stem-like cells from the MHCC97 cell line

Toxicology in Vitro 24 (2010) 538–545

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Replacing Hoechst33342 with Rhodamine123 in isolation of cancer stem-like cells from the MHCC97 cell line Wei-hui Liu 1, Nian-song Qian 1, Ren Li, Ke-feng Dou * Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China

a r t i c l e

i n f o

Article history: Received 12 January 2009 Accepted 6 November 2009 Available online 12 November 2009 Keywords: Cancer stem cells Side population Rhodamine123 Hoechst33342 MHCC97 cell line

a b s t r a c t The side population (SP) is commonly applied to analyzing cancer stem cells (CSCs) biology. However, the Hoechst33342 (Hoechst) dye, which is used for SP isolation, is demonstrated to be toxic to cells. The aim of this study was to identify a dye that might replace or offer an alternative to Hoechst use. In the trial group, by FACS binding Rhodamine123 (Rho), Rholow (low Rho fluorescence) and Rhohigh (high Rho fluorescence) were sorted from the MHCC97 cell line. In the control group, SP (low Hoechst fluorescence) and NSP (high Hoechst fluorescence) were obtained through Hoechst/FACS. The percentages of subpopulations were as following: Rholow 2.1%, Rhohigh 97.9%, SP 1.4%, NSP 98.6%. The proliferative abilities in vitro (growth rate, soft agar clone formation rate), expressions of stem cell markers (early hepatic marker AFP and CSCs marker CD133), and tumorigenicities in vivo were significantly higher in the Rholow and SP cells than those in the Rhohigh and NSP cells, respectively (P < 0.05). In addition, each result was the same between the Rholow and SP cells (P > 0.05). Taken together, Rho/FACS may not be as effective as Hoechst/FACS for CSCs sorting, but given its low toxicity and cost, it may be a useful method for CSCs identification. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Increasing evidence shows that cancers contain a small subset of their own stem-like cells called cancer stem cells (CSCs) (Dalerba et al., 2007; Pardal et al., 2003; Wicha et al., 2006). Identifying CSCs is initially realized in the context of acute myeloid leukemia (Bonnet and Dick, 1997; Lapidot et al., 1994). Such CSCs have also been isolated in several types of solid tumors, such as brain tumors (Singh et al., 2004a,b, 2003), colon cancers (O’Brien et al., 2007; Ricci-Vitiani et al., 2007) and pancreatic cancers (Li et al., 2007). Although the two major characteristics of stem cells (self-renewal and differentiation into multiple lineages) are largely accepted as stem cell attributes, the molecular markers to define stem cells remain controversial. Because of this, side population (SP) is now

Abbreviations: ABC, ATP-binding cassette; AFP, alpha fetoprotein; Bcrp, breast cancer resistant protein; CK, cytokinin; DAB, 3,30 -diaminobenzidine; DMEM, Dulbecco’s minimal essential medium; FACS, fluorescence-activated cells sorting; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid; mAb, monoclonal antibody; MDR1, multidrug resistance transporter 1; MHCC, metastatic hepatocellular carcinoma; P-gp, P-glycoprotein. * Corresponding author. Address: Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle Western Road, Xi’an, Shaanxi Province 710032, China. Tel.: +86 29 84771094; fax: +86 29 84775259. E-mail address: [email protected] (K.-f. Dou). 1 These authors are equally contributed to the article. 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.11.008

commonly applied to the analysis of CSCs biology. While the cells are stained with Hoechst33342 (Hoechst) vital dye, the display of Hoechst fluorescence simultaneously at two emission wavelengths localizes a distinct, small, non-stained cell population that is designated as SP. The isolation of SP is based on the expression of ABC transporters (Goodell et al., 1996). There is mounting speculation that ABC transporters repress the maturation and differentiation of stem cells (Spangrude and Johnson, 1990). Although several subfamilies of genes coding for these transporters have been identified (ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG), the best-known are: members of the ABCB and ABCG subfamilies, such as ABCB1 (MDR1, P-gp) and ABCG2 (Bcrp1) in humans (Choi, 2005). It has been demonstrated that the exclusion of Hoechst by SP cells is an active process mainly involving Bcrp1 (Uchida et al., 2002). Two main criticisms have been raised concerning the use of Hoechst dye as a mean of isolating stem-like cells. Firstly, toxicity: since it is demonstrated that Hoechst interferes with C2C12 cell fusion as long as the dye is present in the nucleus (Adamski et al., 2007), more and more researchers have found that Hoechst staining can affect on cell differentiation. Secondly, instrumentation: Hoechst isolation requires the use of more specialized flow cytometer, in which the ultra-violet (UV) laser device must be installed for fluorescence activation (Montanaro et al., 2004). Thus, it is very expensive to own and operate. In this paper, we try to investigate a new method for stem cells enrichment – Rhodamine123 (Rho)/FACS.

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Rho is always used as a substrate of ABCB1/P-gp transporter to value the toxicity of drugs (Vautier et al., 2008) and to examine the functional activity of P-gp in cultured cells (He and Ji, 2008; He and Liu, 2002; Lin et al., 2007). As a result, no toxicity on cells is found by Rho, even at large dose (1–10 mM) (Ribou et al., 2003). Besides, to perform Rho/FACS, it only needs a common flow cytometer, in which the fluorescence is activated by argon-ion or helium–cadmium laser. The common flow cytometer is a lower cost (both in terms of purchase and maintenance) than cytometers offering UV excitation. Such specific dyes (Hoechst or Rho) can only be excluded out of cells by the ABC transporter, which is a stem-like characteristic. That is to say, only the cells with ABC transporters expression can exclude such dyes. Thus, dye exclusion is a valuable technique to indirectly identify stem-like cells (Wu and Alman, 2008). To conclude, given combining the efflux of Rho with FACS to isolate stem-like cells can avoid cell damage and save expenses, it is a promising and practical method for CSCs biological research. 2. Materials and methods 2.1. Agents test Each compound was screened for possible cytotoxic effect with the Cytotoxicity Detection Kit (Roche Applied Science, Mannheim, Germany). This is a colorimetric assay for the quantification of lactate dehydrogenase activity released from the cytosol of damaged cells into the supernatant. Cytotoxicity was calculated as a percentage of the effect obtained with the positive control (total cell lysis) (Storch et al., 2007). To guarantee the specificity of subpopulations, Verapamil (Sigma Chemical Co., St. Louis, MO) was used for second check. The influence of Verapamil on the fluorescence of the ABC transporter substrates was evaluated by adding increasing concentrations to Rho or Hoechst solution. 2.2. Cells culture and grouping The cells from the MHCC97 cell line (Key Laboratory for Carcinogenesis and Cancer Invasion, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China) were maintained in DMEM culture (Invitrogen, Karlsruhe, Germany), which was supplemented with 10% FCS, 2 mM glutamine, 1 mM HEPES, 100 U/ ml penicillin G and 100 g/ml streptomycin (Invitrogen, Karlsruhe, Germany). These cells (at logarithmic growth phase) were randomly divided into four groups: group A (Hoechst trial group), group B (Hoechst control group), group C (Rho trial group) and group D (Rho control group). All the cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Hoechst/FACS cells sorting As a standard control, the cultured cells were sorted by Hoechst/ FACS. The single-cell suspension was prepared by passing through a 50 lm mesh filter and diluted to a working concentration of 1  106 cells/ml with ice-cold PBS containing 2% FCS. In group A the cells were stained with the Hoechst dye (Sigma Chemical Co., St. Louis, MO) at a concentration of 2, 4, 6, 8 lg/ml (37 °C for 90 min) under mild shaking. In group B the cells were incubated in the presence of Verapamil (50 lM) and Hoechst for 90 min at 37 °C. After washed twice by PBS/2%FBS, the cells were incubated with 10 lg/ml propidium iodide (PI) (Sigma Chemical Co., St. Louis, MO) for exclusion of dead cells. After excitation of the Hoechst dye at 350 nm by UV laser on MoFlow (Cytomation Inc., Fort Collins CO., USA) and measurement of the fluorescence profile in two wavelengths analysis (424 and 650 nm; 434 and 660 nm; 450 and 695 nm), both SP and non side population (NSP) cells were col-

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lected. SP cells showed low staining with Hoechst and NSP cells were more brightly stained. A second 488 nm argon laser (100 mW) was used to excite PI fluorescence for excluding dead cells. Results were analyzed using the Summit v3.1 software (Cytomation, Dako, Glostrup, Denmark). 2.4. Rho staining and cells sorting As a trial group, the cultured cells were sorted by Rho/FACS. The cells in group C was stained with 0.05, 0.1, 0.5, 1 lg/ml Rho (Sigma Chemical Co., St. Louis, MO) at 37 °C for 30 min and washed with PBS/2%FBS twice. The cells in group D, which were used to define fluorescence threshold, were co-incubated in the presence of Verapamil (50 lM) and Rho for 30 min at 37 °C. All the above cells were centrifuged and suspended in PBS/2%FBS with 10 lg/ml PI to extrude dead cells. Flow cytometric analysis of Rho fluorescence at 485 nm was carried out with Aria flow cytometer (Becton Dickinson cooperation, Mountain View, CA). Similar to Hoechst/FACS, two filters were selected to discriminate cell subpopulations. As the Rho dye is activated through the FITC filter (Wang et al., 2006), the combination of FITC and PerCP-Cy5.5 filters was found optimal. The low Rho fluorescence and Verapamil negative was designated as Rholow subpopulation, and the remaining cells were Rhohigh subpopulation. 2.5. Cross detection of cell phenotypes To further compare the effects of Hoechst and Rho on CSCs sorting, the cross detection of SP and Rholow cells was done. After the SP cells were isolated, they were immediately incubated with 0.1 lg/ml Rho (at 37 °C for 30 min) for Rho fluorescence analyzing. Meanwhile, the freshly isolated Rholow cells were also valued by Hoechst dyeing. 2.6. Cell growth test This experiment was applied to value the proliferative ability of the cells from each subpopulation. The cells were adjusted to 1  107/ml and seeded in six well plates (5  105 cells per well). Each kind of cells had 7 parallel samples. The culture media was changed every two days. During 7 days period, in each group, the cells of one parallel sample were trypsinized at 8 o’clock each day. Then the cells were counted under an inverted microscope (BX50-32E01, Olympus, Tokyo, Japan). 2.7. Soft agar clone formation test This experiment was designed to compare the self-renewal capacity of cells from each subpopulation. The 2  DMEM (containing 200 ml/L FCS) were mixed with equal volume agarose (12 mg/ ml) (Sigma Chemical Co., St. Louis, MO) as the bottom-layer agar. The cells from SP, NSP, Rholow and Rhohigh were diluted to 1  104 cells/ml and put on the bottom-layer agars, respectively. Immediately, the cells were covered with the top-layer agars, which consisted of agarose (7 mg/ml) and equal volume 2  DMEM. Culture for 14 days, the number of cell clones was counted. 2.8. Immunocytochemical assay One early hepatic marker was selected to value the maturity of the cells from different subpopulations. First-anti AFP mAb (Zhong-Shan Co., Beijing, China) (1 ll) was diluted with PBS (99 ll) to a working density. The cells from different subpopulations (1  104/ml) were incubated with mAb, colored by DAB (ZhongShan Co., Beijing, China), and stained by hematoxylin. In negative control groups, the PBS was adopted instead of first antibody. These cells were periodically viewed under an inverted microscope. When

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the appearance of Buffy color particles showed in the cytoplasm, the result was identified positive. The positive cells were counted and their percentage was calculated. The percent of AFP positive cells <10% was defined as negative (), 10–25% as positive (+), 25–50% as moderate positive (++), >50% as strong positive (+++). 2.9. Immuno-flow cytometric analysis One CSCs marker was applied to identify the characters of CSCs in each subpopulation. The freshly isolated Rholow, SP, NSP and Rhohigh cells were prepared as single-cell suspensions at a density of 1  106 cells/ml, incubated with 0.075 ml CD133 mAb (ZhongShan Co., Beijing, China) for 30 min at 4 °C, washed in PBS twice, and fixed in 0.1% formaldehyde for flow cytometric analysis. Irrelevant isotype matched mAb was used as negative controls. Dead cells were excluded by PI gating.

ferent concentrations of Hoechst; each concentration activated by three kinds of two-wavelengths modes). As a result, the 8 lg/ml Hoechst greatly affected the cell viability; the 2 and 4 lg/ml Hoechst strategies made the profiles of SP were 8% and 6%, which seemed to get many false positive cells. Therefore, a Hoechst concentration of 6 lg/ml at 37 °C for 90 min was optimal for adherent cells. When the two wavelengths analysis was performed at 450 and 695 nm, the profile of SP was similar to other study (Shi et al., 2008). To avoid misidentification of the Rholow cells, the optimal Rho staining conditions were determined using different concentrations of the dye. Finally, via our preliminary experiments, we found that a Rho concentration of 0.1 lg/ml at 37 °C for 30 min was optimal. The Varapamil, which blocks the transporters responsible for Hoechst or Rho exclusion, is applied to check the purity of the SP or Rholow cells. When the Varapamil was added, the real SP or Rholow cells migrated to the areas with high fluorescence.

2.10. Tumor formation in nude mice The gold standard for measuring tumor initiating potential is to assess the capacity of cells to form tumors when injected into immune-deficient mice (Patrawala et al., 2005). Using this model, we tended to check the differentiating ability of the cells from each subpopulation. Thirty-two male BALB/C nude mice (Fourth Military Medical University, Xi’an, China) were maintained under standard conditions according to the College’s guidelines. The freshly isolated SP, NSP, Rholow and Rhohigh cells (1  106) were injected into the right back of mice, respectively. When the xenograft tumors grew to proper size, the mice were sacrificed and the tumor tissues were collected, fixed in 4% formaldehyde, and embedded in paraffin for H&E staining to assess tumors histology. To avoid subjective bias, each chip was marked with one number within 1–1 to 1–7 (SP), 2–1 to 2–7 (NSP), 3–1 to 3–7 (Rholow) and 4–1 to 4–7 (Rhohigh), and viewed by three different pathologists. Based on the tumor size, no macroscopic tumor was defined as negative (), the diameter of tumor <0.2 cm as positive (+), 0.2–0.5 cm as moderate positive (++), >0.5 cm as strong positive (+++). 2.11. Trials repeat and data analysis Each experiment was performed at least in triplicate on different days, and the data was shown as means ± SD. Randomize principle was adopted to do groups division and two-blind methods were applied to judge all the results. With SPSS13.0 statistical software, one-way ANOVA mean check-up and non parameter Mann– Whitney two-independent samples rank-sum test were used to check whether the discrepancy between groups was significant. A P-value < 0.05 was considered significant. 3. Results 3.1. Subpopulations from the MHCC97 cell line 3.1.1. Agents test Among the tested compounds, only 8 lg/ml Hoechst caused cytotoxic effect >50%. The cytotoxic effect caused by 6 lg/ml Hoechst was 30.8%. Besides, when the concentration of Hoechst was <4 lg/ml, it caused cytotoxic effect <30%. Through statistical analysis, the IC50 of Hoechst was 7.52 lg/ml. For all Rho patterns (the concentrations ranged from 0.05 to 1 lg/ml), no cytotoxic effect was found. There was no quenching effect caused by Verapamil to both Hoechst and Rho solutions. 3.1.2. Optimal Hoechst and Rho staining concentrations Because isolating SP cells lacks the consistency of gating strategies, we tried 12 different conditions detailed in methods (four dif-

3.1.3. Profiles of the subpopulations SP and Rholow cells should be present in the low forward scatter (FSC) and the low side scatter (SSC) fractions. There are two scatter plots reflected the distribution of cells, and a very low profile of SP (Fig. 1A) or Rholow (Fig. 1E) cells was shown. According to PI tests, there were about 30.8% dead cells in the Hoechst stained cells (Fig. 1C), in contrast, there were few dead cells in the Rho stained cells (Fig. 1G). In group A, the percentage of SP was 1.4 ± 0.01% (Fig. 1B). When exclusion of the dye was inhibited by Verapamil in group B, the SP cells almost could not be discriminated from the MHCC97 cell line (Fig. 1D). In group C, the percentage of Rholow in total cells was 2.1 ± 0.02% (Fig. 1F). When the exclusion of Rho dye was inhibited by Verapamil, the Rholow cells could not be discriminated from the MHCC97 cell line either (Fig. 1H). Furthermore, cross detections of cell phenotypes in SP and Rholow were done. The Rholow (Fig. 1I) and SP (Fig. 1K) cells contained much fewer cells than the unsorted cells. The SP cells in Rholow subpopulation was 52.5 ± 0.29% (Fig. 1J), in contrast, the Rholow cells in SP subpopulation was 72.7 ± 0.36% (Fig. 1L). Based on the above results, the ability of Rho staining to separate subpopulations from the MHCC97 cell line is similar to Hoechst staining.

3.2. Proliferative ability of the cells in each subpopulation 3.2.1. Cell growth curve As Fig. 2A shows the Rholow cells with stronger ability of selfrenewal and shorter doubling time than the Rhohigh cells (P < 0.05). The Rholow cells doubled their number in 16.9 ± 0.24 h; the Rhohigh cells doubled in 25.4 ± 0.28 h. Besides, the Rholow cells had a bigger multiplication number than the Rhohigh cells (P < 0.05). The biggest multiple of the Rholow cells was 15.2 ± 0.15 and that of the Rhohigh cells was 12.3 ± 0.18. Thus, the Rholow (Fig. 2B) and Rhohigh (Fig. 2C) cells had greatly different densities 3 days after cultivation. Through statistical assay, the SP cells had little shorter doubling time (16.2 ± 0.23 h) and a slightly bigger multiplication number (15.7 ± 0.21) than the Rholow cells (P > 0.05). Taken together, the Rholow cells had similar ability to the SP cells for self -renewal.

3.2.2. Soft agar cloning assay After culture for 14 days, most of the SP and Rholow cells had formed cell clones. The rate of clone formation in the SP cells was 73.5 ± 0.12%, and that in the Rholow cells (Fig. 2D) was 70 ± 0.11%. In contrast with the SP and Rholow cells, the NSP and Rhohigh cells had much lower clone formation rates (P < 0.01). Such as the clone formation rate in the NSP cells was 25.2 ± 0.06%, and that in the Rhohigh cells (Fig. 2E) was 30.4 ± 0.07%.

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Fig. 1. The profiles of cell subpopulations in the MHCC97 cell line. (A), when total cells were stained with Hoechst (p1), the blue population indicated possible SP cells with low fluorescence. (B), parts of them were demonstrated to be true SP cells (p3), parts were non SP cells (p4). (C), the viable and dead cells were determined by PI. (D), the profile of SP population (p3) decreased in the presence of Verapamil. (E), the distribution of total cells stained with Rho. (F), the Rholow cells were shown as a low Rho fluorescence (p2) and the Rhohigh cells were with high fluorescence (p3). (G), the most cells were viable. (H), when the cells were treated with Verapamil, the fluorescence of the Rholow cells was shifted to a higher level (p3). The distributions of (I) the Rholow cells stained with Hoechst (p1) and (K), the SP cells stained with Rho (p1). (J), the percentage of the SP cells in Rholow (p2). (L), the percentage of Rholow cells in SP (Q3).

3.3. Cell marker expression

3.4. Tumor formation test

3.3.1. Immunocytochemical assay Based on the identification of AFP dyeing grading criteria, we collected and incorporated the same immunocytochemical results. The detailed data have been enumerated in Table 1. The Rholow (Fig. 3A) and SP cells (Fig. 3C) expressed higher AFP than the Rhohigh (Fig. 3B) and NSP (Fig. 3D) cells, respectively (P < 0.01). Besides, the SP cells had similar expression level of AFP to the Rholow cells (P > 0.05) (Fig. 3I). These results demonstrated the cells were immature and from hepatic carcinoma.

Since the mice were injected with cells from different subpopulations, they were checked each day. The xenograft tumors grew to proper size 5 weeks later, within the mice which were injected with SP or Rholow cells, however, we couldn’t find obvious tumor in some mice injected with NSP or Rhohigh cells. We counted the number of tumors in each mouse, measured the size of each tumor and summarized those data in Table 2. The tumor formation rate in mice with SP cells injected was 78.0 ± 1.21%, and that with Rholow cells injected was 66.0 ± 1.03%. In addition, the rate in mice with Rhohigh cells injected was <15.0%. This indicates that the Rholow cells had stronger ability to form tumors in vivo than the Rhohigh cells (P < 0.01); however, the Rholow cells (Fig. 4A) had slightly lower ability to form tumors than the SP cells (P > 0.05) (Fig. 4B). Part of each tumor was made into chips and stained by H&E (Fig. 4C–F) to assess tumor pathology. According to three different pathologists’ judgements, the tumors were all hepatic tumor genesis, which could help us discriminate the neoplasms from other kinds of tumors. After the cells from some tumors were isolated and cultured, we used flow cytometry to analyze the phenotype

3.3.2. Flow cytometry We used CD133 as a probe to quantitatively analyze the profile of stem-like cells in each subpopulation. The percentage of CD133 positive cells in Rholow (Fig. 3E) was 51.84 ± 0.33%, in Rhohigh (Fig. 3F) was 13.36 ± 0.11%, in SP (Fig. 3G) was 53.24 ± 0.42%, and in NSP (Fig. 3H) was 12.70 ± 0.10%. The CD133 expressed significantly different among these subpopulations (P < 0.01) (Fig. 3J).

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Fig. 2. The proliferative abilities of different typed cells. (A), cell growth curve, (B) the Rholow and SP cells from the MHCC97 cell line had a stronger capacity to proliferate than (C) the Rhohigh and NSP cells, respectively. The cell clones formed by (D) the Rholow cells was much bigger than that by (E) the Rhohigh cells. Magnification: panels B–D, 100.

Table 1 The expression of AFP in the cells from each subpopulation. Cell subpopulation

Sample number

AFP

AFP+

AFP++

AFP+++

Rholowa Rhohigh SPb NSP

35 35 35 35

1 ± 0.14 10 ± 0.43 1 ± 0.14 12 ± 0.43

6 ± 0.28 16 ± 0.56 4 ± 0.14 15 ± 0.56

20 ± 0.86 6 ± 0.28 21 ± 0.86 5 ± 0.14

8 ± 0.28 3 ± 0.14 9 ± 0.43 3 ± 0.14

All AFP immunocytochemical data were viewed by three different researchers and expressed as mean ± SD. a Rholow vs. Rhohigh, N = 35, P < 0.05. b SP vs. Rholow, N = 35, P > 0.05.

of those cells. Through our analysis, the tumors generated by the injected cells, were mainly from the SP or Rholow cells.

4. Discussion The cells that exclude Hoechst dye are SP cells. Because the SP cells express high levels of stem cell markers and low levels of differentiating markers (Haraguchi et al., 2006), the SP subpopulation is widely used to enrich stem cells. However, there are two major limitations in the SP research. First one is the toxicity of Hoechst (Kondo et al., 2004). For each cell line, the Hoechst concentration should be optimised to minimize cytotoxicity (Montanaro et al., 2004; Morrison and Kimble, 2006; Platet et al., 2007). Second one is the requirement of an UV laser to excite the Hoechst dye. The flow cytometer equipped with UV sources is expensive to own and operate and is not readily available to many laboratories or institutions. To solve this problem, some researchers (Cabana et al., 2006) tried to design a minimum UV excitation implement for measuring Hoechst. At present, the related endeavor is still undergoing. However, the major toxic drawback of Hoechst remains unsolved. Thus, it may be better to find a replacement of Hoechst in isolating stem-like cells. It is widely accepted that the MHCC97 cell line has heterogeneity, but there are few methods to separate subpopulations from this cell line. We used both Rho/FACS and Hoechst/FACS to enrich CSCs from the MHCC97 cell line. Two years ago, members of our team isolated SP cells from this cell line by Hoechst/FACS. With

lacking of experiences and consideration of SP purity, they used higher Hoechst concentration (20 lg/ml). It was hard for cells to efflux large Hoechst and displayed low fluorescence. As a result, the profile of SP cells was as low as 0.5% (Fan et al., 2007). That is probably the reason why their result (Fan et al., 2007) was different from that shown in this manuscript. Based on the results of cytotoxic effects caused by the two dyes, Hoechst was much more harmful to cells than Rho. The low toxicity of Rho has also been demonstrated in other trials (Ribou et al., 2003). That is to say, the Rho dye avoided one main disadvantage of the Hoechst dye for CSCs isolation. Here comes the key point if Rho is as effective as Hoechst? Our FACS results indicate the percentage of SP was slightly lower than that of Rholow in the MHCC97 cell line. It does not mean that Rho can accumulate more CSCs than Hoechst. The reason may be that the purity of CSCs in Rholow is lower. Based on the cross detections of SP and Rholow cells, the percentage of CSCs in total cells may be 1% (72.7% Rholow cells in 1.4% SP subpopulation; 52.5% SP cells in 2.1% Rholow subpopulation). In other words, the combination of Rho/FACS and Hoechst/FACS may enrich much purer CSCs, which is consistent with others. Early in 1993, with a sequential Hoechst/Rho sorting system, the subfractions of cells that stained most weakly with both dyes were demonstrated to be long-term repopulating primitive hematopoietic cells (Wolf et al., 1993). Again in 2004, Bertoncello and Williams reported ‘‘Hoechst-dull/Rho-dull cells map to the subfraction of SP cells, which possess the highest primitive hematopoietic stem cells (PHSCs) activity” (Bertoncello and Williams, 2004). However, since the cytotoxicity of Hoechst is confirmed in recent years (after 2004) (Adamski et al., 2007), the combination of the two dyes is rare. Based on the analysis of several CSCs characteristics, we found the Rholow cells had similar characters as the SP cells, such as high proliferative ability and high expression of CD133. The use of CD133 has proven to be very successful in the identification of CSCs in both human brain and colon carcinomas (O’Brien et al., 2007; Ricci-Vitiani et al., 2007; Singh et al., 2003, 2004b). Interestingly, about 5 days in culture, Rhohigh and NSP subpopulations reached a plateau in the growth curve The reasons maybe parts of the cells in NSP or Rhohigh became older and older and finally died. Meanwhile, a few of stem cells gave birth to new cells. The number of dead cells was similar to that of the new-born cells. However, the cells from SP and Rholow were mostly stem cells,

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Fig. 3. The expressions of CSCs markers. AFP expressed in (A) the Rholow cells was strongly higher than that in (B) the Rhohigh cells; AFP expressed higher in (C) the SP cells than in (D) the NSP cells. For figures (E)–(H), the black histogram was the isotype control, and the red histogram was the trial. A fluorescent value was set as the regional marker (brunneus bar), which must be just bigger than the fluorescent values of cells in control group. Then the fluorescent value was remembered and used as the regional marker (brunneus bar) in trial group. The positive region was where the fluorescent values were bigger than the set fluorescent value in trial group. The percentages of the CD133 positive cells in (E) Rholow and (G) SP were also higher than those in (F) Rhohigh and (H) NSP, respectively. The detailed expressions of (I) AFP and (J) CD133 are reflected by column charts. Magnification: panels A–D, 400.

which were long lived and could continually differentiate into different typed cells. These cells would continue proliferating for a long time. In addition, we found that the Rholow and SP cells had similar tumor formation rates and the H&E staining confirmed the tissues hepatic genesis. In one word, the Rholow and SP cells shared similar characters both in vivo an in vitro. That is to say, although Rho/FACS is not as effective as Hoechst/FACS to isolate stem cells, given its low cytotoxicity and low cost, it may replace Hoechst/FACS in isolating some typed CSCs.

The supravital dyes Rho and Hoechst have proven to be remarkably powerful probes for the characterization, resolution, isolation and purification of PHSCs (Bertoncello and Williams, 2004; Wu et al., 2007). Several lines of evidence suggest that expressions of two ABC transporters (ABCG2/Bcrp1 and MDR1/P-gp) are responsible for Hoechst and Rho efflux (Uchida et al., 2002; Zhou et al., 2001). In detail, the Hoechst dye exclusion is conferred partly by the expressions of the ABCG2 and ABCG3 transporters (Hirschmann-Jax et al., 2004; Kim et al., 2002), whereas the Rho dye exclu-

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Table 2 The tumor formation rate of distinct typed cells in nude mice. Cell subpopulation low a

Rho Rhohigh SPb NSP

Sample number

Tumor

Tumor+

Tumor++

Tumor+++

8 8 8 8

1 ± 0.11 17 ± 0.36 1 ± 0.11 19 ± 0.36

12 ± 0.51 7 ± 0.21 10 ± 0.23 6 ± 0.20

11 ± 0.28 4 ± 0.15 8 ± 0.24 3 ± 0.12

4 ± 0.14 0 ± 0.00 9 ± 0.21 0 ± 0.11

All the mice were checked carefully and wholly for tumors, and the diameter of each tumor was measured. a Rholow vs. Rhohigh, N = 8, P < 0.01. b SP vs. Rholow, N = 8, P < 0.05.

Fig. 4. Tumor formation capacity of each typed cells. Both (A) the Rholow and (B) SP cells formed different sized tumors. H&E dyeing revealed the tumors formed by (C) the Rholow, (D) Rhohigh, (E) SP and (F) NSP cells were all hepatic carcinoma genesis. Magnification: C–F, 100.

sion is partly mediated by the ABCB1/P-gp protein (Zhou et al., 2001). In recent years, it is widely accepted that multiple transporters are involved in this process (Choi, 2005; Goodell, 2005). The Verapamil has been applied to be one of the inhibitors for such transporters (Iida et al., 2005; Wang et al., 2006). So, to investigate the real situation, it needs to systematically research the distribution of ABC transporters in each subpopulation from the MHCC97 cell line. The members of our team have demonstrated that expressions of Bcl-2 and Bax in the SP and NSP cells from this cell line were different (Fan et al., 2007). Because of lack of some techniques and specialized implement, further research is needed to complete this topic.

5. Conclusions From the above discussions, we get these conclusions: firstly, to isolate CSCs from the MHCC97 cell line, the Rho/FACS is less toxic and expensive than the Hoechst/FACS; secondly, the Rholow cells separated by Rho/FACS has stronger proliferative ability in vitro and tumor formation capacity in vivo than the Rhohigh cells; lastly, the effectiveness of Rho/FACS in enriching CSCs is similar to that of Hoechst/FACS. To conclude, when the Hoechst/FACS is too toxic or expensive to do biological researches of some typed stem cells, the Rho/FACS is still another elective method. To conclude, in circumstances where suspected cancer stem-like cells may be susceptible

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