Measurement of Gene Expression from Single Adherent Cells and

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA...
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Measurement of Gene Expression from Single Adherent Cells and Spheroids Collected Using Fast Electrical Lysis Yuji Nashimoto,† Yasufumi Takahashi,† Takeshi Yamakawa,† Yu-suke Torisawa,† Tomoyuki Yasukawa,† Takahiro Ito-Sasaki,‡ Masaki Yokoo,‡ Hiroyuki Abe,‡ Hitoshi Shiku,*,† Hideki Kambara,§,| and Tomokazu Matsue*,†,‡

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan, Tohoku University Biomedical Engineering Research Organization (TUBERO), Sendai 980-8575, Japan, Central Research Laboratory, Hitachi Ltd., Kokubunji, Tokyo 185-8601, Japan, and Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 µm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 µs between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 µL of the lysed solution was collected using a microinjector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although singlecell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the singlecell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroidssthe aggregated cellular masses formed during the three-dimensional culturesas a model system to isolate small cellular clusters from tissues and organs. Single-cell analysis becomes possible with innovations in instruments, including cell sorters and single-molecule detection with fluorescence microscopes.1,2 In addition, microfluidic cellular devices have achieved remarkable progress in not only the manipulation and culture but also in studying the various cellular * To whom correspondence should be addressed. E-mail: [email protected]. tohoku.ac.jp; [email protected]. † Tohoku University. ‡ Tohoku University Biomedical Engineering Research Organization (TUBERO). § Hitachi Ltd. | Tokyo University of Agriculture and Technology. (1) Andersson, H.; van der Berg, A. Lab Chip 2006, 6, 467-470. (2) Ti, C.; Li, C.-W.; Ji, C.; Yang, M. Anal. Chim. Acta 2006, 560, 1-23. 10.1021/ac071050q CCC: $37.00 Published on Web 08/03/2007

© 2007 American Chemical Society

functions at genomic, proteomic, and metabolomic levels.3,4 The major advantages of using a microsystem are its low cost per run of a single assay and the potential to integrate sequential and parallel operations on a chip. Quake’s group5-7 has recently conducted a single-cell messenger RNA (mRNA) analysis successfully by using a microfluidic device featuring an array of channels, inlets, outlets, pumps, and valves to isolate the mRNA from individual mammalian cells. Using this device, the sequential operations, including capture of a single cell, cell lysis, mRNA isolation, cDNA synthesis, and real-time PCR amplification, can be performed within perfectly closed microfluidic channels; therefore, it is contamination-free.8 However, it might take a considerably long time to realize the goal of an ideal single-cell mRNA analysis because further improvements such as rapidity, ease of use, reliability, sensitivity, and the number of genes to be analyzed in tandem are required for it to become a truly useful microanalytical system for a massively parallel mRNA analysis at a single-cell level. Other problems that need to be solved are the methods of introduction of a single adherent cell into such microsystems and collection of a single cell from a tissue sample while maintaining the original cellular nature and functions. The analysis of the gene expression profiles of individual cells within whole tissues and organs is required for complete understanding of human disease at the cellular level and to gain fundamental knowledge of the mechanisms in cellular transduction, differentiation, development, and malignant transformation.9,10 Gene expression is regulated at the level of individual cells and at different developmental stages even in genetically identical cells. Thus, the development of a microsystem to detect and quantify gene expression in individual cells is becoming increasingly (3) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403-411. (4) Sims, C. E.; Allbritton, N. L. Lab Chip 2007, 7, 423-440. (5) Hong, J. W.; Studer, V.; Hangf, G.; Anderson, W. F.; Quake, S. R. Nat. Biotechnol. 2004, 4, 435-439. (6) Marcus, J. S.; Anderson, W. F.; Quake, S. R. Anal. Chem. 2006, 78, 956958. (7) Huang, Y.; Castrataro, P.; Lee, C.-C.; Quake, S. R. Lab Chip 2007, 7, 2426. (8) Marcus, J. S.; Anderson, W. F.; Quake, S. R. Anal. Chem. 2006, 78, 30843089. (9) Phillips, J. K.; Lipski, J. Autom. Neurosci. Bas. Clin. 2000, 86, 1-12. (10) Liss, B. Nucleic Acids Res. 2002, 30, e89.

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important. At present, the best way to realize single-cell mRNA analysis of tissues and organs might be to use the laser-based microdissection (LMD) system,11 by which tissue slice samples have been quantitatively assessed under physiological conditions (even in liquids) without requiring fixation.12,13 In this case, realtime reverse transcriptase (RT)-PCR is the technique selected most often for gene expression analysis because of its sensitivity and quantifiability, whereas a DNA microarray might not have sufficient sensitivity to detect mRNA at a single-cell level. The drawback of the LMD is its laser apparatus that increases the cost of the system. Capillary electrophoresis (CE) has played a significant role in single-cell analysis and has vigorously promoted this research field since the late 1980s.14-18 Allbritton’s group18 proposed a fast electrical cell lysis system and compared its performance with the laser-based ablation19-21 technique. In their research, a fluorescent dye molecule was introduced into the cell sample before CE analysis. Electrical- and laser-based cell lysis was performed for the single-cell analysis, and the results indicated that the sampling efficiencies of the two techniques were comparable. Unfortunately, a single-cell CE system has not been used in combination with real-time RT-PCR, although a couple of research groups performed an RT reaction.17,22,23 We have been developing a three-dimensional microculture system featuring a 20-nL silicone well array24 for extracellular matrix (ECM)-embedded culture25,26 and spheroid culture.27-29 By electrochemically monitoring the local oxygen profiles near the microwell, the respiratory activities of a single cell in various environmental conditions have been estimated in situ. However, information at the gene or protein level cannot be obtained electrochemically, except by introducing reporter systems such (11) Schutze, K.; Lahr, G. Nat. Biotechnol. 1998, 16, 737-742. (12) Micke, P.; Bjomsen, T.; Scheidl, S.; Stromberg, S.; Demoulin, J.-B.; Ponten, F.; Ostman, A.; Lindahl, P.; Busch, C. J. Pathol. 2004, 202, 130-138. (13) Stich, M.; Thalhammer, S.; Burgemeister, R.; Friedemann, G.; Ehnle, S.; Luthy, C.; Schutze, K. Pathol. Res. Pract. 2003, 199, 405-409. (14) Woods, L. A.; Gandhi, P. U.; Ewing, A. G. Anal. Chem. 2005, 77, 18191823. (15) Anazawa, T.; Matsunaga, H.; Yeung, E. S. Anal. Chem. 2002, 74, 50335038. (16) Zhong, W.; Yeung, E. S. Anal. Chem. 2003, 75, 4415-4422. (17) Matsunaga, H.; Anazawa, T.; Yeung, E. S. Electrophoresis 2003, 24, 458465. (18) Han, F.; Wang, Y.; Sims, C. E.; Bachman, M.; Chang, R.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2003, 75, 3688-3696. (19) Sims, C. E.; Meredith, G. D.; Krasieva, T. B.; Berns, M. W.; Tromberg, B. J.; Allbritton, N. L. Anal. Chem. 1998, 70, 4570-4577. (20) Wang, Y.; Young, G.; Bachman, M.; Sims, C.; E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 2356-2366. (21) Salazar, G. T.; Wang, Y.; Young, G.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2007, 79, 682-687. (22) Zabzdyr, J. L.; Lillard, S. H. Anal. Chem. 2001, 73, 5771-5775. (23) Zabzdyr, J. L.; Lillard, S. H. Electrophoresis 2005, 26, 137-145. (24) Torisawa, Y.; Kaya, T.; Takii, Y.; Oyamatsu, D.; Nishizawa, M.; Matsue, T. Anal. Chem. 2003, 75, 2154-2158. (25) Torisawa, Y.; Shiku, H.; Kasai, S.; Nishizawa, M.; Matsue, T. Int. J. Cancer 2004, 109, 302-308. (26) Torisawa, Y.; Shiku, H.; Yasukawa, T.; Nishizawa, M.; Matsue, T. Biomaterials 2005, 26, 2165-2172. (27) Torisawa, Y.; Takagi, A.; Nashimoto, Y.; Yasukawa, T.; Shiku, H.; Matsue, T. Biomaterials 2007, 28, 559-566. (28) Torisawa Y.; Takagi, A.; Shiku, H.; Yasukawa, T.; Matsue, T. Oncol. Rep. 2005, 13, 1107-1112. (29) Torisawa, Y.; Chueh, B.-h.; Huh, D.; Ramamurthy, P.; Roth, T. M.; Barald, K. F.; Takayama, S. Lab Chip 2007, 7, 770-776.

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as β-galactosidase30-34 and alkaline phosphatase.35,36 The human mammary epithelial cell HMT-3522 series was laminin-sensitive in its morphological and genetic characteristics. Bissell’s group has developed an assay system in which HMT-3522 cells are cultured in a three-dimension reconstructed basement membrane to reversibly control the polarity of the mammary epithelial cells.37-40 In a previous study, we succeeded in clarifying the correlation between the respiratory activity and the cellular polarity in the ECM-embedded microculture system41 by using the human mammary epithelial cell series, namely, HMT-3522 s-1 and T4-2. The purpose of the present research is to discuss whether it is possible to quantitatively collect mRNA at a single-cell level by using the fast electrical cell lysis method. Real-time PCR was used to quantify the collected mRNA. A normal capillary suction method was also used. Here, we show that the fast electrical cell lysis is practically applicable for quantitative mRNA analysis of samples collected from a monolayer. The gene expression analysis for single spheroids is also reported in order to compare the variation in gene expression in different cell types and to semiquantitatively discuss the transcription levels. EXPERIMENTAL SECTION Chemicals and Materials. Glass capillary (1.5-mm o.d., 0.86mm i.d.; Harvard Apparatus), type 1 collagen (Research Institute for the Functional Peptide, Yamagata, Japan), Matrigel (BD Biosciences, San Jose, CA), 3,3,4,4,5,5,6,6,6-nonafluorohexyltrichlorosilane (Shin-etsu Chem. Ind. Co., Ltd.), RNeasy Micro Kit (Qiagen), QuantiTect reverse transcription kit (Qiagen), LightCycler FastStart DNA Master (Roche), and all other chemicals were used as received. Microcapillary Probe Fabrication. Fabrication of Microcapillary Probe for Cell Suction (Probe 1). A glass capillary tube (1.5-mm o.d., 0.86-mm i.d.; Harvard Apparatus) was pulled with a capillary puller (model PN-3; Narishige) and cut with precision scissors to form a microcapillary tip with an outer diameter of 3-10 µm for single-cell collection and of 30-100 µm for single-spheroid collection. The surface of the microcapillary probe was modified with fluorosilane LS-912 (3,3,4,4,5,5,6,6,6nonafluorohexyltrichlorosilane; Shin-etsu Chem. Ind. Co., Ltd.) (30) Scott, D. I.; Ramanathan, S.; Shi, W. P.; Rosen, B. P.; Daunert, S. Anal. Chem. 1997, 69, 16-20. (31) Biran, I.; Klimentiy, L.; Hengge-Aronis, R.; Ron, E. Z.; Rishpon, J. Microbiology 1999, 145, 2129-2133. (32) Nagamine, K.; Onodera, S.; Kurihara, A.; Yasukawa, T.; Shiku, H.; Asano, R.; Kumagai, I.; Matsue, T. Biotechnol. Bioeng. 2007, 96, 1008-1013. (33) Nagamine, K.; Onodera, S.; Torisawa, Y.; Yasukawa, T.; Shiku, H.; Matsue, T. Anal. Chem. 2005, 77, 4278-4281. (34) Kaya, T.; Nagamine, K.; Matsui, N.; Yasukawa, T.; Shiku, H.; Matsue, T. Chem. Comm. 2004, 248-249. (35) Kelso, E.; McLean, J. M.; Cardosi, M. F. Electroanalysis 2000, 12, 490494. (36) Torisawa, Y.; Ohara, N.; Nagamine, K.; Kasai, S.; Yasukawa, T.; Shiku, H.; Matsue. T. Anal. Chem. 2006, 78, 7625-7631. (37) Weaver, V. M.; Petersen, O. W.; Wang, F.; Larabell, C. A.; Briand, P.; Damsky, C.; Bissell, M. J. J. Cell Biol. 1997, 137, 231-245. (38) Wang, F.; Weaver, V. M.; Petersen, O. W.; Larabell, C. A.; Dedhar, S.; Briand, P.; Lupu, R.; Bissell, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1482114826. (39) Liu, H.; Radisky, D. C.; Wang, F.; Bissell, M. J. J. Cell Biol. 2004, 164, 603-612. (40) Park, C. C.; Zhang, H.; Pallavicini, M.; Gray, J. W.; Baehner, F.; Park, C. J.; Bissell, M. J. Cancer Res. 2006, 66, 1526-1535. (41) Torisawa, Y.; Nashimoto, Y.; Yasukawa, T.;. Shiku, H.; Matsue, T. Biotechnol. Bioeng. 2007, 97, 615-621.

by gas-phase silanization under a nitrogen atmosphere for 2 h at room temperature. The fluorosilane coating process was essential to prevent the adsorption of cytosol and nucleic acids on the inner/ outer glass surface of the probe. Before use, the microcapillary probe was washed with 99.5% ethanol, dried, and irradiated with UV in a clean bench (Oriental Giken Inc.) for more than 30 min. Probe Fabrication for Fast Electrical Cell Lysis (Probe 2). A glass capillary tube (1.5-mm o.d., 0.86-mm i.d.; Harvard Apparatus) was pulled with a capillary puller (model PN-3; Narishige). The pulled capillary was then coated with Pt/Ti by a sputtering evaporator (Anelva Corp.; L-232S-FH, RF200, 280-nm thickness) and subsequently insulated with 900-nm-thick parylene C (Daisan Kasei Ltd.) by an evaporator (PD2110; Parylene Japan K.K., Specialty Coating Systems).42 The top of the capillary probe was cut out to form a Pt-ring electrode with an outer diameter of 5-10 µm for single-cell collection and of 50-100 µm for singlespheroid collection. The inner diameters of the capillary probes resulted in 4-8 µm and 40-80 µm, respectively. The electrode was connected with a copper wire (3-mm o. d.) using Ag paste (Sanyo Vacuum Industries). The processes preceding use, including fluorosilane coating, washing, and UV irradiation, were the same as mentioned above. The scanning electron microscopy (SEM) images of the microring electrode probe for the fast electrical cell lysis are shown in Supporting Information Figure S-1. Preparation of Cells. Monolayer Culture. The human breast cancer cell line (MCF-7) was donated by the Cell Resource Center for Biomedical Research (Tohoku University). The nonmalignant human mammary epithelial cell line HMT-3522-s-1 (s-1) and its malignant form (T4-2) were obtained from American Type Culture Collection. An RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 50 µg‚mL-1 penicillin (Gibco), and 50 µg‚mL-1 streptomycin (Gibco) was used for MCF-7 culture. A DMEM-F12 medium (Gibco) containing 250 ng‚mL-1 insulin (Boehringer Mannheim), 10 µg‚mL-1 transferrin (Sigma), 2.6 ng‚mL-1 sodium selenite (Collaborative Research), 0.1 nM estradiol (Sigma), 1.4 µM hydrocortisone (Collaborative Research), and 5 µg‚mL-1 prolactin (Sigma) was used for culturing s-1 and T4-2 cells. The s-1 cells were propagated in the presence of 10 ng‚mL-1 epidermal growth factor (EGF; Collaborative Research), and the T4-2 cells were propagated in the absence of EGF.37,41 The bottom of a 35-mm polystyrene culture dish (BD Biosciences) was coated with type 1 collagen solution (Research Institute for the Functional Peptide) by incubation for more than 30 min. The cells were seeded onto a collagen-coated dish as a single-cell suspension. Three-Dimensional Culture on Reconstructed Basement Membrane (On-Top Culture).43 The bottom of a 35-mm polystyrene culture dish was initially coated with 30 µL of Matrigel (BD Biosciences). The final surface concentration of the protein was adjusted to ∼300 µg‚cm-2. The Matrigel coated on the culture dish was solidified at 37 °C for 10 min. Single cells were seeded on the Matrigel layer with a cell suspension containing (1-5) × 104 cells/mL that was made up to 50 µL in a medium containing 3% Matrigel. The single cells were adherent onto the Matrigel

layer after 30-60-min incubation at 37 °C. The culture dish was further filled with the medium containing 3% Matrigel. Cells proliferate and form clusters after 3-6 days in three-dimensional culture and subsequently form spheroids (MCF-7, T4-2 after 3 days) or spheroids with acini-like structures (s-1 after 10 days). T4-2 cells were cultured in the absence or presence of 80 nM tyrphostin AG 1478, an inhibitor against EGFR that formed spheroids with an acini-like polarized structure (T4-2 REV) during 3-10 days of culture.38,41 For the samples in the fast electrical cell lysis, the monolayer culture and the on-top culture were performed on the indium-tin oxide (ITO) electrode (30 mm × 30 mm; Corning Japan Co.) instead of in a 35-mm polystyrene culture dish. The ITO electrode was electrically connected with a copper wire (3-mm o.d.) using Ag paste (Sanyo Vacuum Industries). Cell Extraction and Gene Expression Analysis. Figure 1 shows the schematic views of mRNA collection from a single adherent cell using two types of microcapillary probes. In the first method, a microcapillary probe44,45 (probe 1) was used to pick single cells from a monolayer cultured in medium (RPMI-1640 or DMEM-F12) under microscopic observation with Olympus IX 71. The position of the probe was controlled using an oil-derived three-axis manual micromanipulator (MNO-203; Narishige). In the second method, a microcapillary probe with a Pt-ring electrode (probe 2) was used. The probe was positioned 20 µm above the targeted single cell cultured on the ITO electrode. The probe and ITO electrodes were connected to the output terminals of ECM 2001 Electro Cell Manipulator (BTX-Harvard Apparatus). An electric pulse applied for a single adherent cell was set at +40 V (versus ITO electrode) for a 10-µs period. The conditions for the fast electrical cell lysis were practically similar to those optimized by Allbritton et al.18 although in our case, the period of the pulse was less than 1/10th of that reported by their group. The medium was exchanged for 0.2 M sucrose for the electrical cell lysis in our experiment.

(42) Takahashi, Y.; Hirano, Y.; Yasukawa, T.; Shiku, H.; Yamada, H.; Matsue, T. Langmuir 2006, 22, 10299-10306. (43) Debnath, J.; Muthuswamy, S. K.; Brugge, J. S. Methods 2003, 30, 256268.

(44) Chiang, L. W. J. Chromatogr., A 1998, 806, 209-218. (45) Yamamura, S.; Kishi, H.; Tokimitsu, Y.; Kondo, S.; Honda, R.; Ramachandra, Rao, S.; Omori, M.; Tamiya, E.; Muraguchi, A. Anal. Chem. 2005, 77, 80508056.

Figure 1. Schematic views of mRNA collection from a single adherent cell using a microcapillary probe. (a) Capillary suction: A glass microcapillary was positioned above the cells, and a single cell and its surrounding medium was aspirated using a manual microinjector. (b) Electrolysis: A microring electrode probe was positioned above the cells cultured on an ITO electrode. Then, an electric pulse was applied between the two electrodes. Immediately after the electrical cell lysis, the surrounding solution (0.2 M sucrose) was aspirated.

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Figure 2. Sequential photographs of the collection of a single adherent cell by capillary suction (a) and electrolysis (b). A capillary probe was positioned above a single cell (a-1) negatively pressurized with a microinjector (a-2, a-3). Before (b-1) and 1 s after (b-2) applying an electric pulse of magnitude 40 V in a 10-µs period between the probe and the ITO electrodes. The targeted single cell was electrically lysed and aspirated by the electrode probe (b-3, 2 s after applying the electric pulse).

The collected solution with a volume of less than 1 µL containing a single cell or the lysed cell of was transferred into a 0.2-mL PCR tube. A 75-µL lysis buffer (Lysis Buffer RLT from RNeasy Micro Kit Qiagen) solution was further added into the PCR tube and vortexed. The RLT buffer functions as deactivation of RNase and stabilization of RNA. A 5-µL carrier RNA (Qiagen) was added in order to prevent the loss of the objective mRNA. Total RNA purification was performed according to the protocols for RNeasy Micro Kit (Qiagen). The RT reaction was carried out to synthesize first strand cDNA according to the protocols for the QuantiTect reverse transcription kit (Qiagen) at 42 °C for 30 min (RT reaction) followed by 95 °C for 3 min (deactivation of RTase). The synthesized cDNA sample solutions with a final volume of 20 µL were stored at -80 °C. Real-time PCR was performed using the LightCycler 1.5 System (Roche) according to the LightCycler Fast Start DNA Master kit (Roche) with a total volume of 20 µL in glass capillaries. A 2-µL cDNA sample, 1.6 µL of 25 mM MgCl2, 1 µL of 10 µM forward (Fw) primer, 1 µL 10 µM reverse (Rv) primer, 2 µL of SYBR Green, and 12.4 µL of H2O were added. After the initial denaturation at 95 °C for 10 min, 45 PCR cycles were performed with denaturation at 95 °C for 10 s, annealing at the annealing temperature of the individual primer pair as mentioned below for 10 s, and extension at 72 °C for 9 s. Primers for glyceraldehyde3-phosphate dehydrogenase (GAPDH; GenBank Access No. M33197), β1-integrin (GenBank Access No. NM_133376), epidermal growth factor receptor (EGFR; GenBank Access No. NM_005228), and phosphatidylinositol 3-kinase (PI3K; GenBank Access No. NM_181504) were designed and synthesized by Nihon Gene Research Laboratories Inc. The actual sequences, apricon sizes, and annealing temperatures of the primers were listed as follows. GAPDH (Fw) 5′-TGAACGGGAAGCTCACTGG-3′, (Rv) 5′TCCACCACCCTGTTGCTGTA-3′, 307 bp, 62 °C; β1-integrin (Fw) 5′-GTC CAA CCT GAT CCT GTG TC-3′, (Rv) 5′-GCA ACC ACA CCA GCT ACA AT-3′, 167 bp, 66 °C; EGFR (Fw) 5′-ATG AGA TGG AGG AAG ACG-3′, (Rv) 5′-GTA GCA TTT ATG GAG AGT GAG-3′, 115 bp, 60 °C; PI3K (Fw) 5′-ACT CCC TCA ATG TCA CAC TA-3′, (Rv) 5′-AGC TCA ATT CAC AGA TCA GA-3′, 148 bp, 61 °C. The expression level of each targeted gene was normalized 6826

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to the expression level of the housekeeping gene GAPDH. Realtime PCR was performed for at least two genes, including GAPDH, using the 2-µL cDNA samples obtained from the source solution (20 µL). The copy number of GAPDH was estimated using a standard plasmid, including the human GAPDH sequence designed and synthesized by Nihon Gene Research Laboratories Inc. We also performed bulk measurement using 104-106 cells as the initial sample. In this case, the RNeasy Mini Kit (Qiagen) was used for RNA purification. For the monolayer cultured cell sample, the medium (RPMI-1640 or DMEM-F12) of the culture dish was exchanged for the RLT lysis buffer (Qiagen). RESULTS AND DISCUSSION Single-Cell Extraction and Real-Time Reverse Transcription PCR. The aim of this research is to quantify the mRNA expression level in a single adherent cell isolated using two types of capillary probessa normal glass capillary (probe 1) and a Ptring electrode probe (probe 2). Probe 2 was used to evaluate whether fast electrical cell lysis was applicable for mRNA analysis. We first performed single-cell manipulation in the MCF-7 monolayer system. Figure 2 shows sequential photographs of the collection of a single adherent MCF-7 cell by the capillary suction method (a) and the electrical cell lysis method (b). In the capillary suction method, a capillary probe was positioned above a single cell (a-1) and a negative pressure was applied with a microinjector (a-2, a-3) to gather the cell and the surrounding medium measuring less than 1 µL. For the electrical cell lysis method, a capillary electrode probe (probe 2) was microfabricated by Pt-sputtering evaporation and parylene C coating42 as an insulating layer. The MCF-7 cell monolayer was formed on an ITO electrode coated with type I collagen. Before the electrical cell lysis, the medium was exchanged for an isotonic 0.2 M sucrose solution. The capillary electrode probe was set 20 µm away from the ITO substrate by using an oil pressure-driven XYZ manipulator. When an electric pulse of ∼40 V was applied for 10 µs, the targeted single cell was punctured within several seconds (Figure 2b-2; also see movies in Supporting Information). Immediately after cell puncture, the cytosolic solution was aspirated in a manner similar

Figure 3. Copy number of GAPDH mRNA as a function of the cell number collected by capillary suction (left) and electrolysis (right). Filled diamonds indicate the individual results obtained with real-time PCR. Open circles indicate the mean values for the corresponding cell numbers.

to that followed in the capillary suction method. The collected cell or cytosol solution was poured into a PCR tube, and the mRNA was analyzed according to a standard RT-PCR protocol for a single cell. A fluorescence signal originating from the single cell was obtained during the real-time PCR. The shape and peak in the melting curve were well correlated with the signal obtained for the bulk of cellular samples with 105-106 cells (Figure S-2, Supporting Information). The culture medium without any cells was assigned as a negative control, and no noticeable peak was observed. These findings clearly indicate the specific amplification of the GAPDH mRNA. Next, we evaluated the relation between the number of cells collected using the probes and the copy number of GAPDH mRNA estimated by real-time PCR analysis. The simple capillary suction and electrical cell lysis methods were used again. The GAPDH mRNA copy number was calculated from the calibration line obtained by using the standard GAPDH sample, i.e., a plasmid containing human GAPDH sequence. Figure 3 shows the copy number of GAPDH mRNA as a function of the number of cells collected by capillary suction (left) and electrical cell lysis (right). Although the data points were scattered, the slope of the leastsquares analysis for the mean value (open circles) indicated 2576 GAPDH mRNA copies/cell with a coefficient of determination (R2) of 0.940. The R2 value for all the data points (filled diamonds) was 0.441, indicating that the GAPDH expression in a single cell depended largely on the individual cellular status. A similar phenomenon was reported by Quake and co-workers8 for dispersed individual cells by using a multichannel microfluidic device. The average copy number in a single cell is also in good agreement with the value reported by Quake et al. The process of fluorosilanization onto the capillary probe surface is significantly effective. In the case of using the probe without silanization, the GAPDH mRNA copy number was found to be critically decreased; therefore, the mRNA collection efficiency was greatly lowered because of undesired adsorption of mRNA onto the capillary surface. Furthermore, a negative control, i.e., a solution without cells, often showed a broad increase in fluorescence during the real-time PCR operation and the following melting curve analysis suggesting the formation of primer dimers. For the electrical cell lysis shown in Figure 3 (right), the slope and R2 value for mean data were 2599 copies/cell and 0.906, respectively. The R2 value for all the data points was 0.742. There is no apparent difference in the collection efficiencies of the mRNA

between the capillary suction and electrical cell lysis methods. The fast electrical cell lysis method used in this study is basically the same as that reported by Allbritton et al.18 They utilized a longer pulse period in a nonisotonic solution and did not perform mRNA analysis. We have shown for the first time that the fast electrical cell lysis procedure is available for mRNA analysis at the single-cell level. There have been several other reports demonstrating mRNA analysis by using CE microsystems at the single-cell level. In those reports, RNA or DNA fragments were fluorescently detected with highly sensitive microscopes.15,16 Our research, on the contrary, has utilized real-time PCR to quantitatively assess the expression of the objective gene and has clarified the copy number of the GAPDH gene within a single cell. For the electrical cell lysis, the target cell does not have to be perfectly destroyed. The mRNA can be collected only if the cellular membrane is broken down to release cytosol from the cell. Therefore, we compared the two capillary suction methods, with and without an electric pulse. Capillary suction of the adherent single cell can be applied for the limited cell types and not for the cell types with much stronger adhesions. Fortunately, MCF7, HMT-3533 s-1, and T4-2 were valid and therefore appropriate as a model system to compare the two methods. Furthermore, the capillary suction method takes a longer time to remove an adherent cell than the electrical cell lysis. We added several movie files in Supporting Information to clearly show the individual adherent cell are certainly collected. From the movies of the electrical cell lysis, the diffusion of the cytosol after the electric pulse seems to be conspicuous. Nevertheless, the results estimating the copy number of the GAPDH mRNA suggest that the loss of the mRNA by electrical cell lysis is not evident. The volume of the collected sample, ∼1 µL, is significantly larger than the volume (∼2 pL) of the cell and the surroundings estimated from the diffusion distance. Application to β1-Integrin Expression Analysis. GAPDH is a housekeeping gene expressed constitutively under any condition and often selected as a model mRNA analyte for singlecell analysis. In this section, we focus on the gene expression of β1-integrin because integrin triggers many signal transduction pathways and relays information across the ECM and the intracellular interfaces. For instance, malignant transformation, cellular polarity, proliferation, and apoptosis are all controlled by the cell adhesion molecules, including integrins. Here, we collected β1-integrin mRNA from several types of cells, such as MCFAnalytical Chemistry, Vol. 79, No. 17, September 1, 2007

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Figure 4. Relative expression levels of the β1-integrin gene normal to GAPDH for a single adherent cell (MCF-7 (gray), T4-2 (filled), s-1 (open)) cultured in a monolayer. Messenger RNAs were collected from bulk (105-106 cells, left), capillary suction (middle), or electrolysis (right).

7, T4-2, and s-1 cells. The individual cells of these cell types cultured as a monolayer were collected by either the capillary suction (probe 1) or electrical cell lysis (probe 2) method. The expression levels of the β1-integrin in the MCF-7, T4-2, and s-1 cells collected using probe 1 were characterized normal to that of GAPDH. Figure 4 shows the relative expression level of β1-integrin from single MCF-7, T4-2, and s-1 cells collected by simple capillary suction and electrical cell lysis. As a control, mRNA analysis was also performed using the bulk of each cell type (105-106 cells) cultured within the monolayer in a culture dish. The result obtained from the bulk cellular sample clearly indicates that the MCF-7 cell expresses β1-integrin at significantly low levels, as compared to the s-1 or T4-2 cells. In the single-cell mRNA analysis using capillary suction and electrical cell lysis, the expression levels of β1-integrin for the MCF-7 cell were found to be 1/8th to 1/12th of those for the T4-2 and s-1 cells, reinforcing that there was no apparent difference between the collection methods. These results were quantitatively in good agreement with those obtained by bulk mRNA analysis. A similar tendency of very low β1-integrin expression for MCF-7 was observed in the literature at bulk cell levels.40 However, the gene expression spectrum would change drastically when the surrounding environmental conditions change. In fact, it is well-known that the gene expression behavior of an identical cell type can change during differentiation or malignant transformation. Therefore, tools to isolate an arbitrary single cell from a relatively complicated heterogeneous cellular body are useful for the detailed analysis of cellular functions. In the following section, our methods are applied to a three-dimensional culture system. Application to Analysis of Gene Expression in Spheroids. The three-dimensional cell culture is expected to simulate the in vivo-like microenvironmental conditions of mammarian cells in order to maintain their original characteristics and gene expression profiles. Many reports have demonstrated that the cellular microenvironment of a three-dimensional culture is similar to the in vivo cellular microenvironment and is significantly different from that of a conventional monolayer culture. This cellular nature originates from the cell-cell and cell-ECM interactions; there6828 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

fore, the conventional pretreatment to prepare a single-cell suspension is not suitable for the analysis of gene expression profiles of the three-dimensionally cultured cells. Here, we demonstrate the collection of mRNA from a single MCF-7 spheroid formed in a three-dimensional culture by using electrical cell lysis. The spheroid was formed according to the on-top culture method (See Experimental Section) as a model system to isolate small cellular clusters from tissues. The electric pulse for spheroid lysis generally requires a higher magnitude of voltage than that required for the single-cell lysis shown above. In fact, an electric pulse of 40 V for a 10-µs period was insufficient to break a spheroid with a diameter of 100 µm. An electric pulse of 100 V for a 10-µs period successfully destroyed a spheroid of the same size. Figure 5 shows a sequential microscopic view of mRNA collection from a single MCF-7 spheroid by the electrical cell lysis method with an electric pulse of 100-300 V and a 10-µs period. This pulse magnitude is larger than that used for the electrical single-cell lysis shown above because the distance between the probe and the ITO electrodes is larger (∼100 µm), and the electric field becomes broader inside the spheroid space measuring a 50-µm radius. It required 1-2 s for the cells to be destroyed on application of the 10-µs period electric pulse (Figure 5b). The copy number of the GAPDH mRNA collected from the single MCF-7 spheroid has been estimated as 2.1 × 105. This is practically well correlated with the copy number (3.1 × 105) calculated on the basis of the average GAPDH mRNA copy number per cell (2500 copies) and the number of cells existing in the spheroid (125 cells), which is estimated from the volume (size) of the spheroid.27,28 Furthermore, the expression of β1integrin was evaluated for a single MCF-7 spheroid by the collection methods. The relative expression levels of β1-integrin in a single MCF-7 spheroid collected by capillary suction or electrical cell lysis method were listed in Table 1, together with the results obtained by bulk measurements ( ∼8000 spheroids or 105-106 cells). There is no difference in the β1-integrin expression levels between the two methods, suggesting that the fast electrical cell lysis method is also suitable to collect mRNA in a three-dimensional culture system. Application to the Evaluation of Cellular Polarity. In this section, on-top culture was applied to the human mammary epithelial HMT-3522 cell series, namely, s-1 and T4-2. HMT-3522 is a model system to reversibly control polarity in vitro. Bissell’s group found that laminin is essential for regulating the expression levels and activity of EGFR and β1-integrin in cultured mammary epithelial cells.37-40 When cultured in Matrigel, the nonmalignant s-1 cells arrest the growth and form polarized mammoplasty-like structures (called acini). In contrast, the malignant T4-2 cells continue to proliferate and form apolar structures similar to the structures formed by primary tumor cells. Besides, T4-2 cells treated with tyrphostin AG1478, an inhibitor of EGFR, formed a polarized structure (T4-2 REV) because their proliferation activity was restricted (See photographs in Supporting Information Figure S-6).38,41 Our previous study showed that the respiratory activity per cell also changed depending on the cellular polarity. However, no information on cellular functions at the transcriptional level

Figure 5. (a-c) Sequential microscopic view of the mRNA collection from a single MCF-7 spheroid by electrolysis. Before (a) and 2 s after (b) applying voltage pulse (100 V, 10 µs), and (c) after suction. Table 1. Relative Expression Levels of β1-Integrin, EGFR, and PI3K Normal to GAPDH in a Single Spheroid of MCF-7, T4-2, T4-2 REV, and s-1 Collected with Capillary Suction or Electrolysis, Together with the Results Obtained by Bulk Measurements β1-integrin

EGFR

PI3K

MCF-7 T4-2 T4-2REV s-1

0.0445 ((0.0078) 1.0434 ((0.1117) 1.7795 ((0.1793) 0.5398 ((0.0815)

Bulk not measured 2.4415 ((0.6450) 3.0756 ((0.3455) 0.8016 ((0.0607)

not measured 5.9565 ((2.2769) 12.8744 ((1.6917) 5.7611 ((0.4786)

MCF-7 T4-2 T4-2 REV s-1

Capillary Suction 0.0990 ((0.0462) not measured 0.5686 ((0.2212) 0.8348 ((0.2718) 2.1100 ((0.8674) 0.9255 ((0.0508) 0.3799 ((0.2281) 0.5369 ((0.0517)

not measured 1.5159 ((1.2168) 9.6372 ((4.6193) 1.7496 ((1.5654)

MCF-7 T4-2 T4-2 REV s-1

0.0678 ((0.0089) 0.4808 ((0.1278) 1.1822 ((0.7369) 0.5397 ((0.1996)

Electrolysis not measured 0.9255 ((0.5296) 1.2032 ((0.7768) 0.3735 ((0.4677)

not measured 2.5950 ((1.2971) 5.9746 ((0.7374) 1.2284 ((1.7356)

was obtained. We selected tyrosine kinase-related genes, namely, EGFR and PI3K, in addition to GAPDH and β1-integrin in order to characterize these cells. EGFR is a transmembrane tyrosine kinase required for normal mammary development and location and is recognized as a potential target for cancer therapy. PI3K is a key mediator that regulates cell orientation and is activated by the growth factor-responsive tyrosine kinases, including EGFR.39 The relative gene expressions of β1-integrin, EGFR, and PI3K normal to GAPDH in a single spheroid (T4-2, T4-2 Rev, s-1) after an on-top culture for 6-10 days collected by capillary suction and electrolysis together with the gene expression in bulk spheroids were listed in Table 1. The three gene expression profiles in bulk spheroids changed depending on the cell types. The expression of β1-integrin and EGFR in T4-2 cells was higher than that in s-1 cells even though the two cell types originated from the identical cell line, i.e., HTM-3522. In T4-2REV cells, the three genes were significantly expressed when compared to T4-2 cells. The general tendency of the gene expression profiles did not largely depend on the mRNA collection methods (Table 1, capillary suction and electrical cell lysis); the order of the gene expression level was T4-2REV > T4-2 > s-1 for β1-integrin and EGFR and T4-2REV . T4-2 ∼ s-1 for PI3K. However, the exact levels of gene expression depend on the experimental conditions. The EGFR expression levels in T4-2REV cells collected by capillary suction appeared to be lower. The values of the gene expression levels in cells obtained by capillary suction and electrical cell lysis tended to be lower compared to those obtained from the bulk measurement. This might be due to the reliability of the collection method or different expression levels for individual spheroids. For the

mRNA collection from single spheroids, the size of the sample, the size of the probe tip, and the probe-sample distance were larger than for the monolayer system. The diffusion of the cytosol may affect the mRNA collection efficiency critically for the spheroids rather than for the monolayer of adherent cells. However, we evaluated the copy number of GAPDH mRNA from single spheroid indicating that there is no evidence of the serious loss of the GAPDH mRNA during the whole analysis procedures. As a final possibility, the β1-integrin mRNAs are reduced due to the localization at the surface of the spheroid where the electric field is highest.46,47 In the present stage, we conclude that both capillary suction and electrical cell lysis allow semiquantitative comparison in the three-dimensional culture system although further improvement is necessary for precise quantitative discussions. Moreover, the Pt-ring electrode probe can be used to measure the local oxygen concentration in order to estimate the respiratory activity of the single spheroid based on scanning electrochemical microscopy27,28,41 before collecting mRNA from the same spheroid as shown in Supporting Information Figure S-7. We have demonstrated the mRNA analysis of single spheroids where the respiration activity was estimated based on SECM technique. However, systematic comparison of the gene expression profiles with the respiration activity has not been performed yet. There are still several problems to do the critical comparison. For example, the desirable Pt-ring electrode radius for the estimation of respiration activity with SECM may be less than 10 µm whereas the probe is too small to collect mRNAs from spheroids with 50200-µm radius. CONCLUSION Messenger RNA collected from single adherent cells was analyzed based on real-time RT-PCR using two types of microcapillary probessa normal capillary and a Pt-ring capillary. In both methods, the fluorosilane coating was essential in order to prevent the adsorption of cytosol and nucleic acids on the inner/outer glass surface of the probe. We have first shown that the fast electrical cell lysis method is practically applicable for a quantitative mRNA analysis of single adherent cells. The quantitative comparison of gene expression was possible for not only GAPDH but also β1-integrin mRNAs from single cells cultured in a monolayer. The expression of β1-integrin in MCF-7 cells was 1/8th-1/12th of that in T4-2 and s-1 cells, regardless of the RNA collection methods. In the three-dimensional culture system, the mRNAs of EGFR and PI3K were collected in addition to those of (46) Gowrishankar, T. R.; Weaver, J. C. Proc. Natl. Acd. Sci. U.S.A. 2003, 100, 3203-3208. (47) Agarwal, A.; Zundans, I.; Weber, E. A.; Olofsson, J.; Orwar, O.; Weber, S. Anal. Chem. 2007, 79, 3589-3596.

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GAPDH and β1-integrin from single spheroids formed by the ontop culture method. The electric pulse required for mRNA collection from spheroids was found to be slightly stronger than that required for that from a monolayer culture system. Further, the gene expression analysis of single spheroids allows comparison of the gene expression profiles of different cell types. ACKNOWLEDGMENT This work was partly supported by a Grant-in-Aid for Scientific Research on Priority Areas (445) “Lifesurveyer” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; by Grants-in-Aid for Scientific Research (18101006 and 19750055) from MEXT; and by a Program Research Grant from the Center for Interdisciplinary Research, Tohoku University.

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SUPPORTING INFORMATION AVAILABLE SEM image of the Pt-ring electrode probe, time courses of realtime PCR operation and melting curves for different RNA collection methods, microscopic views of spheroids, and oxygen reduction current profile near the single MCF-7 spheroids measured using the Pt-ring electrode probe. Movie files for the collection of monolayered MCF-7 cells using capillary suction and electrical cell lysis. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 22, 2007. Accepted June 28, 2007. AC071050Q