Cytomics: a Multiparametric, Dynamic Approach to Cell Research

Toxicology in Vitro 21 (2007) 176–182 www.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 2006 Available online 22 July 2006

Abstract

Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external inXuences. Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based multiparametric methods and the event-integrating 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 technologies based 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 isolation of stem cell populations. In our experience, Xow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic assays leads to a real-time kinetic approach based on sequential examination of diVerent single cells from a population undergoing a dynamic process, the in Xuxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment. © 2006 Elsevier Ltd. All rights reserved.

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

1. Cytomics and cytomes: single-cell based analysis and drug screening, since they approach complex cell-to- in complex systems cell and cell-to-environment interactions, in assay formats that are both information-rich and technically convenient. Genomics, proteomics and metabolomics provide out- In this context, where “-omics” meet Systems Biology, cyto- standing technical contributions to Cell Biology but mics represents a novel analytical strategy aimed to deter- become limited when single or scarce cells are examined or mine multiple biochemical features (the molecular fast cellular processes followed kinetically (Figeys, 2004). In phenotype) in single cells and may be deWned as the cytom- addition, the importance of epigenetic changes (Plass, 2002) etry of complex cellular systems. and the modulatory inXuence of cell environment (Abrous In analogy with other -omics (genomics/genome; proteo- et al., 2005) recommend to combine “-omic” studies with mics/proteome, and so on) the objective of cytomics are the functional analysis (Bernas et al., 2006). Because of this, heterogeneous cellular systems known as the cytomes. The cell-based assays are increasingly sought for basic research cytomes can be understood as the heterogeneous cellular systems and functional components of the pluricellular organisms. Since the functional heterogeneity of the cyto- * Corresponding author. Tel.: +34 963 28 9680; fax: +34 963 28 9671. mes results from both the genome and extracellular envi- E-mail address: [email protected] (J.-E. O’Connor). ronment, cytomics can be considered as a discipline that

0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.07.003 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 177 links genomics and proteomics to cell and tissue function, susceptibility or resistance. The advantages of FCM derive as modulated by external inXuences. Of special importance from its multiparametricity that provides multiple, simulta- is the cell-by-cell basis of cytomic analysis, an approach neous targets to assess cell lesion or death in selected cell that allows to resolve heterogeneous systems and avoids the populations, either as end-point or kinetic measurements. loss of information that characterizes bulk technologies, in which average values are obtained from large number of 2.2. Confocal Xuorescence microscopy cells or from tissue homogenates. For a deeper, comprehensive view of cytomics and their Confocal Xuorescence microscopy is advantageous to relevance to biomedicine, see Valet (2005) and Bernas et al. conventional Xuorescence microscopy, as it narrows the (2006). Weld depth, eliminates out-of-focus blur and allows to produce serial optical stacks from thick specimens (z-axis reso- 2. Technical and analytical features of cytomic technologies lution). Confocality may be applied to image single cells from Wxed or living preparations labelled with appropriate Inherent to cytomics are the use of sensitive, scarcely Xuorescent probes. This technique allows sophisticated cell invasive, Xuorescence-based methods and the integrating information based on spatial- and time-resolved Xuores- concept of individual cell analysis to understand the com- cence measurements and as such is being increasingly used plexity and behaviour of tissues and organisms. Due to the for scientiWc and technological applications (Rubart, 2004; availability of large number of Xuorescent markers and the Ivorra et al., 2006). Usually, CLSM yields better image multiplicity of Xuorescence detectors interfaced to the dedi- quality but the imaging frame rate is slow while SDCM cated instrumentation, cytomic assays may be multipara- may produce video rate imaging, which is required for metric, polychromatic and multiplexed. Fluorescence-based eYcient dynamic observations (Maddox et al., 2003). How- measurements may be qualitative and quantitative and can ever, the new confocal systems function like hybrids allow- be obtained as the result of single end-point measurement ing to obtain both high speed and good resolution. or kinetic, sequential measurements. While these features are common to all cytomic technologies, there are impor- 2.3. Laser scanning cytometry tant speciWc diVerences depending on whether the quantitative cell Xuorescence data are extracted together with cell LSC is a microscope-based, scanning cytoXuorimeter morphology in image-based cytomics (Eils and Athale, that combines the advantages of Xow and image cytometry. 2003) or from Xuorescence-pulse analysis in Xow-based It allows multiparametric analysis performed directly by cytomics (O’Connor et al., 2001). Among the current measuring the Xuorescence of individual cells in solid-state cytomic technologies, Xow cytometry (FCM), confocal preparations, such as monolayers, smears, imprints, cyto- laser scanning microscopy (CLSM), spinning-disk confocal spins or tissue sections in several supports including slides, microscopy (SDCM) and laser scanning cytometry (LSC) dishes and multiwell plates. LSC provides increased sensi- are of established relevance. Other cytomic technologies tivity and speciWcity compared to traditional microscopic based on single-cell based image analysis and powerful bio- techniques and a similar structure for data analysis to Xow informatic tools (high-content screening bioimaging, HCS- cytometry, albeit at lower data acquisition velocity. In addi- B) have been recently introduced for drug discovery and tion, it allows relocation of the coordinates of analyzed toxicity testing, as they can provide both high-content and cells of interest following slide restaining. Finally, single high-troughput analysis. Finally, laser capture microdissec- cells identiWed by their Xuorescence measurements can be tion (LCM), a preparative technique for obtaining pure individualized from histogram or dot-plot displays and cells from speciWc microscopic regions of tissue sections, shown as single-cell pictures or combined in picture galler- can also be considered among cytomic technologies. ies for further analysis (Juan and Cordon-Cardo, 2001).

2.1. Flow cytometry 2.4. High-content screening bioimaging

This methodology requires that cells (or microscopical An HCS-B system is typically composed by a motorized biological particles) are in suspension. FCM allows the Xuorescence microscope, a CCD camera that captures the simultaneous quantiWcation of multiple Xuorescence emis- images, and a digitizing system that stores the images in a sions in the same cell, arising from Xuorescent markers, and large-capacity computer, with each step controlled by soft- scattered light related to morphology, revealing key cellular ware for image acquisition and analysis. When applied to functions or structures (O’Connor et al., 2001). The velocity high-troughput screening (HTS) cell-based assays this of analysis can be up to thousands of single-cells per second novel technical concept allows cell research at large scale and individual cells from heterogeneous subpopulations using multiwell plates and analyzing in bulk millions of can be physically isolated on the basis of their Xuorescence cells per experiment. These systems may examine up to one or light scatter properties. The multiparametric capacity of single well per second and combine both high-temporal and FCM permits to quantify the eVects induced by the expo- spatial resolution. HCS-B is very eYcient for complex anal- sure to a toxic agent, providing a direct proof of cellular yses that involve combinations of diVerent cell types and 178 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 concentrations of agonists and antagonists, varying times of exposure as well as the type and number of parameters studied on each condition. In addition, the possibility of maintaining stable conditions of temperature and CO2 allows live cell functional assays. The detection in individual cells provides additional information on the responses of heterogeneous subpopulations that may diVer in their developmental stage, cell cycle phase, transfection status or other parameters. However, individual cell analysis may require special measurements (neurite outgrowth, nuclear localization, etc.), that reduce the speed required to be qual- iWed as both HTS and HCS (Abraham et al., 2004; Borchert et al., 2005).

2.5. Laser capture microdissection

Meaningful proteomic and genomic analysis demand the preparation of homogeneous cell populations. Flow-cytom- Fig. 1. Summary of the main processes susceptible of being analyzed by etry cell sorting may be used to purify a particular cell type cytomic techniques in the investigation of the interactions between drugs/ in suspension but not easily from solid tissue samples. To toxicants and cells. The boxes represent major aspects of the interaction. improve cell isolation, several microdissection methods In italics, particular phenomena that are frequently explored by cytomic have been developed, but are often time-consuming and techniques. imprecise. LCM has been developed to automate and stan- dardize microdissection, increasing reproducibility and 3.1. IdentiWcation and selection of speciWc cell subpopulations accuracy of selecting targeted cells from a complex tissue for subsequent molecular analysis. LCM systems consist of Cytomic assays allow to identify and eventually to an inverted microscope Wtted with a low-power near-infra- purify a particular cell subpopulation of a complex cytome, red laser. Tissue sections are mounted on standard glass by means of Xuorescent staining of surface markers, intra- slides, and a transparent, 100-m thermoplastic Wlm is cellular functional activities, morphological features or, placed over the dry section. The laser energy melts Wlm in a more frequently, a combination of the above. As an exam- precise location, binding it to the targeted cells, so that indi- ple of this, by implementing a battery of assays we have vidual cells or a cluster of cells can be selected. After the recently demonstrated signiWcant functional diVerences appropriate cells have been selected, Wlm with adherent among hepatoma cell subpopulations previously cloned by cells is removed, and the non-selected tissue remains in con- us using a Xow cytometry-based cell sorter on the basis of tact with the glass slide. Cells isolated by LCM can be diVerential expression of P-glycoprotein in the parental extracted and submitted to a range of molecular analytical hepatoma (O’Connor et al., 2005a). methods. Thus, mRNA measurements and cDNA micro- Lately, the multiparametric capacity of cytomic technol- arrays of LCM-puriWed cells from microdissected tissues ogies is proven very useful for the identiWcation, character- allow to compare loss of heterozygosity, detection of muta- ization and isolation of stem cell populations, including tions and gene expression proWles between various cell embryonic, adult and tumoral stem cells. This is of special types within a tissue. Mass spectrometric sequencing, pep- relevance, in view of the increasing interest in the cytotoxic- tide mass Wngerprinting, in-gel zymography, and Western ity on stem cell populations in toxicological and pharmaco- blot have been used to identify proteins of interest. These logical context (Hou et al., 2005). An essential requirement approaches are particularly advantageous in identifying the for FCM in this area is the identiWcation of the so-called diVerences between expression levels in normal, developing side population (SP), a fraction enriched in totipotent stem and diseased tissues (Curran et al., 2000; Zieziulewicz et al., cells in bone marrow (Goodell et al., 1996), human cancers 2003). including leukemia, solid tumors and primary cultures, and some adult normal tissues (Challen and Little, 2006). In 3. Cytomics in pharmacology and toxicology in vitro these models, when cells are labelled with the membrane- permeant DNA binding dye Hoechst 33342, a very small Cytomic analysis may be easily integrated among the fraction of cells extrudes this dye via ABCG2/BCRP1 trans- essential strategies to study the interaction xenobiotic-cells porter and forms a dim tail extending from the normal cell for basic research, industrial development and the evalua- populations, that allows to purify them via cell sorting. tion of therapeutic and toxic eVects of chemicals and bio- Technically, SP cells are deWned by the decreased Xuores- logical compounds. In general, the aims of applying cence emissions (red, FL7, and blue, FL6) of Hoechst 33342. cytomics to pharmacology and toxicology can be summa- The participation of the transporter in the eZux of Hoechst rized as follows (Fig. 1): 3342 in SP cells is demonstrated by the disappearance of G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182 179

Fig. 2. An example of the sensitivity of Xow cytometry to detect and to characterize functionally a small subpopulation of relevant cells. (A) Detection of the 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 demonstrated by 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).

SP cells following incubation with the eZux blocker verap- numbers of cells per second, thus evidencing incipient or amil (Fig. 2). minoritary toxic eVects (Alvarez-Barrientos et al., 2001; Gómez-Lechón et al., 2003). 3.2. IdentiWcation of speciWc target cells or cells susceptible to drugs or xenobiotics 3.4. Characterization of drug and xenobiotic mechanisms

The multiparametric capacity of cytomic methods Because of their multiparametric analytical power, their allows to identify the expression of receptors speciWc for temporal (FCM) and topological (CLSM, HCS-B) resolu- given drugs or the presence of structural and functional tar- tion and the easy interaction with other “-omics”, cytomic gets for a drug or xenobiotic within a cell population. In strategies are frequently applied to explore the mechanisms other cases, relevant parameters are related to the uptake, of actions of drugs and toxics in human, animal and micro- retention, biotransformation and eZux of xenobiotic com- bial cell models for biomedical (Gómez-Lechón et al., pounds. From such parameters, the sensitivity of a particu- 2002), biotechnological (Perlman et al., 2004) and environ- lar cell type can be inferred. Most directly, cytomic assays mental studies (Lage et al., 2001). can reveal on the whole cytomes or in speciWc subpopulations the eVects produced by the exposure to the drug or 3.5. Multiplexed analysis of soluble analytes xenobiotic, thus providing evidence for cellular susceptibility or resistance (Lage et al., 2001; Herrera et al., 2003). A recent development of Xow cytometry, multiplexed Cytomic assays can be also applied to determine the sensi- assays consist in detecting separately but simultaneously tivity of eukaryotic or prokaryotic cells (Fig. 3) that have soluble analytes (usually proteins or nucleic acid sequences) been submitted to genomic manipulation, which makes the bound speciWcally by aYnity onto reactive Xuorescent cytomic approach an interesting tool to search for new microspheres of deWned optical properties. The design of models in cytotoxicity in vitro (Herrera et al., 2003). such assays allows to quantify simultaneously several analytes in small volumes of sample. Because of this, multi- 3.3. Detection and quantiWcation of toxicity plexed assays are advantageous over many conventional biochemical, immunological or molecular bulk standard Cytomic techniques are widely used for quantitative and techniques (Khan et al., 2004; Fuja et al., 2004). qualitative analysis of cell and organ toxicity (Alvarez-Bar- rientos et al., 2001). The main advantage of the cytomic 3.6. High-troughput and high-content screening approach derives from their multiparametric capacity (high-content assays), that provides multiple targets and The development of current systems of ultrasensitive endpoints to assess sublethal lesion and death in speciWc detection of Xuorescence, together with the fast rate of data cell subpopulations. Thus, cytomic endpoints may repre- acquisition provided by interfaced computing systems, sent early or late marker parameters along the cytotoxic allows the application of cytomic assays to robotized pro- process. On the other hand, the high velocity of many cedures of both high-content and high-troughput screening cytomic strategies allows the sequential analysis of large of compound libraries, based on cellular Xuorescence in 180 G. Herrera et al. / Toxicology in Vitro 21 (2007) 176–182

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 Escherichia coli, and regulate other downstream genes by sensing hydrogen peroxide (oxyR) or superoxide (sodA, sodB). (B) E. coli B WP4 tester strains that have been made deWcient in oxyR, sodA and sodB genes exhibit increased levels of intracellular oxidative stress when analyzed by Xow cytometry using the superoxide-sensitive Xuorochrome hydroethidine, as described in Herrera et al. (2003). (C) OxyR mutants of E. coli B WP4 are more sensitive 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 of hydroxyl generation in the oxyR mutants over wild type cells is suggested by the enhanced production of Xuorescent rhodamine 123 from the parental leuko dye (DHRh123). samples deposed in multiwell plates (Perlman et al., 2004; use succinate or glucose as metabolic fuels (Juan et al., Smith and Giorgio, 2004). 1996).

3.7. The in Xuxo level of kinetic analysis by Xow cytometry 4. May cytomics provide in vitro alternative methods for toxicity assessment? The dynamic nature of functional cytomic assays is exempliWed by a unique real-time kinetic approach, the in FCM is applied widely to the analysis of in vitro and Xuxo level (O’Connor et al., 2005b), based on sequential ex vivo of many toxicity cellular markers albeit it never has examination of diVerent single cells from a population been applied as a systematic and normalized unique strat- undergoing a dynamic process, that is triggered when egy, in the form of an in vitro alternative method for chem- cells of interest are being examined in the Xow cytometer ical risk assessment. However, the recent developments in (Fig. 3). Using the in Xuxo strategy, Xow cytometry has automatization and bioinformatics interfaced to FCM allowed us to focus on a highly speciWc biochemical pro- (Smith and Giorgio, 2004) and HCS-B instruments (Perl- cess, the activity of the Na+/H+ transporter of plasma man et al., 2004) can be implemented into robotic proce- membrane (Dolz et al., 2004). In the other hand, an early dures based on 96-well format assays in equipments application of the in Xuxo assay provided us a novel available in growing number of laboratories. approach to calculate classical parameters of enzyme During the development of the European Contract “An kinetics and to use them to reveal similarities and dis- evaluation of the reproducibility and transferability of Xow crepancies between normal rat hepatocytes and rat hepa- cytometric and confocal microscopic endpoints in an toma for the mitochondrial bioenergetic processes that in 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 Xow Very recently, our laboratory has been enrolled in the cytometry (the so-called “primary toxicity cytomic panel”, EC sixth FP Integrated Project “A-Cute-Tox: Optimization PTCP) reveals toxic eVects in models of acute, sustained and pre-validation of an in vitro test strategy for predicting and delayed exposure of tubular renal cells to nephrotoxins acute human toxicity” (www.acutetox.org). This ambitious (cadmium chloride, cyclosporin an cis-platin) in sub-lethal project (37 partners from 13 EC countries) is aimed to concentrations (Alvarez-Barrientos et al., 2001). Our results develop a simple and robust assay strategy to predict suggest that cytomic functional assays may detect speciW- human acute systemic toxicity that could eventually replace cally early or transient changes in the process of cytotoxic- current regulatory assays on whole animals. One of the spe- ity, which makes these assays advantageous over other tests ciWc objectives of A-Cute-Tox is to explore innovative tools limited to the quantiWcation of cell death as endpoint of and cell systems to identify new endpoints and strategies cytotoxicity. In the second part of the mentioned research that anticipate better human and animal toxicity. Incorpo- contract we showed how the application of PTCP allows to ration of cytomics to this project may deWne new endpoints separate basal- and biotransformation-dependent cytotox- of in vitro cytotoxicity to be incorporated into the predic- icity by means of the comparison of the cytotoxic eVects of tive model or to provide new alerts and correctors of toxic- two neuropharmacological substrates of cytochrome ity. CYP2D6 (mianserin and imipramine) on the cell line V79 In the Wrst part of this project we have been able of min- transfected stably with CYP2D6 and its control, mock- iaturizing (96-well format) the cultures of rat and human transfected cell line (Martínez-Romero et al., 2004). The cell lines, as well as of adjusting the experimental conditions Xow cytometric data on nephrotoxicity and metabolism- for toxic treatment, cell resuspension, Xuorescent staining dependent toxicity generated in this study were consistent and Xow cytometric assay with the new Xow cytometers with those obtained by confocal microscopy, another pow- interfaced to 96-well plate sample loaders. This format of erful cytomic technology, in an independent laboratory assay is comparable to that used for most of the current cel- (Alvarez-Barrientos et al., 2001, 2003). Thus, for certain lular assays of reference for in vitro cytotoxicity (Fig. 4). In samples, FCM appears as a versatile tool that allows to this context, we have deWned the settings and calibration of approach diVerent levels of cellular complexity to reveal the Xow cytometers for this type of assay and established cytotoxic eVects and mechanisms in vitro or ex vivo, even in the correlation in the rat hepatoma Fao between a Xow complex, heterogeneous cell populations. cytometric 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. The established cell lines FAO (rat hepatoma), SH-SY5Y (human neuroblastoma) and A.704 (human kidney adenocarcinoma) were seeded in plastic 96-well X plates and incubated in 10% FCS containing medium for 24 h (50% con uence) in CO2 incubator at 37 °C. Then, the indicated concentrations of digoxin in 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 cytometric strategy 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- Herrera, G., Martínez, A., Blanco, M., O’Connor, J.E., 2003. Functional siderations, we believe that both the technology (Xow assays of oxidative stress using genetically engineered Escherichia coli cytometry) and the strategy (functional cytomic assays) strains. Current Protocols in Cytometry, 11.16.1–11.16.9. Hou, M., Chrysis, D., Nurmio, M., Parvinen, M., Eksborg, S., Söder, O., may succeed in accomplishing meaningfully the proposed Jahnukainen, K., 2005. 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