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Rapid recognition and isolation of live colon cancer stem cells by using metabolic labeling of azido sugar and magnetic beads Lingbo Sun, Hongxia Fu, Yanru Li, Xinrui Duan, and Zhengping Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00154 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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Analytical Chemistry

Rapid recognition and isolation of live colon cancer stem cells by using metabolic labeling of azido sugar and magnetic beads Lingbo Sun, Hongxia Fu, Yanru Li, Xinrui Duan*, Zhengping Li* Key laboratory of Applied Surface and Colloid Chemistry Ministry of Education, Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, 620 Xi Chang’an Street, Xi’an, Shaanxi, 710119, P. R. China. ABSTRACT: New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis and resistance for radio/chemo-therapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercial available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and mRNA level of colon stem cells maker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

INTRODUCTION Even we have documented cancer as a disease in ancient medical texts, it is still the leading cause of morbidity and mortality all over the world.1 Metastasis and therapy (chemotherapy/radiation) resistance are the major cause of death from cancer.2-4 In the hierarchical scheme of normal tissues and organs, stem cells have the unique character of self-renewal and differentiation. Stem cells are located at the apex of this scheme and drives organogenesis.5 Like normal tissues, tumors are also hierarchically organized. Only a subset cell population in tumor could self-renew and differentiate into all the cell types, namely cancer stem cells (CSCs).6 Current cancer therapies are developed to target bulk tumors but CSCs are resistant to both chemotherapies and radiation therapies, thus CSCs may be responsible for tumor recurrence.6 CSCs population also show much higher capacity to metastasize than non-CSCs.1 Developing a therapy that is specifically targeting CSCs has a very tempting potential to provide a way against metastasis and resistance of current therapies, even a cure for cancer. In order to develop such therapy, further understanding and characterization of these cells are needed, which firstly required rapid and efficient identification/isolation method for CSCs. Recognition of specific proteins on cell surface of CSCs with antibodies and isolation by flow cytometry or magnetic cell sorting are the most popular tools for isolating these cells from cell lines or freshly harvested primary tumors.6 In the flow cytometry-based cell sorting (namely, fluorescence-activated cell sorting, FACS), cells are usually labelled with fluorescent dyes tagged antibodies and sorted individually based on its fluorescence and light

scattering, however, it requires sophisticated instrument and is time-consuming.6 On the other hand, magnetic cell sorting (namely, magnetic-activated cell sorting, MACS) uses superparamagnetic nano/micro-particles tagged antibodies which allows to enrich targeted cells in parallel only with the help of a permanent magnet. 7,8 Thus MACS is much cheaper and faster for cells isolation than FACS. However, both FACS and MACS are based on the recognition of cell surface marker with an antibody, which may influence activity and biology of the cells.5 Moreover the expression level of these surface markers tightly depends on tumor microenvironment and digestion protocols, their expressions are not always exclusively linked to a functional stem cell phenotype, even within same tumor types.9 Compared to recognition of CSCs with specific proteins on cell surface, recognition and isolation of CSCs by using intracellular markers can suffer much less influence cellular activity and biology. Aldehyde dehydrogenase (ALDH) activity as an intracellular marker is used to isolate CSCs from various cell lines and tumors.10,11 Higher expression of ATP-binding cassette transporters (ABC transporter) also has been used to isolate breast CSCs. In a typical experiment, CSCs exclude the Hoechst 33342 dye via their ABC transporters and stain poorly.12,13 Later the finding of fluorescent riboflavin accumulation in CSCs due to the expression of ABC transporter sub-family G member 2 (ABCG2) allows to isolate them across from different solid tumors without dye staining.9,14,15 Since these recognition methods rely on the ability of fluorescent detection to discriminate CSCs, FACS is necessarily required to

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achieve cell sorting.6 In other words, these recognition methods still remain the FACS’s limitations. Developing of alternative recognition methods may solve the aforementioned problems. Since CSCs are likely derived from transformed adult stem cells and are thought to share many characteristics with their parental population, including sphere formation ability in serumfree medium and quiescent slow-cycling phenotype.16 Sphere formation culture takes the advantage of only CSCs could survive and proliferate under serum-free culture condition to form discrete clusters of cells, which termed “tumor spheres”.17 However, sphere formation culture is time consuming and high levels of growth factors may bias the differentiation potential of the cultured cells.18-21 Based on quiescent slow-cycling phenotype, the nature of lower rate of protein synthesis of these quiescent cells was used in our previous work, an alkyne bearing analogue of methionine (homopropargylglycine, HPG) could specifically label non-CSCs in colon cancer cell lines via selectively labelling nascent synthesized proteins.22 This method cannot isolate live CSCs due to the specific culture condition of metabolic labeling and the negative labeling nature can hardly realize the rare cells isolation. Here we propose a method by metabolic labelling cancer cells with azido sugars, which can use normal culture medium during labelling. Azido sugars can be easily incorporated into glycans via cellular synthesis machinery and azido groups will be displayed on cell surface, 23,24 which gives the possibility of isolating azido sugars labelled cells with simple MACS. In order to achieve positive labelling, both of CSCs and non-CSCs were labelled with azido sugars first. Since CSCs have lower rate of protein synthesis, after withdraw of the azido sugars from the cell culture medium, azido sugars would be kept longer time in the cellular proteome of CSCs than non-CSCs. Also we could use natural sugars to compete with incorporated azido sugars to enlarge the contrast and lower the background. Previous reports on the metabolic labeling of glycans with azido sugars in small animals paved the road toward labeling tumors in vivo.23,27 By using copper-free azide-alkyne reaction to tag magnetic beads on the cell surface,27 we could achieve accurate detection and enrichment of live CSCs. Current design is superior in following points: 1) the recognition is based on the quiescence nature of CSCs rather than single or few protein markers. Quiescence is a fundamental phenotype of CSCs, which support the accurate recognition; 2) Azido groups display on cellular surface that allows isolating CSCs rapidly via rapid click reaction and simple MACS; 3) positive isolation can be achieved by using a pulse-chase labelling strategy. In this study, we try to isolate colon CSCs from two colon cancer cell lines, HCT-116 and HT-29. We chose the peracetylated N-α-azidoacetylmannosamine (Ac4ManNAz) as azido sugar for metabolic labeling. N-αazidoacetylmannosamine (ManNAz) can be converted to sialic acids and incorporated into sialoglycans including sialylated proteins and sialylated lipids via cellular metabolic machinery.23 Ac4ManNAz is more efficient than free ManNAz

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due to its easy entry into cells by passive diffusion, later the acetyl groups are cleaved by non-specific esterases within cells.27 CSCs exist in many colon cancer cell lines including HCT-116 and HT-29.28 As shown in Scheme 1a, metabolic incorporation of peracetylated N-αazidoacetylmannosamine (Ac4ManNAz) and its natural counterpart peracetylated N-acetyl-mannosamine (Ac4ManNAc) have been used to distinguish normal cancer cells and quiescent CSCs in a heterogeneous population. First, Ac4ManNAz were incorporated into all cells, and then we added Ac4ManNAc to compete with Ac4ManNAz during metabolic process. Due to the lower rate of protein metabolic of quiescent CSCs, Ac4ManNAz will be kept only in CSCs cells not in non-CSCs after appropriate incubation time of Ac4ManNAc. As shown in Scheme 1b, we added dibenzocyclooctyne-PEG12-biotin conjugate (DBCO-biotin) first to react with the azido group in Ac4ManNAz on the surface of cancer cells though copper-free click chemistry.29 The magnetic beads (Dynabeads Biotin Binder) that bear biotin recognition component were added later to bind cells containing Ac4ManNAz. With the help of a permanent magnet, a subpopulation with quiescent property was isolated from colon cancer cell lines. Careful comparison of sphere formation efficiency, soft-agar colony formation efficiency, and gene expression of colon stem cells maker Lgr5 between the isolated population and the leftover population were performed to confirm the enrichment.28

Scheme 1. a) Schematic representation of discrimination of CSCs using the metabolism of Ac4ManNAz and Ac4ManNAc; b) CSCs isolation by using Dynabeads Biotin Binder; insert image shows the copper-free alkyne-azide cycloaddition reaction between azido group and DBCOPEG12-Biotin (Dibenzocyclooctyne-PEG12-biotin conjugate), and the subsequent binding with Dynabeads Biotin Binder.

EXPERIMENTAL SECTION


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Chemicals and Materials. Colon cancer cell line HCT116 and HT-29 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F12, Ac4ManNAz, Dynabeads Biotin Binder and SYBR Green were purchased from Life technologies. Fetal bovine serum (FBS), 0.25% trypsin/EDTA, dibenzocyclooctyne-PEG12-biotin conjugate (DBCO-biotin), bovine serum albumin (BSA), epidermal growth factor, basic fibroblast growth factor, Agarose (low gelling temperature, BioRegent), ROX-Refenrece Dye were obtained from Sigma-Aldrich. N-acetyl-D-mannosamine (ManNAc) was purchased from Aladdin (Shanghai, China). PenicilinStreptomycin Solution was purchased from Hyclone. Dylight 488-Avidin was brought from Boster Biological Technology (Wuhan, China). Cell Apoptosis PI Dection Kit was purchased from Nanjing KeyGen Biotech. Co. Ltd (Nanjing, China). RNAprep Pure Cell/Bacteria Kit, TIANScript RT Kit, HotMaster Taq DNA polymerase were obtained from TIANGEN Biotech Co., Ltd (Beijing, China). dNTPs and custom synthesized primers were obtained from TaKaRa Biotechnology Co., Ltd (Dalian, China). Primers used in the mRNA level analysis including GAPDH (Forward 5’ ACC ACA GTC CAT GCC ATC AC 3’, Reverse 5’ TCC ACC ACC CTG TTG CTG TA 3’) and LGR5 (Forward 5’ CTC CCA GGT CTG GTG TGT TG 3’, Reverse 5’ GAG GTC TAG GTA GGA GGT GAA G 3’).28 All Other reagents were obtained commercially and used without further purification. Ac4ManNAc was prepared according to the procedure in the literature.25 Cell culture. Cells were maintained in DMEM and supplemented with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin in a 5% CO2 humidified incubator at 37℃. Ac4ManNAz and Ac4ManNAc were dissolved in DMSO resulting 10 mM and 100 mM stock solution, respectively. Fluorescence microscopy and Flow cytometry. 1×105 cells were seeded on 35-mm petri dish. Ac4ManNAz (50 μM) treated cells were grown for 3 day, then cells washed twice with PBS and treated with Ac4ManNAc in this culture condition. After 2 day treatment, cells were detached by treating with 0.25% trypsin/EDTA. Cells were washed with labeling buffer I (PBS, pH 7.4 containing FBS (1%) and BSA (1%)) and transferred to round bottom tubes (1×106 cells/sample) and washed with labeling buffer I. After cells were incubated with DBCO-biotin (50μM) in labeling buffer I for 1 hour at room temperature, the cells were washed with cold labeling buffer II (PBS, pH 7.4 containing BSA (1%)) and then incubated with Dylight 488-Avidin (2.5 μg/mL) for 15 min at 4 ℃ in the dark. After three washes with cold PBS, the fluorescence of the labelled cells was detected by laser confocal fluorescent microscope (Olympus FV1200) and flow cytometry (BD Accui C6). Magnetic-activated cell sorting(MACS). 1×105 cells were seeded on 35-mm petri dish. Ac4ManNAz (50 μM) treated cells were grown for 3 day (one dish treated with 2% DMSO before adding Ac4ManNAz, and the 2% DMSO were existing the whole culture process), then cells washed twice with PBS and treated with Ac4ManNAc in

this culture condition. After two day’s treatment, cells were detached by 0.25% trypsin/EDTA. Cells were washed with labeling buffer I and transferred to round bottom tubes (1×106 cells/sample) and washed with labeling buffer I. Next cells were incubated with DBCO-biotin (20 μM for HCT-116 or 50 μM for HT-29) in labeling buffer I for 1 hour at room temperature. The cells were washed with cold labeling buffer I, and incubated with Dynabeads Biotin Binder for 20min (HCT-116) or 30 min (HT-29). After these steps, cells were isolated by NdFeB magnet (N42 40×40×20 mm). Both numbers of both isolated and leftover cells were counted by a hemocytometer. Sphere formation efficiency assays and soft-agar colony formation assays. Two type of sample are prepared, one is Ac4ManNAz treated for 3 days (with samples of normal culture and 2% DMSO culture, respectively), cells were detached by treating with 0.25% trypsin/EDTA; and the other is cells after MACS (with 2 samples of the cells which dynabeads captured and the cells dynabeads not captured). All the cells were resuspended as single cell solution. For sphere formation efficiency assays (SFE), cells were washed with PBS and resuspended in tumor sphere culture medium (serum-free DMEM/F12 and supplemented with 20 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 100 U/mL penicillin, 100 U/mL streptomycin and 0.5% BSA). Cells were seeded in 6 well tissue culture plate (per-coated with 1% low gelling temperature agarose) at density of 1×104 cells/mL. Cells were cultured in 5% CO2 at 37℃ for 6 days. Spheres were counted under microscope. For soft-agar colony formation assay, 2500 cells from each sample were seeded in one well of 6 well plate in 0.35% low gelling temperature agarose with culture medium on top of solidified 0.5% low gelling temperature agarose with culture medium. Cells were culture in 5% CO2 at 37℃ for 2 weeks. 1 mL 0.005% crystal violet was added to visualize the colonies before counting. Colonies were counted by naked eye. Images of soft agar assay were obtained by using gel imaging system (GelDoc XR+, Bio-Rad). Gene expression analysis. We used reversetranscription quantitative polymerase chain reaction (RTPCR) to evaluate gene expression level by measuring relative quantity of mRNA of each gene. Total RNA was extracted from the cells using RNAprep Pure Cell/Bacteria Kit, and subsequently reverase-transcribed with TIANScript RT Kit. Quantitative PCR was performed for 50 cycles of PCR (95℃ for 10 s, 55℃ for 20 s, 65℃ for 30 s) with the system containing HotMaster Taq DNA Polymerase (0.025U/μL), HotMaster Taq DNA buffer (1×), dNTP (2 mM), SYBR Green (1×), ROX-Refenrece Dye (1×), Forward Primer (10 μM), Reverse Primer (10 μM). Data analysis was performed using the 2-∆∆CT method for relative quantification.30 All samples were normalized to GAPDH as the house keeping gene. Each experiment was performed in triplicate. Error bars represent standard deviation.

RESULTS AND DISCUSSION



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Copper-free version of azide-alkyne reaction is a powerful tool to label and detect proteins. We used Ac4ManNAz, an azide-containing biomolecule, to incorporate into cellular proteins. The Ac4ManNAz is an appropriate substrate for the cellular glycosylation machinery and it rapidly reacts with strained alkynes under physiological condition (namely strain promoted azide-alkyne cycloaddition, SPAAC) that does not require a toxic metal catalyst.31,32 The reactivity of the cyclooctyne can be enhanced by installing difluoro group or biphenyl rings.33,34 We choose commercial available dibenzocyclooctynebiotin conjugating with a PEG spacer (DBCO-biotin) for our cell studies.

Figure 1. Metabolic labeling of HCT-116 with 50 μM Ac4ManNAz for 72 hours (a, b), after treated with 800 μM Ac4ManNAc for 48 hours (c, d). (a, c) Fluorescence microscopy images of HCT-116 cells; (b, d) Flow cytometry histogram of blank cells (black) and metabolic labelled cells (red). Scale bar is 120 µm.

Firstly, we tested the incorporation of Ac4ManNAz in colon cancer cell line HCT-116. Cells were treated with Ac4ManNAz for 3 days in culture medium. And the azido groups were visualized by DBCO-biotin and Dylight-488 Avidin.29 As shown in Figure 1a and 1b, both the fluorescence microscopy images and flow cytometry histogram confirmed all cells were completely labelled with azido groups. Then Ac4ManNAc was added to compete with incorporated Ac4ManNAz in order to discriminate the slow cycling CSCs and non-CSCs. Under appropriate incubation time and concentration, two distinguishable populations should be observed under fluorescent microscopy and flow cytometry histogram. We evaluated the influence of concentration of Ac4ManNAc with 24 hours incubation time by using fluorescent microscopy. As shown in Figure S1, the results indicated percentage of bright cells decreased as the increasing of the

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Ac4ManNAc concentration, but the decreasing tendency almost reached plateau when the concentrations were higher than 500 µM. In order to obviously distinguish the CSCs and non-CSCs, we increase the time and concentration. As shown in Figure 1c and 1d, two distinguishable populations with slight overlap appeared after treating cells with 800 μM Ac4ManNAc for 48 hours. However longer incubation time (72 hours) resulted in the disappearance of the fluorescence of all populations (Figure S2). The overlap might from the uniformity of the cell growth rates or metabolic rates of ManNAz among CSCs as well as non-CSCs.

Figure 2. HCT-116 cells treated with or without 2% DMSO. a) soft agar colony formation assay (CFE) of HCT-116. 2500 cells per well in six well plate were incubated for 2 weeks at 37 ℃ in 5% CO2; b) images of CFE assay, Scale bar is 3.5 mm; c) sphere formation efficiency assays (SFE). 1×106 cells per well in six well plate were incubated for 6 days at 37 ℃ in 5% CO2. Inserted images show the tumor sphere formed in these conditions; Scale bar is 100 µm. d) Relative Lgr5 mRNA levels of two populations by RT-qPCR analysis. Gapdh was used as housekeeping gene; Error bars represent standard deviation from triplicate experiments. Cells are treated with Ac4ManNAz for 3 days.

Based on these results, we could be able to achieve live colon CSCs isolation by simply replacing Dylight-488 avidin with magnetic beads.14,15 The commercial available Dynabeads Biotin Binder were used as magnetic beads in subsequent experiments. In order to facilitate the development of the magnetic isolation protocol, 2% DMSO treated cells as negative control was introduced into our system. Many reports indicated that DMSO treatment could induce the differentiation of stem cells.35,36 Before we used it as negative control, we compared the tumor sphere formation efficiency (SFE) and soft-agar colony formation efficiency (CFE) of DMSO treated cells versus normal culture cells. SEF and CFE were well-established in vitro assay for identifying CSCs.37,38 Sphere formation and clonogenic efficiency were calculated as the number of spheres or colonies divided by the number of cells seeded. CSCs could survive and proliferate under serumfree culture condition to form discrete clusters of cells are


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termed “tumor spheres”. SCF efficiency is associated with tumorigenicity in vivo.17-21 As we expected, results in Figure 2a, 2b, and c showed that the SFE and CFE were dramatically decreased in DMSO treated cells. Later we measured mRNA level of colon CSCs marker Lgr5 by reverse-transcription quantitative PCR (RT-qPCR) to further confirm the decrease of CSCs population.28,39 CSCs have been identified by using several markers, such as CD133, CD44 and CD166, but their expression are not exclusively linked to the CSCs phenotype.40-42 Wnt signals constitute a dominant force preserving the normal fate of colon stem cells.43 Studies confirmed that Lgr5 is a preponderant colon stem cell marker of stem-cell associated Wnt target genes, and Lgr5+ cells are the stem cells of the colon, it is more specific than other stem cell markers and the Lgr5 gene was expressed in a unique fashion in approximately 80 selected Wnt target genes.44-46 As shown in Figure 2d, the Lgr5 mRNA level of DMSO treated cells is distinctly decreased.

Figure 3. MACS of 2% DMSO non-treated and treated HCT116 cells and validation of isolated cells (+) and leftover cells (-) from untreated HCT-116. a) percentage of Dynabeads captured cells from 2% DMSO treated and non-treated cells, respectively. b) SFE assay of two populations. 1×106 cells were cultured in agarose coated 6-well plate for 6 days in serum-free culture medium at 37 ℃ and 5% CO2. Inserted images show the tumor spheres formed in these conditions, scale bar is 100 µm; c) Soft agar colony formation assay (CFE) of two populations. 2500 cells per well in 6-well plate were cultured in 0.35% soft agar for 2 weeks at 37 ℃ and 5% CO2. d) Relative Lgr5 mRNA levels of two populations by RT-PCR analysis. Gapdh was used as housekeeping gene. Error bars represent standard deviation from triplicate experiments.

Then magnetic-activated cell-sorting (MACS) proceeded based on the results mentioned above.47 Necessary optimization of isolation efficiency and nonspecific adsorption were performed before MACS according to the manufacturer’s instruction. 4 million Dynabeads were used to separate CSCs from 1 million colon cancer cells that treated with Ac4ManNAz and Ac4ManNAc. Then triplicated experiments were conducted with the binding time of 20 min and 20 μM DBCO-Biotin (Figure 3a). As

we expected, under this condition, only few percent of cells were isolated from negative control (DMSO treated cells) and fifty-four percent of cells were isolated from untreated cells. If we compare Figure 1d with Figure 3a, we can observe the percentage of fluorescent cell population (55%, V1-R in Figure 1d) and the percentage of the isolated cells (54%) are very close. In other word, the isolation efficiency for fluorescence positive cells is around 98%. Thus, we could separate two populations as long as significant difference can be observed on flow cytometry. After MACS, SFE and CFE assays were used to testify the two populations of cells which dynabeads captured and not captured form normal cultured colon cancer cells HCT-116. The efficiency of sphere formation of the isolated cells is significantly higher than the leftover cells (Figure 3b). The insert images in Figure 3b shows the HCT-116 formed spherical aggregates under serum-free condition. The clonogenic ability in soft agar of dynabeads captured cells are also significant higher than the leftover cells (Figure 3c). The images of colonies formed in soft agar were present in Figure S3. These results indicated that colon CSCs are successful enriched by simply magnetic isolation without any cellular surface makers involved. Then analysis of mRNA level of Lgr5 by RT-qPCR were used to confirm the enrichment of CSCs, as shown in Figure 3d, much higher mRNA level of Lgr5 was observed in dynabeads isolated population than leftover population (~4 folds). To verify whether our method can be used in other colon cancer cell lines, we isolated two different populations from colon cancer cell line HT-29. DMSOtreated HT-29 cells were used as negative control to optimize the isolation conditions (Figure S4). Beads binding time 30 min and 50 μM DBCO-Biotin were chosen due to the highest isolation percentage ratio between HT-29 and DMSO-treated HT-29. As shown in Figure 4, Lgr5 mRNA levels of these isolated cells are much higher than the leftover cells (~4 folds), also very close to the difference of mRNA level of Lgr5 between DMSO-treated and untreated HT-29 cells (Figure S5). Under this condition, around eighty percent of cells were isolated from untreated cells and twenty percent of cells were isolated from DMSOtreated cells, which may due to higher percentage of colon CSCs in HT-29 that HCT-116.48,49

Figure 4. MACS of 2% DMSO non-treated and treated HT29 cells and validation of isolated cells (+) and leftover cells (-) from HT-29. a) MACS result from HT-29; b) Relative Lgr5 mRNA levels of isolated cells and leftover cells. Gapdh was used as housekeeping gene. HT-29 cells were treated with 50 μM Ac4ManNAz for 3 days and then treated with 800 μM Ac4ManNAc for 2 days in culture medium before isolation.



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Error bars represent standard deviation from triplicate experiments.

CONCLUSIONS In conclusion, difference of metabolic rate of azido sugar Ac4ManNAz has been successfully used to distinguish CSCs and cancer cells. Because of the slower protein synthesis rate of CSCs, we isolated them by metabolic labelling and magnetic-activated cell sorting without the involvement of any specific cellular markers. The putative CSCs were isolated by using commercial available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and gene expression of colon stem cells maker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

ASSOCIATED CONTENT Supporting Information. Figure S1-S5 are presented in Electronic Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

* [email protected]; [email protected] Author Contributions

All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the National Natural Science Foundation of China (21305083, 21335005) and Fundamental Research Funds for the Central Universities (GK201303003).

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