Article pubs.acs.org/ac
Autophagic Subpopulation Sorting by Sedimentation Field-Flow Fractionation Thomas Naves,*,† Serge Battu,†,‡ Marie-Odile Jauberteau,† Philippe J.P. Cardot,†,‡ Marie-Hélène Ratinaud,† and Mireille Verdier† †
Université de Limoges, Institut 145 GEIST, EA 3842 “Homéostasie cellulaire et pathologies”, Faculté de Médecine, 2 rue du Dr Marcland, 87025 Limoges Cedex, France ‡ Faculté de Pharmacie, Laboratoire de Chimie Analytique et Bromatologie, 87025 Limoges Cedex, France ABSTRACT: The development of hypoxic areas often takes place in solid tumors and leads cells to undergo adaptive signalization like autophagy. This process is responsible for misfolded or aggregated proteins and nonfunctional organelle recycling, allowing cells to maintain their energetic status. However, it could constitute a double-edged pathway leading to both survival and cell death. So, in response to stress such as hypoxia, autophagic and apoptotic cells are often mixed. To specifically study and characterize autophagic cells and the process, we needed to develop a method able to (1) isolate autophagic subpopulation and (2) respect apoptotic and autophagic status. Sedimentation field-flow fractionation (SdFFF) was first used to monitor physical parameter changes due to the hypoxia mimetic CoCl2 in the p53 mutated SKNBE2(c) human neuroblastoma cell line. Second, we showed that “hyperlayer” elution is able to prepare autophagic enriched populations, fraction (F3), overexpressing autophagic markers (i.e., LC3-II accumulation and punctiform organization of autophagosomes as well as cathepsin B overactivity). Conversely, the first eluted fraction exhibited apoptotic markers (caspase-3 activity and Bax increased expression). For the first time, SdFFF was employed as an analytical tool in order to discriminate apoptotic and autophagic cells, thus providing an enriched autophagic fraction consecutively to a hypoxic stress.
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whereas the mutated p53 SKNBE2(c) cell line also engaged an autophagic signaling. Indeed, in response to stress such as hypoxia, autophagic and apoptotic processes are often mixed, both involved in tumor cell fate, tumor progression, and therapy issues. Autophagy is an intracellular process leading to limited morphological cell changes, while apoptosis is both an internal and external process leading to important morphological changes. Then, in order to specifically characterize the autophagic cells and process, it could be of first importance to develop a cell-sorting method able to isolate this subpopulation from the hypoxic SKNBE2(c) cell line, respecting apoptotic or autophagic status. In this way, we suggested the use of sedimentation field-flow fractionation (SdFFF). Cell separation methods, allowing isolation and purification of specific subpopulations, have played an increased role in many lifescience domains such as cell biology, cellular therapies, and clinical diagnosis. 11−14 Aside from FACS (Fluorescent Activated Cell Sorting), MASC (Magnetic Activated Cell Sorting), capillary electrophoresis, dielectrophoresis, etc., which are efficient methods based on cell labeling and electrical mobility of cells, many other cell separation techniques based on physical criteria such as size and density (e.g., elutriation,
euroblastoma, the most common extracranial solid cancer in childhood exhibits hypoxic areas, due in part to anarchic cell growth, oxygen consumption, and poorly formed tumor blood vessels.1,2 This leads cells to stabilize the hypoxiainducible factor 1 (HIF-1), the transcriptional factor responsible for the adaptation of tumor cells to the hypoxic environment, frequently associated with aggressiveness, tumor progression, and chemoresistance.3,4 For a few years, autophagy appeared to be a mechanism upregulated during hypoxia, which can favor tumor cells survival and growth.5 Macroautophagy (commonly named autophagy) is a conserved lysosomedependent catabolic pathway responsible for macromolecules and organelles degradation. This process targets and holds part of cytoplasm in double-membrane vesicles named autophagosomes, which finally fuse with lysosomes. This pathway is highly controlled by ATG genes, involved in the autophagosome formation.6 Whereas the rate of this process is low in basal conditions performing housekeeping functions, it could be greatly enhanced during metabolic stresses, such as nutriment or oxygen starvation, in order to maintain the energetic status of the cell.7 However, under severe stress it has been observed that autophagy could also be associated with cell death.8 Our previous study9 showed that cobalt chloride, a salt largely used to mimic hypoxia,10 induced apoptosis via the mitochondrial pathways, Bax expression, and caspase-9 and -3 activation in a p53 wild-type neuroblastoma cell line, SHSY5Y, © 2012 American Chemical Society
Received: July 18, 2012 Accepted: September 24, 2012 Published: September 24, 2012 8748
dx.doi.org/10.1021/ac302032v | Anal. Chem. 2012, 84, 8748−8755
Analytical Chemistry
Article
SdFFF, Cell Elution, and Subculture Conditions. The SdFFF separation device used in this study was previously described and schematized.34,41 Channel dimensions were 818 × 12 × 0.175 mm with two 50 mm V-shaped ends with a measured total void volume of 1772 ± 6.00 μL (n > 6). The channel rotor radius was measured at r = 14.82 cm. Sedimentation fields were expressed in units of gravity, 1g = 980 cm/s2, and calculated as previously described. Cleaning and decontamination procedures, control of rotation speed, and the chromatographic and acquisition device have been previously described.34,41 The optimal elution conditions were determined experimentally and were as follows: flow injection through the accumulation wall of a 100 μL SKNBE2(c) cell suspension (3.5 × 106 cells/ml); flow rate, 0.8 mL/min; mobile phase, sterile phosphate-buffered saline (PBS, pH 7.4) (Invitrogen); and external field strength, 10.00 ± 0.01 g. Figure 1 summarizes the protocol used to prepare the different subpopulations. After a
microfluidic devices, SdFFF, etc.) have shown their effectiveness as gentle, noninvasive, and tagless cell-sorting methods.11−19 In the case of SdFFF, these advantages are based on the drastic limitation of cell−solid phase interactions by the use of (1) a specific separation device: an empty ribbonlike channel without a stationary phase and (2) the “Hyperlayer” elution mode, a size/density-driven separation mechanism.20−24 The principle of cell separation is based on physical criteria such as size and density,20,21,25 and depends on the differential elution of species submitted by the combined action of (1) a parabolic profile generated by flowing a mobile phase through the channel and (2) an external field applied perpendicularly to the flow direction.21 In SdFFF, a multigravitational external field is generated by rotation of the separation channel in a rotor basket, constituting one of the most complex devices used in FFF separation.11,21 Since the report of Caldwell et al.,20 FFF and related technologies have been used in many biological fields.11,17,18,26 In the past decade, we developed both prototypes and applications for SdFFF cell sorting in many fields such as neurology, oncology, and stem cells.27−34 Different aspects in oncology have been evaluated including (1) the monitoring of the biological events induction such as apoptosis or differentiation,28,35,36 (2) the cell sorting of specific subpopulations such as preapoptotic37,38 or differentiated cells,33,39 (3) kinetics of biological events using both monitoring and cell separation capacities of SdFFF.38,40,41 Others aspects in oncology concern the isolation of specific phenotypes such as immature and cancer stem cells.29,34 As was done for two other important events in cancer therapy, apoptosis and differentiation, the aim of the present study was to investigate the capacity of SdFFF to sort an enriched autophagic cell population.
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MATERIALS AND METHODS Reagents. The sterile ready-to-use 0.1 M stock solution of cobalt chloride (CoCl2) and 3-methyladenine (3MA) were purchased from Sigma-Aldrich (Lyon, France). Caspase-3 (AcDEVD-AMC) and the cathepsin-B substrate (z-RR-AMC) were supplied by Bachem (Weil am Rhein, Germany). Other chemicals used were of the purest grade available from Sigma Aldrich and Euromedex (Mundolsheim, France). Mouse monoclonal antialpha-tubulin (TU-02) was purchased from Santa Cruz (Tebu-bio, Le Perray-en-Yvelines, France), polyclonal rabbit anti-MAP-LC3 was obtained from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France). HRP-labeled secondary antibodies were purchased from DakoCytomation (Trappes, France) and secondary Alexa fluor 488TM conjugated antibodies (diluted 1:1000) provided by Molecular Probes (Invitrogen, Saint Aubin, France). Cell Line, Cell Culture, and Treatment. The SKNBE2(c) human neuroblastoma cell line was graciously provided by Pr Castrasena of Navarre University. Cells were maintained in RPMI 1640 supplemented with heat-inactivated 10% fetal bovine serum, 10 mM sodium pyruvate (Invitrogen), 2 mM glutamine (Invitrogen), and an antibiotic mixture (penicillin/ streptomycin, Invitrogen) at 37 °C in a 5% CO2/95% airhumidified incubator. For different analyses, the cells were seeded at 2.5 × 105 cells/mL and harvested with versene (Invitrogen). CoCl2 treatment was performed on 80% confluent cells, at a final dose of 500 μM, from 0 to 24 h before analysis, as previously described.9 For the 3-MA treatment, cells were first incubated with a 2 mM drug for 4 h before adding CoCl2.
Figure 1. Preparation of different SKNBE2(c) cell populations ranging from controls to CoCl2 populations exposed to 500 μM CoCl2 for 15 h. For elution conditions, see Materials and Methods. End run (ER) (stop of rotation, mean field = 0 g) and release peak (RP) of trapped material. 8749
dx.doi.org/10.1021/ac302032v | Anal. Chem. 2012, 84, 8748−8755
Analytical Chemistry
Article
the differences between treatments and the respective controls was analyzed using a one-way analysis of variance (ANOVA) followed by t-tests, and the values are expressed as the mean ± SD.
48 h subculture (80% confluence), cells were treated with either 500 μM CoCl2 (treated cells) or nothing (control) and incubated for an additional 15 h. Then, the cells (treated and control) were harvested and eluted by SdFFF, leading to representative fractograms shown in Figure 1. The elution of treated cells resulted in the separation of four cell fractions collected and designated as follows: (1) TP for total peak (starting with F1, ending with F3) and (2) Fn for fraction number for F1, F2, and F3 (Figure 1). To obtain a sufficient quantity of cells for autophagy analysis, successive cumulative SdFFF fraction collections were performed (10−15, CV of retention time
