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Journal of Chromatography B, 963 (2014) 40–46

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom epage: www.elsev ier .com/ locate /chromb

edimentation field flow fractionation monitoring ofn vitro enrichment in cancer stem cells by specificerum-free culture medium

arole Mélina, Aurélie Perrauda,b, Christophe Bounaix Morand du Puchc, Elodie Loumc,téphanie Giraudc, Philippe Cardota,d, Marie-Odile Jauberteaua, Christophe Lautrettec,erge Battua,d,∗, Muriel Mathonneta,b

Université de Limoges, Institut 145 GEIST, EA 3842 “Homéostasie cellulaire et pathologies”, Faculté de Médecine, 2 rue du Dr Marcland, 87025 Limogesedex, FranceCHU de Limoges, Service de chirurgie digestive générale et endocrinienne, 2 rue Martin Luther King, 87042 Limoges Cedex, FranceOncomedics, 1 Avenue d’Ester, 87069 Limoges, FranceUniversité de Limoges, Institut 145 GEIST, EA 3842 “Homéostasie cellulaire et pathologies”, Faculté de Pharmacie, Laboratoire de Chimie Analytique etromatologie, 87025 Limoges Cedex, France

r t i c l e i n f o

rticle history:eceived 10 March 2014ccepted 16 May 2014vailable online 27 May 2014

a b s t r a c t

The development of methods to enrich cell populations for cancer stem cells (CSC) is urgently neededto help understand tumor progression, therapeutic escape and to evaluate new drugs, in particular forcolorectal cancer (CRC). In this work, we describe the in vitro use of OncoMiD for colon, a CRC-specificprimary cell culture medium, to enrich CRC cell lines in CSC. Sedimentation field flow fractionation (SdFFF)

eywords:ell enrichmentolorectal cancerefined mediumancer stem cells

was used to monitor the evolution of subpopulations composition. In these models, medium induced aloss of adherence properties associated with a balance between proliferation and apoptosis rates and,more important, an increased expression of relevant CSC markers, leading to specific SdFFF elution profilechanges.

© 2014 Elsevier B.V. All rights reserved.

edimentation field flow fractionation

. Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-elated mortality in the Western world [1,2]. None of thereatment options currently available is curative in patients withdvanced cancer. For those, mortality rate approaches 100% dueo the propensity for early metastatic spread, and also becausehe disease is highly resistant to radiation and chemotherapy3].

As for other cancers, CRC development and resulting therapeu-ic efficiency are primarily based on tumor heterogeneity [4]. Inhis context, one of the main questions concerns cancer stem cells

CSC) [2]. CSC, or cancer-initiating tumor cells, have been identifiedn several types of tumors, including CRC, through the expressionf specific markers associated with the ability to self-renew and to

∗ Corresponding author at: Laboratoire de Chimie Analytique et Bromatologie,aculté de Pharmacie, 2 rue du Dr Marcland, Université de Limoges, 87025 Limogesedex, France. Tel.: +33 5 55 43 59 79; fax: +33 5 5543 5858.

E-mail address: serge.battu@unilim.fr (S. Battu).

ttp://dx.doi.org/10.1016/j.jchromb.2014.05.039570-0232/© 2014 Elsevier B.V. All rights reserved.

generate all types of differentiated cells composing the tissue of ori-gin [5]. The CSC hypothesis predicts that these subpopulations areresponsible for driving tumor growth [3], but also for therapeuticfailures characterized by cancer recurrence, emergence of resis-tant clones, and therapeutic escalation detrimental to the patient[5]. Phenotypic characterization of CSC in CRC is still debated. Ini-tially, CD133 was identified as a CSC marker [6–8], but other studiesshowed that CD133 expression is present in a large majority oftumor cells (reviewed in [9]). Alternatively, co-expression of differ-ent molecules presented as immature cell markers, such as CD44and EpCAM, is now used to target suspected stem cell subpopula-tions [10–15]. In addition to phenotypic characterization, the majordifficulty in studying CSC resides in the isolation of these potentialbiological targets, because of their low numbers. Indeed, in CRC,CSC account for only a small fraction of total tumor cells and varywidely based on the marker used: from 2.5% [2] to 11.8% [6] whenmeasuring CD133 expression, and 5.4% with a double detection of

both CD44 and EpCAM [16]. Interestingly, CSC can be found in CRCin vitro models, namely cell lines [13,15], where they ensure self-renewal. Therefore, such models may provide an easily-accessedsource to study this specific tumor cell subpopulation.

C. Mélin et al. / J. Chromatogr. B 963 (2014) 40–46 41

Table 1Characteristics of the three selected cell lines according to stage and patient (sex and age). n: Data not available/reference: ATCC.

Cell line Derived from pTNM stage Dukes’ stage Patient (sex/age)

WiDr Primary adenocarcinoma of the rectosigmoid colon, derivative of HT29 n n Female/78n

cfcabadicmeegwmiuaCsc

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SW480 Moderatly differentiated adenocarcinoma of descending coloColo205 Ascites metastasis from colon adenocarcinoma

For cell sorting, one strategy to obtain CSC from cell linesould be the use of specific serum-free culture media. Indeed,etal calf serum (FCS) is a component of the vast majority ofurrently employed media. However, several concerns have beenssociated with its use (reviewed in [17]), the most importanteing the extremely variable composition of serum batches. As

consequence, FCS-containing media may introduce phenotypicifferences in susceptible cultivated cells and radically alter exper-

mental reproducibility. For several decades, there has thus been aonstantly growing interest in serum-free or chemically definededia. Employing a carefully controlled formulation offers sev-

ral advantages. First, it ensures experiment reliability by virtuallyliminating medium variation. Also, chemically defined media areenerally designed to fulfill requirements of particular cell types,hich may vary greatly from one tissue of origin to another. Suchedia may favor the selection of a subpopulation of interest dur-

ng culture. Increasing CSC frequency within a larger population, bysing serum-free or chemically defined culture media is a valuablepproach [18]. Isolation of subpopulations significantly enriched inSC by that mean is an attractive perspective, already supported byeveral reports on glioma [19,20] and various other tissues such asolon, rectum, breast and prostate [21].

In order to monitor the effect of a series of defined mediumn CSC enrichment, and because CSC phenotypic characterizations highly controversial, the development of a label-free methodppears essential. Monitoring methods that rely on intrinsic bio-hysical properties of cells, rather than gene expression profiles,ay prove useful for this application.It is the case for field flow fractionation (FFF) [22,23] and, more

pecifically, sedimentation FFF (SdFFF), which is a gentle, non-nvasive and tagless method particularly well suited for stem cellorting [13,24–26] and for monitoring biological events [27–35].ts advantages are based on the drastic limitation of cell–solidhase interactions by the use of (i) an empty ribbon-like chan-el without a stationary phase; and (ii) the “Hyperlayer” elutionode, a size/density-driven separation mechanism [23,36–41]. Cell

eparation depends on the differential elution of species submit-ed to the combined action of a parabolic profile generated byowing a mobile phase through the channel and a multigrav-

tational external field (generated by rotation of the channel)pplied perpendicularly to the flow direction [22,36,37,42,43]. Inhe last decade, we developed applications for SdFFF cell sor-ing in many fields such as neurology, oncology and stem cells13,22,24,25,30,31,33,35,44–47]. Different uses of SdFFF in oncol-gy have been explored such as the induction of chemical apoptosis,ifferentiation or autophagy [28,30,32,35,45,47,48], and differentspects of these mechanisms have been studied from the monitor-ng of induced biological events [29,31,34,45,48] to the isolation ofSC [13,45].

The goal of the present study was (1) to monitor by SdFFF theffect of a serum-free CRC-specific defined medium, named Onco-iD for colon (Oncomedics Defined Medium for colon [49]); and

2) to determine the capacity of OncoMiD for colon to produce aomogenous CSC cell subpopulation from CRC cell lines. To achieve

hese objectives, we chose to combine both strategies: monitoringf population changes through evolution of SdFFF profiles and, inarallel, validation of subpopulation identities with relevant CSCarkers. We used a panel of CRC cell lines, classified from low to

II B Male/50IV D Male/70

high tumor grade (pTNM stages for pathology Tumor Nodes Metas-tases): WiDr, SW480 and Colo205.

Results showed for the first time that the defined mediumOncoMiD for colon, generated in these CRC models, morphologicalchanges, a balance between proliferative and apoptotic activities,and an increased expression of CSC-like markers, suggesting thatthis medium is a suitable tool to favor emergence of CSC-likesubpopulations. These biological changes were associated with bio-physical modifications which could be quickly monitored by SdFFFprofiles.

2. Material and methods

2.1. Cell lines and culture conditions.

WiDr, SW480 and Colo205 (human CRC cells) were obtainedfrom the American Type Culture Collection (ATCC) (Table 1), andcultured according to their recommendations using what we termas Classic Medium (CM). Colo205 and SW480 were cultured in RPMIsupplemented with 10% inactivated fetal calf serum, 1 mM sodiumpyruvate, 100 IU/ml penicillin and 100 �g/ml streptomycin. TheWiDr line was cultured in MEM medium supplemented with10% FCS, 1% non-essential amino acids, 1 mM sodium pyruvate,100 IU/ml penicillin and 100 �g/ml streptomycin. Cells were main-tained at 37 ◦C in a humidified 5% CO2 environment. All analyseswere performed after 72 h of culture in each medium. Cell sus-pensions were obtained through trypsinization (0.5% trypsin for5 min), followed by a centrifugation at 1500 rpm (5 min) and usedfor different experiments.

All culture reagents were purchased from Invitrogen Gibco(Invitrogen, Grand Island, NY, USA) and OncoMiD for colon(Oncomedics Defined Medium for colon, Oncomedics, Limo-ges, France) was produced, quality-controlled and provided byOncomedics.

2.2. SdFFF device and cell elution conditions

The SdFFF separation device used in this study was previ-ously described and schematized [13]. Channel dimensions were818 × 12 × 0.175 mm with two 50 mm V-shaped ends with a mea-sured total void volume of 1772 ± 6.00 �L (n > 6). The channel rotoraxis distance was measured at r = 14.82 cm. Sedimentation fieldswere expressed in units of gravity, 1 g = 980 cm/s2, and calculatedas previously described [13]. Cleaning and decontamination pro-cedures, control of rotation speed, as well as chromatographic andacquisition device were previously described [13]. SdFFF elutionof CRC cell suspensions (100 �l/2.5 × 106 cells/ml) was performedunder previously defined optimal elution conditions (Hyperlayerelution mode) [13], and was 8 g/0.8 ml/min. Monitoring of culturecondition effects on populations was performed by comparison offractograms obtained under Classic (reference profile) and Onco-MiD for Colon media. Size was measured using a Coulter Counter

as previously described [13]. The experimental retention ratioRobs = void time versus retention time = t0/tR (measured by firstmoment method) [13,41], was used to determine the average veloc-ity, retention order, and elution mode (Table 2).

42 C. Mélin et al. / J. Chromatogr. B 963 (2014) 40–46

Table 2Optimal elution conditions, retention ratio Robs, cell diameter, and the calculated cell elevation value “s” for the different CRC cell lines cultured under different conditions:Classic Medium (CM) and OncoMid for Colon medium. Results are expressed as mean ± S.D. (n = 3). Cell diameter was measured by Coulter Counter, and CRC cell lines aredisplayed according to the international pTNM (pathology Tumor Node Metastasis) stage classification.

CellLines Robs Diameter (�m) Radius (�m) (CM) s (�m) (CM)

CM OncoMiD CM OncoMiD

WiDr 0.427 ± 0.015 0.488 ± 0.014 13.29 ± 0.03 13.25 ± 0.11 6.64 14.235 ± 0.0 ± 0.

2

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2

wB(t

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SW480 0.482 ± 0.007 0.546 ± 0.024 13.4Colo205 0.383 ± 0.003 0.406 ± 0.015 13.2

.3. Antibodies

Anti-CD44 (#3570) and anti-EpCAM (#2929) were purchasedrom Cell Signaling (Beverly, MA, USA). Anti-Ki-67 (M7240) wasurchased from Dakocytomation (Glostrup, Denmark).

.4. Indirect immunofluorescence

Cells grown on 12-mm coverslips (VWR, Strasbourg, France)

ere rinsed twice in phosphate buffered saline (PBS, pH 7.4, GibcoRL, Cergy-Pontoise, France), fixed with 4% para-formaldehydePFA, Sigma–Aldrich, Saint Quentin Fallavier, France) at roomemperature for 20 min and permeabilized during 30 min with

ig. 1. Cell morphology of WiDr, SW480 and Colo205 in various culture media. Cells were and F) for 72 h. Magnification 200×.

16 13.52 ± 0.24 6.73 14.0630 13.46 ± 0.25 6.60 11.17

0.1% Triton X-100 (Sigma–Aldrich, Saint Quentin Fallavier, France).Nonspecific binding was prevented by an incubation with PBS-2% bovine serum albumin (BSA, Sigma–Aldrich, Saint QuentinFallavier, France) blocking solution at room temperature for 30 min.Coverslips were then incubated overnight at 4 ◦C in blocking solu-tion containing the primary antibodies (1:50 for CD44; 1:250 forEpCAM). Following this step, coverslips were incubated at roomtemperature for 2 h with Alexa Fluor 594 nm-conjugated secondaryantibodies diluted 1:5000 in PBS (goat anti-rabbit IgG Alexa Fluor

594 nm, A11012; goat anti-mouse IgG Alexa Fluor 594 nm, A11005;Invitrogen, Grand Island, NY, USA). After three washes in PBS, nucleiwere stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:10,000in PBS, Sigma–Aldrich) for 5 min at room temperature. Finally,

cultured in Classic Medium (CM) (A, B and C) or in OncoMiD for Colon medium (D,

atogr. B 963 (2014) 40–46 43

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Fig. 2. Representative fractograms of WiDr, SW480 and Colo 205 CRC cell lines.Profiles for Classic Medium (CM) (reference profile, in blue) and OncoMiD for Colonmedium (in red) are displayed. Elution conditions: channel thickness: 175 �m; flow

6

C. Mélin et al. / J. Chrom

overslips were intensively washed with PBS then inverted onlides and mounted with Dako Fluorescent Mounting MediumS3023, Dakocytomation, Glostrup, Denmark). Negative con-rols were cells incubated with irrelevant normal mouse IgGSigma–Aldrich) used at the same dilution as primary antibodies.ictures were captured using a Leica microscope and a Leica digitalamera. Images were processed using Leica IM500 Image ManagerWetzlar, Germany).

.5. Apoptosis assay

To measure apoptosis, cytoplasmic soluble nucleosomes wereetected using a calorimetric assay, Cell Death Detection ELISAPLUS

it (Roche Molecular Diagnostic, Pleasanton, CA, USA) accordingo the manufacturer’s instructions. Absorbance values were mea-ured at 405–490 nm dual wavelengths. The absorbance obtainedor controls was normalized to a value of 1. All experiments wereerformed in triplicate and repeated at least two times.

.6. Statistical analysis

A statistical analysis of differences was carried out by analysisf variance (ANOVA) using StatView version 5.0 software (Abacusoncepts, SAS Institute Inc., Cary, NC, USA). P-values less than 0.05Fisher’s PLSD test) were considered to indicate significance.

. Results and discussion

Considering the great difficulty to isolate and study CSC, whichre responsible for the CRC development, methods permittingnrichment and monitoring of such cells are urgently needed. Forhis reason we decided to test the effects of OncoMiD for Colon

edium on cell lines commonly used as CRC models: WiDr andW480 are primary CRC-derived cell lines, whereas the Colo205ine is a model of advanced CRC obtained from ascites (Table 1)13,50].

.1. Cell morphology

Cell morphologies were compared after maintaining cells inncoMiD for Colon or Classic media (CM). As observed in Fig. 1, cellsetached from plates and formed clusters that were more numer-us and visible to the naked eye for WiDr and Colo205. Remarkably,W480 cells cultured in OncoMiD for colon also produced cyto-lasmic extensions. Thus, following culture in the defined medium,wo distinct subpopulations formed for each cell line: adherent andonadherent clustering cells.

.2. SdFFF elution and monitoring

Recently, SdFFF has been used to study some major phenomenanvolved in cancer therapy such apoptosis, differentiation and CSC,n particular for monitoring induction of biological events [27–32].

any applications arose from these results such as (i) screening aeries of molecules and their doses, (ii) studies of biological pro-esses on limited cell subpopulations such as CSC [44,45], andiii) monitoring of new biological models used in various life sci-nce fields such as the effect of culture media, which is, for therst time, the purpose of this study. Indeed, if culture conditionsesulted in morphological changes, we could expect an evolutionf elution profiles between basal and OncoMiD for Colon medium.

sing “Hyperlayer” elution mode as previously described [13], eachell line displayed similar profiles with two major peaks, the firstorresponding to unretained species (void volume peak, Robs ≈ 1),he second corresponding specifically to the cell population with

injection of 100 �L cell suspension (2.5 × 10 cells/mL); spectrophotometric detec-tion at � = 254 nm; flow rate: 0.80 mL/min and external multi-gravitational field:8.00 ± 0.01 g. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

Robs < 1 (Fig. 2). For each line, we observed an evolution in the elu-tion profile, with a shift of the main peak toward a lower retentiontime, associated with an increased Robs (Fig. 2, Table 2).

It has been demonstrated that under the “Hyperlayer” mode,samples are lifted away from the accumulation wall and as conse-quence, s which is the average distance from the center of the cell tothe channel wall [39], should be greater than particle radius r, calcu-lated from the mean cell diameter (Coulter Counter measurement).S was calculated by using the following eq. (39):

s = Robs × ω

6(1)

in which ω is the channel thickness (175 �m). According to the

results of size analysis displayed in Table 2, we confirmed the“Hyperlayer” elution of these CRC cell lines but also that cul-ture in OncoMiD for Colon medium did not induce a significantincrease in cell diameter. According to the “Hyperlayer” elution

44 C. Mélin et al. / J. Chromatogr. B 963 (2014) 40–46

F ltureda labelinn

mwwfftDe[(ctwawaaii

cNc

3a

a

ig. 3. Cell proliferation status according to culture conditions. All cell lines were cunalyzed for adherent cells by microscopy and indirect immunofluorescence. Ki-67

uclei were stained with DAPI (blue fluorescence). Magnification 200×.

ode [36–41,51], cells are focused away from the accumulationall, in a thin layer corresponding to their equilibrium positionhere the external field is exactly balanced by hydrodynamic

orces. At equivalent density, large particles generate more liftorces and are focused in faster streamlines to be eluted first. Onhe other hand, gravitational forces increase with particle density.enser particles are thus focused near the accumulation wall to beluted last. Cell shape and rigidity also influence cell elution order52]. As demonstrated by elution profiles (Fig. 2) and Robs valuesTable 2), we clearly observed that independently of the cell lineonsidered, culture in OncoMiD for Colon medium led to a change inhe elution profile, with a shift to the low retention time associatedith increased Robs values. This is particularly important for WiDr

nd SW480. Nevertheless, these increased Robs were not associatedith increased cell diameters. Since “Hyperlayer” mode is a size-

nd density-dependent elution mode, the change in retention ratiot constant size would actually correspond to a significant changen density, and the decreased retention time observed would thusndicate a decrease in cell density.

These results demonstrated that SdFFF was able to monitor thehange in cell population induced by OncoMiD for Colon medium.ext, we determined if it was associated with increased CSContents by phenotypical and functional analyses.

.3. Biological characterization: proliferative activity, apoptosis

nd CSC markers expression

Cell proliferative activity was measured by Ki-67 staining withn antibody recognizing a 395-kDa nuclear protein expressed

in Classic Medium (CM) or OncoMiD for Colon medium for 72 h. Proliferation wasg was performed using Alexa Fluor 594 secondary antibody (red fluorescence) and

during cell-cycle phases (G1, S, G2, and M). After culture in Clas-sic Medium, staining was homogeneous (Fig. 3, panels A, E and I).The same observation was made for adherent cells after culture inOncoMiD for Colon medium (Fig. 3, panels C, G and K). For WiDr, weobserved a small increase in labeling (Fig. 2C versus A), suggesting agreater proliferation. Non-adherent cells emerging in OncoMiD forColon medium were also analyzed, and no staining was observedcorresponding to a lack of proliferation (data not shown).

Apoptosis was measured by Cell Death Detection ElisaPLUS forthe three cell lines studied and compared with those measuredafter culture in Classic Medium (Fig. 4). WiDr was not affectedby medium change, while SW480 had a lesser apoptosis ratio inOncoMiD for Colon medium. In contrast, for Colo205, the apoptosisratio was higher in OncoMiD for Colon medium, suggesting a morepronounced sensitivity for apoptosis in this advanced CRC stagemodel.

The identification of CSC is based on the expression of specificmarkers, such as CD44 and EpCAM. Our previous works recentlyhighlighted that CSC from CRC expressed CD44 and EpCAM [13].Expression of these markers was studied by indirect fluorescencein cells maintained in both media. As observed, all three cell linesdisplayed increased expression of both markers following culturein OncoMiD for Colon medium (Fig. 5). This was more particularlytrue for non-adherent cell clusters, especially for WiDr cells whosemembranes staining was even more pronounced.

We observed that this defined medium induced a slight increasein proliferation for adherent cells. More importantly, those adher-ent CRC lines formed non-adherent cell clusters that did notundergo proliferation. Apoptosis rates were different between

C. Mélin et al. / J. Chromatog

Fig. 4. Apoptosis state according to culture condition. All cell lines were culturedfor 72 h in Classic Medium (CM) or OncoMiD for Colon medium. Apoptosis was ana-lyzed by Cell Death Detection ElisaPLUS according to manufacturer’s instructions.Histograms show mean ratio of apoptotic cells + - SEM of two independent exper-iments. *p < 0.05; **p < 0.01; ***p < 0.001, when compared to Classic Medium (CM)condition.

Fig. 5. Cell line characterization by indirect immunofluorescence. Cells were characterizeClassic Medium (CM) or OncoMiD for Colon medium. Labeling was performed using AlexDAPI (blue fluorescence). Magnification 200×.

r. B 963 (2014) 40–46 45

adherent subpopulations as a function of the cell line: CRC early-stage cell lines WiDr and SW480 were not globally affected, whilethe advanced-stage cell line, Colo205, was more engaged in apo-ptosis following culture in this defined medium. We propose thata more rapid cell turnover is responsible for this observation forColo205 cells. Finally, cells from all three selected lines culturedin OncoMiD for Colon medium, were analyzed for expression ofrelevant CSC markers CD44 and EpCAM, displayed significantlyincreased expression of both markers, which suggests that thedefined medium induced a shift in composition of the subpopu-lations, favoring an increased proportion of immature cells.

Then, according to the biological characterization of cells cul-tured in OncoMiD for Colon medium, which showed increases inboth formation of non-adherent cell clusters and expression of CSCmarkers, which could explain the modified retention time in SdFFFelution, corresponding to an increased proportion of CSC. For exam-ple, in OncoMiD for colon, CD44+ WiDr cells represented 95% ofthe population and 100% for the EpCAM+ cells whereas in ClassicMedium, they corresponded to 40% and 18% respectively.

The defined medium, intended for selective culture of tumorcells, modified the phenotype of a portion of the total populationin all three cell lines. This may have caused the emergence of non-adherent floating cells forming clusters or colospheres. In previous

studies [13,24,45,46,53], we usually observed that immature andstem cells were the most retained species and eluted in the lastpart of fractogram. In this way, we expected an increase in reten-tion time and thus a decreased Robs for cells cultured with OncoMiD

d by using undifferentiated cell markers (CD44 and EpCAM) after 72 h of culture ina Fluor 594 (red fluorescence) secondary antibodies, and nuclei were stained with

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or Colon medium compared to Classic Medium. On the contrarye observed the opposite results. In our previous study on CRC

ells lines cultured in basal conditions, quiescent CSC subpopu-ations were mainly eluted in the last part of fractograms (moreense or smaller cells), while CSC eluted first in the HCT116 cell

ine [13]. HCT116 CSC appeared as less quiescent that those elutedn the end of fractograms for WiDr, SW480 or Colo205 cells. In fact,ur previous results indicated, with other studies, a possible bal-nce between quiescent and cycling states in CSC subpopulations54,55]. Then, reduced retention times could indicate an increasedctivated CSC pools, or it could be the result of a combination ofomplex variations in the cellular morphology, density or rigidity.

. Conclusion

In conclusion, our work strongly suggests that cells from CRCell lines cultured in OncoMiD for Colon medium were initially ableo proliferate (adherent state). Then, an intermediate state with

balance between proliferation/apoptosis was reached, with thebility to form non-adherent clusters that strongly expressed CD44nd EpCAM. These characteristics are common properties of CSC.onsequently, OncoMiD for Colon medium appears to be a valu-ble tool to enrich in vitro cell cultures in undifferentiated cellsomprising both activated (responsible for tumor growth) and qui-scent (responsible for resistance) CSC. This enrichment could beue to cellular de-differentiation of a portion of cells from the CRC

ines. SdFFF could be used to monitor changes in physical propertiesf subpopulations and validate the emergence of CSC. Further-ore, SdFFF could quickly objectivize and monitor effects of cultureedium in order to select, develop and validate their composition.The possibility to specifically enrich CSC populations within

ommonly-used tumor models is of interest to design new stud-es and to further investigate the role of CSC in cancer initiation,rogression, and resistance to treatments. Such approaches mayventually pave the way for the identification of new CSC-specificiological targets leading to the development of therapeutic drugs.

cknowledgements

Authors are grateful to Dr J. Cook-Moreau and Dr C. Wil-on for corrections in the preparation of this manuscript. Thexpenses for this work were defrayed in part by the Ministère de’Éducation Nationale, de la Recherche et de la Technologie, theonseil Régional du Limousin, and by the Ligue contre le CancerComité du Limousin). The funders had no role in study design,ata collection and analysis, decision to publish, or preparation ofhe manuscript.

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