Toxicology in Vitro 21 (2007) 176–182www.elsevier.com/locate/toxinvit

Cytomics: A multiparametric, dynamic approach to cell research

Guadalupe Herrera, Laura Diaz, Alicia Martinez-Romero, Angela Gomes, Eva Villamón, Robert C. Callaghan, José-Enrique O’Connor ¤

Laboratorio de Citómica, Unidad Mixta de Investigación CIPF-UVEG, Centro de Investigación Príncipe Felipe,Avda. Autopista del Saler, 16, 46013 Valencia, Spain

Received 8 April 2006; accepted 13 July 2006Available online 22 July 2006

Abstract

Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigationof multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the scienceof single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by externalinXuences. Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based multiparametric methods and the event-inte-grating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies,Xow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologiesbased on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both high-content and high-troughput. The multiparametric capacity of cytomics is very useful for the identiWcation, characterization and isolationof stem cell populations. In our experience, Xow cytometry is a powerful and versatile tool that allows quantitative analysis of single mol-ecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomicassays leads to a real-time kinetic approach based on sequential examination of diVerent single cells from a population undergoing adynamic process, the in Xuxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for tox-icity assessment.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Cytometry; Fluorescence; Toxicology; Pharmacology; In vitro

1. Cytomics and cytomes: single-cell based analysisin complex systems

Genomics, proteomics and metabolomics provide out-standing technical contributions to Cell Biology butbecome limited when single or scarce cells are examined orfast cellular processes followed kinetically (Figeys, 2004). Inaddition, the importance of epigenetic changes (Plass, 2002)and the modulatory inXuence of cell environment (Abrouset al., 2005) recommend to combine “-omic” studies withfunctional analysis (Bernas et al., 2006). Because of this,cell-based assays are increasingly sought for basic research

* Corresponding author. Tel.: +34 963 28 9680; fax: +34 963 28 9671.E-mail address: eoconnor@ochoa.Wb.es (J.-E. O’Connor).

0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2006.07.003

and drug screening, since they approach complex cell-to-cell and cell-to-environment interactions, in assay formatsthat are both information-rich and technically convenient.In this context, where “-omics” meet Systems Biology, cyto-mics represents a novel analytical strategy aimed to deter-mine multiple biochemical features (the molecularphenotype) in single cells and may be deWned as the cytom-etry of complex cellular systems.

In analogy with other -omics (genomics/genome; proteo-mics/proteome, and so on) the objective of cytomics are theheterogeneous cellular systems known as the cytomes. Thecytomes can be understood as the heterogeneous cellularsystems and functional components of the pluricellularorganisms. Since the functional heterogeneity of the cyto-mes results from both the genome and extracellular envi-ronment, cytomics can be considered as a discipline that

G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 177

links genomics and proteomics to cell and tissue function,as modulated by external inXuences. Of special importanceis the cell-by-cell basis of cytomic analysis, an approachthat allows to resolve heterogeneous systems and avoids theloss of information that characterizes bulk technologies, inwhich average values are obtained from large number ofcells or from tissue homogenates.

For a deeper, comprehensive view of cytomics and theirrelevance to biomedicine, see Valet (2005) and Bernas et al.(2006).

2. Technical and analytical features of cytomic technologies

Inherent to cytomics are the use of sensitive, scarcelyinvasive, Xuorescence-based methods and the integratingconcept of individual cell analysis to understand the com-plexity and behaviour of tissues and organisms. Due to theavailability of large number of Xuorescent markers and themultiplicity of Xuorescence detectors interfaced to the dedi-cated instrumentation, cytomic assays may be multipara-metric, polychromatic and multiplexed. Fluorescence-basedmeasurements may be qualitative and quantitative and canbe obtained as the result of single end-point measurementor kinetic, sequential measurements. While these featuresare common to all cytomic technologies, there are impor-tant speciWc diVerences depending on whether the quantita-tive cell Xuorescence data are extracted together with cellmorphology in image-based cytomics (Eils and Athale,2003) or from Xuorescence-pulse analysis in Xow-basedcytomics (O’Connor et al., 2001). Among the currentcytomic technologies, Xow cytometry (FCM), confocallaser scanning microscopy (CLSM), spinning-disk confocalmicroscopy (SDCM) and laser scanning cytometry (LSC)are of established relevance. Other cytomic technologiesbased on single-cell based image analysis and powerful bio-informatic tools (high-content screening bioimaging, HCS-B) have been recently introduced for drug discovery andtoxicity testing, as they can provide both high-content andhigh-troughput analysis. Finally, laser capture microdissec-tion (LCM), a preparative technique for obtaining purecells from speciWc microscopic regions of tissue sections,can also be considered among cytomic technologies.

2.1. Flow cytometry

This methodology requires that cells (or microscopicalbiological particles) are in suspension. FCM allows thesimultaneous quantiWcation of multiple Xuorescence emis-sions in the same cell, arising from Xuorescent markers, andscattered light related to morphology, revealing key cellularfunctions or structures (O’Connor et al., 2001). The velocityof analysis can be up to thousands of single-cells per secondand individual cells from heterogeneous subpopulationscan be physically isolated on the basis of their Xuorescenceor light scatter properties. The multiparametric capacity ofFCM permits to quantify the eVects induced by the expo-sure to a toxic agent, providing a direct proof of cellular

susceptibility or resistance. The advantages of FCM derivefrom its multiparametricity that provides multiple, simulta-neous targets to assess cell lesion or death in selected cellpopulations, either as end-point or kinetic measurements.

2.2. Confocal Xuorescence microscopy

Confocal Xuorescence microscopy is advantageous toconventional Xuorescence microscopy, as it narrows theWeld depth, eliminates out-of-focus blur and allows to pro-duce serial optical stacks from thick specimens (z-axis reso-lution). Confocality may be applied to image single cellsfrom Wxed or living preparations labelled with appropriateXuorescent probes. This technique allows sophisticated cellinformation based on spatial- and time-resolved Xuores-cence measurements and as such is being increasingly usedfor scientiWc and technological applications (Rubart, 2004;Ivorra et al., 2006). Usually, CLSM yields better imagequality but the imaging frame rate is slow while SDCMmay produce video rate imaging, which is required foreYcient dynamic observations (Maddox et al., 2003). How-ever, the new confocal systems function like hybrids allow-ing to obtain both high speed and good resolution.

2.3. Laser scanning cytometry

LSC is a microscope-based, scanning cytoXuorimeterthat combines the advantages of Xow and image cytometry.It allows multiparametric analysis performed directly bymeasuring the Xuorescence of individual cells in solid-statepreparations, such as monolayers, smears, imprints, cyto-spins or tissue sections in several supports including slides,dishes and multiwell plates. LSC provides increased sensi-tivity and speciWcity compared to traditional microscopictechniques and a similar structure for data analysis to Xowcytometry, albeit at lower data acquisition velocity. In addi-tion, it allows relocation of the coordinates of analyzedcells of interest following slide restaining. Finally, singlecells identiWed by their Xuorescence measurements can beindividualized from histogram or dot-plot displays andshown as single-cell pictures or combined in picture galler-ies for further analysis (Juan and Cordon-Cardo, 2001).

2.4. High-content screening bioimaging

An HCS-B system is typically composed by a motorizedXuorescence microscope, a CCD camera that captures theimages, and a digitizing system that stores the images in alarge-capacity computer, with each step controlled by soft-ware for image acquisition and analysis. When applied tohigh-troughput screening (HTS) cell-based assays thisnovel technical concept allows cell research at large scaleusing multiwell plates and analyzing in bulk millions ofcells per experiment. These systems may examine up to onesingle well per second and combine both high-temporal andspatial resolution. HCS-B is very eYcient for complex anal-yses that involve combinations of diVerent cell types and

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concentrations of agonists and antagonists, varying timesof exposure as well as the type and number of parametersstudied on each condition. In addition, the possibility ofmaintaining stable conditions of temperature and CO2allows live cell functional assays. The detection in individ-ual cells provides additional information on the responsesof heterogeneous subpopulations that may diVer in theirdevelopmental stage, cell cycle phase, transfection status orother parameters. However, individual cell analysis mayrequire special measurements (neurite outgrowth, nuclearlocalization, etc.), that reduce the speed required to be qual-iWed as both HTS and HCS (Abraham et al., 2004; Borchertet al., 2005).

2.5. Laser capture microdissection

Meaningful proteomic and genomic analysis demand thepreparation of homogeneous cell populations. Flow-cytom-etry cell sorting may be used to purify a particular cell typein suspension but not easily from solid tissue samples. Toimprove cell isolation, several microdissection methodshave been developed, but are often time-consuming andimprecise. LCM has been developed to automate and stan-dardize microdissection, increasing reproducibility andaccuracy of selecting targeted cells from a complex tissuefor subsequent molecular analysis. LCM systems consist ofan inverted microscope Wtted with a low-power near-infra-red laser. Tissue sections are mounted on standard glassslides, and a transparent, 100-�m thermoplastic Wlm isplaced over the dry section. The laser energy melts Wlm in aprecise location, binding it to the targeted cells, so that indi-vidual cells or a cluster of cells can be selected. After theappropriate cells have been selected, Wlm with adherentcells is removed, and the non-selected tissue remains in con-tact with the glass slide. Cells isolated by LCM can beextracted and submitted to a range of molecular analyticalmethods. Thus, mRNA measurements and cDNA micro-arrays of LCM-puriWed cells from microdissected tissuesallow to compare loss of heterozygosity, detection of muta-tions and gene expression proWles between various celltypes within a tissue. Mass spectrometric sequencing, pep-tide mass Wngerprinting, in-gel zymography, and Westernblot have been used to identify proteins of interest. Theseapproaches are particularly advantageous in identifying thediVerences between expression levels in normal, developingand diseased tissues (Curran et al., 2000; Zieziulewicz et al.,2003).

3. Cytomics in pharmacology and toxicology in vitro

Cytomic analysis may be easily integrated among theessential strategies to study the interaction xenobiotic-cellsfor basic research, industrial development and the evalua-tion of therapeutic and toxic eVects of chemicals and bio-logical compounds. In general, the aims of applyingcytomics to pharmacology and toxicology can be summa-rized as follows (Fig. 1):

3.1. IdentiWcation and selection of speciWc cell subpopulations

Cytomic assays allow to identify and eventually topurify a particular cell subpopulation of a complex cytome,by means of Xuorescent staining of surface markers, intra-cellular functional activities, morphological features or,more frequently, a combination of the above. As an exam-ple of this, by implementing a battery of assays we haverecently demonstrated signiWcant functional diVerencesamong hepatoma cell subpopulations previously cloned byus using a Xow cytometry-based cell sorter on the basis ofdiVerential expression of P-glycoprotein in the parentalhepatoma (O’Connor et al., 2005a).

Lately, the multiparametric capacity of cytomic technol-ogies is proven very useful for the identiWcation, character-ization and isolation of stem cell populations, includingembryonic, adult and tumoral stem cells. This is of specialrelevance, in view of the increasing interest in the cytotoxic-ity on stem cell populations in toxicological and pharmaco-logical context (Hou et al., 2005). An essential requirementfor FCM in this area is the identiWcation of the so-calledside population (SP), a fraction enriched in totipotent stemcells in bone marrow (Goodell et al., 1996), human cancersincluding leukemia, solid tumors and primary cultures, andsome adult normal tissues (Challen and Little, 2006). Inthese models, when cells are labelled with the membrane-permeant DNA binding dye Hoechst 33342, a very smallfraction of cells extrudes this dye via ABCG2/BCRP1 trans-porter and forms a dim tail extending from the normal cellpopulations, that allows to purify them via cell sorting.Technically, SP cells are deWned by the decreased Xuores-cence emissions (red, FL7, and blue, FL6) of Hoechst 33342.The participation of the transporter in the eZux of Hoechst3342 in SP cells is demonstrated by the disappearance of

Fig. 1. Summary of the main processes susceptible of being analyzed bycytomic techniques in the investigation of the interactions between drugs/toxicants and cells. The boxes represent major aspects of the interaction.In italics, particular phenomena that are frequently explored by cytomictechniques.

G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 179

SP cells following incubation with the eZux blocker verap-amil (Fig. 2).

3.2. IdentiWcation of speciWc target cells or cells susceptibleto drugs or xenobiotics

The multiparametric capacity of cytomic methodsallows to identify the expression of receptors speciWc forgiven drugs or the presence of structural and functional tar-gets for a drug or xenobiotic within a cell population. Inother cases, relevant parameters are related to the uptake,retention, biotransformation and eZux of xenobiotic com-pounds. From such parameters, the sensitivity of a particu-lar cell type can be inferred. Most directly, cytomic assayscan reveal on the whole cytomes or in speciWc subpopula-tions the eVects produced by the exposure to the drug orxenobiotic, thus providing evidence for cellular susceptibil-ity or resistance (Lage et al., 2001; Herrera et al., 2003).Cytomic assays can be also applied to determine the sensi-tivity of eukaryotic or prokaryotic cells (Fig. 3) that havebeen submitted to genomic manipulation, which makes thecytomic approach an interesting tool to search for newmodels in cytotoxicity in vitro (Herrera et al., 2003).

3.3. Detection and quantiWcation of toxicity

Cytomic techniques are widely used for quantitative andqualitative analysis of cell and organ toxicity (Alvarez-Bar-rientos et al., 2001). The main advantage of the cytomicapproach derives from their multiparametric capacity(high-content assays), that provides multiple targets andendpoints to assess sublethal lesion and death in speciWccell subpopulations. Thus, cytomic endpoints may repre-sent early or late marker parameters along the cytotoxicprocess. On the other hand, the high velocity of manycytomic strategies allows the sequential analysis of large

numbers of cells per second, thus evidencing incipient orminoritary toxic eVects (Alvarez-Barrientos et al., 2001;Gómez-Lechón et al., 2003).

3.4. Characterization of drug and xenobiotic mechanisms

Because of their multiparametric analytical power, theirtemporal (FCM) and topological (CLSM, HCS-B) resolu-tion and the easy interaction with other “-omics”, cytomicstrategies are frequently applied to explore the mechanismsof actions of drugs and toxics in human, animal and micro-bial cell models for biomedical (Gómez-Lechón et al.,2002), biotechnological (Perlman et al., 2004) and environ-mental studies (Lage et al., 2001).

3.5. Multiplexed analysis of soluble analytes

A recent development of Xow cytometry, multiplexedassays consist in detecting separately but simultaneouslysoluble analytes (usually proteins or nucleic acid sequences)bound speciWcally by aYnity onto reactive Xuorescentmicrospheres of deWned optical properties. The design ofsuch assays allows to quantify simultaneously several ana-lytes in small volumes of sample. Because of this, multi-plexed assays are advantageous over many conventionalbiochemical, immunological or molecular bulk standardtechniques (Khan et al., 2004; Fuja et al., 2004).

3.6. High-troughput and high-content screening

The development of current systems of ultrasensitivedetection of Xuorescence, together with the fast rate of dataacquisition provided by interfaced computing systems,allows the application of cytomic assays to robotized pro-cedures of both high-content and high-troughput screeningof compound libraries, based on cellular Xuorescence in

Fig. 2. An example of the sensitivity of Xow cytometry to detect and to characterize functionally a small subpopulation of relevant cells. (A) Detection ofthe side population (SP) of totipotent progenitor stem cells in mouse bone marrow. SP cells are deWned by the decreased Xuorescence emissions (red, FL7,and blue, FL6) of the vital DNA stain Hoechst 33342. (B) The participation of the ABCG2 transporter in Hoechst 33342 eZux in SP cells is demonstratedby the disappearance of SP cells following incubation with the eZux blocker verapamil. Plots represent a single experiment using the MoFlo cell sorter(Dako) and following the procedure described in Goodell et al. (1996).

180 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182

samples deposed in multiwell plates (Perlman et al., 2004; use succinate or glucose as metabolic fuels (Juan et al.,

Fig. 3. Correlation between genomic modiWcation of bacterial strains (A) and functional cytomic assays by means of end-point (B) or in Xuxo (C)Xow cytometric determination of intracellular oxidative stress. (A) OxyR, soxR and soxS are essential genes for antioxidant defence in Escherichiacoli, and regulate other downstream genes by sensing hydrogen peroxide (oxyR) or superoxide (sodA, sodB). (B) E. coli B WP4 tester strains thathave been made deWcient in oxyR, sodA and sodB genes exhibit increased levels of intracellular oxidative stress when analyzed by Xow cytometryusing the superoxide-sensitive Xuorochrome hydroethidine, as described in Herrera et al. (2003). (C) OxyR mutants of E. coli B WP4 are more sensi-tive to hydroxyl radical than wild type controls when exposed to a source of hydroxyl radical (a combination of hydrogen peroxide plus copper sul-phate), as shown by in Xuxo assay with the oxidant-sensitive Xuorogenic substrate dihydrorhodamine 123 (DHRh123). The increased rate ofhydroxyl generation in the oxyR mutants over wild type cells is suggested by the enhanced production of Xuorescent rhodamine 123 from the paren-tal leuko dye (DHRh123).

Smith and Giorgio, 2004).

3.7. The in Xuxo level of kinetic analysis by Xow cytometry

The dynamic nature of functional cytomic assays isexempliWed by a unique real-time kinetic approach, the inXuxo level (O’Connor et al., 2005b), based on sequentialexamination of diVerent single cells from a populationundergoing a dynamic process, that is triggered whencells of interest are being examined in the Xow cytometer(Fig. 3). Using the in Xuxo strategy, Xow cytometry hasallowed us to focus on a highly speciWc biochemical pro-cess, the activity of the Na+/H+ transporter of plasmamembrane (Dolz et al., 2004). In the other hand, an earlyapplication of the in Xuxo assay provided us a novelapproach to calculate classical parameters of enzymekinetics and to use them to reveal similarities and dis-crepancies between normal rat hepatocytes and rat hepa-toma for the mitochondrial bioenergetic processes that

1996).

4. May cytomics provide in vitro alternative methods for toxicity assessment?

FCM is applied widely to the analysis of in vitro andex vivo of many toxicity cellular markers albeit it never hasbeen applied as a systematic and normalized unique strat-egy, in the form of an in vitro alternative method for chem-ical risk assessment. However, the recent developments inautomatization and bioinformatics interfaced to FCM(Smith and Giorgio, 2004) and HCS-B instruments (Perl-man et al., 2004) can be implemented into robotic proce-dures based on 96-well format assays in equipmentsavailable in growing number of laboratories.

During the development of the European Contract “Anevaluation of the reproducibility and transferability of Xowcytometric and confocal microscopic endpoints in anin vitro nephrotoxicity and in vitro metabolism models” we

G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 181

showed that a compact set of functional assays by Xowcytometry (the so-called “primary toxicity cytomic panel”,PTCP) reveals toxic eVects in models of acute, sustainedand delayed exposure of tubular renal cells to nephrotoxins(cadmium chloride, cyclosporin an cis-platin) in sub-lethalconcentrations (Alvarez-Barrientos et al., 2001). Our resultssuggest that cytomic functional assays may detect speciW-cally early or transient changes in the process of cytotoxic-ity, which makes these assays advantageous over other testslimited to the quantiWcation of cell death as endpoint ofcytotoxicity. In the second part of the mentioned researchcontract we showed how the application of PTCP allows toseparate basal- and biotransformation-dependent cytotox-icity by means of the comparison of the cytotoxic eVects oftwo neuropharmacological substrates of cytochromeCYP2D6 (mianserin and imipramine) on the cell line V79transfected stably with CYP2D6 and its control, mock-transfected cell line (Martínez-Romero et al., 2004). TheXow cytometric data on nephrotoxicity and metabolism-dependent toxicity generated in this study were consistentwith those obtained by confocal microscopy, another pow-erful cytomic technology, in an independent laboratory(Alvarez-Barrientos et al., 2001, 2003). Thus, for certainsamples, FCM appears as a versatile tool that allows toapproach diVerent levels of cellular complexity to revealcytotoxic eVects and mechanisms in vitro or ex vivo, even incomplex, heterogeneous cell populations.

Very recently, our laboratory has been enrolled in theEC sixth FP Integrated Project “A-Cute-Tox: Optimizationand pre-validation of an in vitro test strategy for predictingacute human toxicity” (www.acutetox.org). This ambitiousproject (37 partners from 13 EC countries) is aimed todevelop a simple and robust assay strategy to predicthuman acute systemic toxicity that could eventually replacecurrent regulatory assays on whole animals. One of the spe-ciWc objectives of A-Cute-Tox is to explore innovative toolsand cell systems to identify new endpoints and strategiesthat anticipate better human and animal toxicity. Incorpo-ration of cytomics to this project may deWne new endpointsof in vitro cytotoxicity to be incorporated into the predic-tive model or to provide new alerts and correctors of toxic-ity.

In the Wrst part of this project we have been able of min-iaturizing (96-well format) the cultures of rat and humancell lines, as well as of adjusting the experimental conditionsfor toxic treatment, cell resuspension, Xuorescent stainingand Xow cytometric assay with the new Xow cytometersinterfaced to 96-well plate sample loaders. This format ofassay is comparable to that used for most of the current cel-lular assays of reference for in vitro cytotoxicity (Fig. 4). Inthis context, we have deWned the settings and calibration ofthe Xow cytometers for this type of assay and establishedthe correlation in the rat hepatoma Fao between a Xowcytometric test of cell viability (propidium iodide exclusion)

Fig. 4. Analysis of digoxin cytotoxicity in three established cell lines by a Xow cytometric 96-well automated assay of propidium iodide exclusion. Theestablished cell lines FAO (rat hepatoma), SH-SY5Y (human neuroblastoma) and A.704 (human kidney adenocarcinoma) were seeded in plastic 96-wellplates and incubated in 10% FCS containing medium for 24 h (50% conXuence) in CO2 incubator at 37 °C. Then, the indicated concentrations of digoxinin 5% FCS containing medium were added and the plates kept for further 24 h. Then, cells were trypsinized and resuspended in their original wells in 5%FCS containing medium, in the presence of 5 �g/mL propidium iodide (PI) and then analyzed automatically with the Cytomics FC500 MPL multiwell-plate Xow cytometer (Beckman–Coulter). Data points represent triplicate wells from two separate experiments. The insert graph shows the Xow cytometricstrategy to diVerentiate live cells (PI excluding cells) from apoptotic and necrotic by means of a plot correlating cell size (forward scatter) and PI uptake.Notice that neuroblastoma cells are much more sensitive to digoxin than the hepatoma or renal adenocarcinoma cells.

182 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182

and the Neutral Red uptake assay. For all the above con-siderations, we believe that both the technology (Xowcytometry) and the strategy (functional cytomic assays)may succeed in accomplishing meaningfully the proposedresearch project.

Acknowledgements

Sponsored by EC sixth FP projects A-Cute-Tox (LSHB-CT-2004-512051) and Predictomics (LSHB-CT-2004-504761), the Generalitat Valenciana (GV04B-143) and thePrograma de Medicina Regenerativa de la Comunidad Val-enciana. E.V. is a Bancaixa post-doctoral fellow. A.G. isrecipient of a Leonardo fellowship.

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