Single Nuclei Raman Spectroscopy for Drug Evaluation - Analytical

Nov 4, 2011 - Detection of cellular changes at single-cell level has a great potential for biomedical and biopharmaceutical applications. Raman spectr...
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Single Nuclei Raman Spectroscopy for Drug Evaluation Hsin-Hung Lin,†,‡,§ Yen-Chang Li,† Chih-Hao Chang,†,^ Chun Liu,† Alice L. Yu,*,† and Chung-Hsuan Chen*,† †

Genomics Research Center, Academia Sinica, Taipei, Taiwan Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan § Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan ^ Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan ‡

ABSTRACT: Detection of cellular changes at single-cell level has a great potential for biomedical and biopharmaceutical applications. Raman spectroscopy is an important tool for single-cell molecular imaging analysis. Raman spectroscopy can provide time-resolved information of the selected biomolecular distributions inside a single cell without the need of chemical labeling. In this study, we monitored the cellular responses to antineoplastic drug at a single cell basis with Raman spectroscopy. We demonstrated that single nuclei Raman spectroscopy has the ability to detect and identify nuclear changes related to cytotoxicity at lower concentrations and in shorter time span than conventional cell based assays. Thus, this strategy of using Raman spectroscopy of single, isolated nuclei may be very valuable for rapid and sensitive detection of cellular changes in response to chemotherapeutic agents.

biological molecules, such as flavins, porphyrins, and structural proteins, contribute to fluorescence. In contrast, nearly all biological molecules are Raman active with fingerprint spectral characteristics. Therefore, Raman spectroscopy can overcome some of the limitations of fluorescence spectroscopy for disease diagnosis or detection of cytotoxic responses. Raman microspectrometry has been used to detect apoptotic events occurring in the cytoplasm and nuclei at the single cell level.10 12 At the initial stage of our study, we attempted to capture early cytotoxic events in response to doxorubicin at single cell level by Raman spectrometry. However, no significant changes in the DNA and protein associated Raman spectra of the whole cell treated with doxorubicin were observed until 48 h (data not shown). Thus Raman spectrometry of whole cell at single cell level did not offer any advantage over the conventional cytotoxicity assays. This was consistent with the previous report that decreases in the DNA and protein associated Raman peaks of the whole cell undergoing apoptosis was not evident until 48 h.10 In addition to Raman spectrometric analysis at the single cell level, changes in cellular processes were detected by Raman spectrometry, based on algorithm analyses of the entire cell population, such as principal components analysis (PCA), artificial neural network, and hierarchical cluster analysis (HCA).8,12 17 However, these analyses can be cumbersome and time-consuming. Besides, they can not decipher changes in the individual cells nor detect biochemical alterations early in the

arious optical spectroscopy techniques, such as fluorescence, Raman scattering, and infrared absorption spectroscopy, are, to certain extent, sensitive to biochemical composition and have been shown to be useful in discriminating various forms of malignant tissues.1,2 Raman spectroscopy is a laser-based spectroscopic analysis of molecular vibrations and molecule-specific light scattering. Thus, it allows nondestructive composition analysis, yielding a molecular fingerprinting of biological tissues or cells under investigation.3 7 To facilitate biomedical application of Raman spectrum, many studies have focused on the identification of Raman active compounds and biological materials (i.e., toxins, viruses, or intact bacterial cells/spores).6,7 In recent years, Raman spectroscopy has been used to interrogate single, nonaffixed cell to yield a biomolecular fingerprint of the cell, or its subcellular components of interest without altering its biology. A few studies used Raman spectroscopy to detect cytotoxic changes in response to adverse effects of chemicals and physical agents on the cells, causing specific time-dependent biochemical changes associated with the process of cell death.8,9 Alterations in cellular processes were reported to be associated with changes in Raman spectrum, based on statistical analysis of the entire cell population. However, such cellular alterations were not reflected by Raman spectrum at the single cell level. When coupled with confocal microscopy, Raman spectroscopy can be applied to imaging biomolecule distribution in an individual cell. Furthermore, combined with laser tweezers,6 several individual cells can be analyzed sequentially. Raman spectroscopy can probe materials without fluorescence since Raman scattering arises from perturbations of the molecules that induces vibrational or rotational transitions. Only a very limited number of

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r 2011 American Chemical Society

Received: July 24, 2011 Accepted: November 4, 2011 Published: November 04, 2011 113

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course of apoptosis.8,12 According to Pijanka et al, the spectra of single nuclei exhibit spectral signatures different from those of the whole cell.14 Since initial apoptotic changes occur in the nucleus, we reasoned that study of the isolated nuclei may minimize the interfering signals from cytoplasm and thereby prevent the Raman spectra of the nuclei from being hidden in the whole cell signature. Therefore, we adopted the strategy of single nuclei Raman spectroscopy to detect time-resolved nuclear changes in response to chemotherapeutic agents, doxorubicin and vinblastine. The results demonstrated that this method offers a more sensitive and faster detection of cytotoxicity than conventional cytotoxicity assays.

Raman Spectroscopy and Data Treatment. Raman spectra were acquired by a commercial micro-Raman spectrometer (LabRam-INV, HOBIRA JOBIN YVON, France) equipped with an 514 nm laser source (Innova 90C, Coherent Inc., U.S.A.) to excite the Raman scattering. The power applied to the sample was approximately 10 mW, to prevent sample from photon-heating damage. A 100 oil-immersed objective (UPlanSApo, NA: 1.4, Olympus), which produced a small laser spot size (∼1 μm) was used to collect Raman signals and focus laser on the individual cell nucleus. Other experimental parameters included the following: grating adjusted to 1800 g/mm, confocal hole set to 300 μm, at least 20 accumulations of 5 s at different positions on each nucleus, and the spectra were recorded in the 500 2000 cm 1. All experimental conditions were kept constant for all measurements. For data capture and processing, a certain area within the sample is first chosen for the laser focus to interrogate different regions of the sample and divided into small squares, which can be considered Raman pixels. Raman spectroscopy is performed on each of these defined Raman pixels, and following background subtraction and normalization of the data, each pixel is assigned an intensity value based on the strength of a particular Raman peak of interest. By repeating this procedure for different Raman bands, Raman spectra showing the spatial distribution of biomolecules, such as DNA, proteins, and lipids can be constructed. Therefore, the absolute value after construction of Raman spectra is obtained without the baseline. Raman data acquisition and preprocessing of preliminary data such as baseline subtraction, smoothing, and spectrum analysis were carried out by Labspac5.0 software. All nucleus samples were kept in PBS during data acquisition. Over 100 nuclei of cells from each group were measured. All Raman intensity was corrected by subtracting the spectra of the cells treated with the drug by the spectra of untreated cells. Cytotoxicity Assay. Cell viability was determined by a quantifying reduction of the fluorogenic indicator Alamar Blue according to the manufacturer’s instructions (AbD Serotec, Oxford, U.K.). Cells (5  103) were allowed to attach to 96-well tissue culture plates and then exposed to doxorubicin or vinblastine. At various time intervals, Alamar Blue was added to the medium and 3 4 h later fluorescence was determined in a Spectramax M2 plate reader (Molecular Devices, Sunnyvale, CA). It has previously been shown that oxidized Alamar Blue is taken up by cells and is reduced by intracellular dehydrogenases, and the water-soluble product is excreted into the medium from which changes in fluorescence emission (590 nm) are utilized as an index of cytotoxicity. Cell Synchronization Thymidine Double Block. The cell cycle of HL60 was arrested at G1/S boundary by incubation with an inhibitor of DNA synthesis, thymidine (2.5 mM) for 25 h and allowed to enter S phase by removing the inhibitor. Eight hours later, the cells were treated by 2.5 mM thymidine again for another 24 h, rendering most cells synchronized at S phase. The cells were harvested, washed with normal medium, plated onto flasks, and incubated in the humidified 5% CO2 incubator at 37 C. Statistical Analysis. Histogram data were given as mean ( standard deviation (SD). Comparison between anticancer drug treated group and untreated group was performed by Student’s ttest. Analysis of the differences in time course study between drug treated group and control was performed by the use of mixed model. A P-value less than 0.05 was considered statistically significant. All data analyses were performed using SAS statistical software for Windows, version 9.1 (SAS institute, Cary, NC).

’ EXPERIMENTAL METHODS Reagents. Doxorubicin hydrochloride was obtained from Parmacia & Upjohn (Milan, Italy). Vinblastine sulfate was obtained from Sigma-Aldrich (Steinheim, Germany). Cell Preparation. CCRF-CEM (Human T cell lymphoblastlike cell line) and HL60 (a human promyelocytic leukemia cell line) cell lines were obtained from American Type Culture Collection (Rockville, MD). Leukemia cell line HL60/ADR, which was derived from the parent HL60 cells by transfecting with the MRP-1 gene, a member of the ATP-binding cassette (ABC) family of drug transporters, was generously provided by Dr. Michael Kelner (University of California in San Diego, CA). HL60/ADR exhibited a multidrug resistance phenotype associated with MRP-1 overexpression.18,19 CEM/VLB were selected by growing CEM cells in the continuous presence of sublethal concentrations of vinblastine. Cells were cultured at 37 C in RPMI 1640 medium supplemented with 10% calf serum, 1% L-glutamine, and 1% standard penicillin/streptomycin (10.000 IU/ml) under a 5% CO2 atmosphere (all from Invitrogen, Carlsbad, CA). Treatment of Cells with Doxorubicin and Vinblastine. Working solutions of doxorubicin hydrochloride were made by diluting a 4 mM stock in dimethyl sulfoxide (DMSO; Sigma) with PBS. HL60 and HL60/ADR cells in flasks with complete media were treated with indicated concentration of the drug for designated time intervals at 37 C. Negative control samples were treated with the corresponding volume of DMSO/PBS; the final DMSO concentration was e0.25%. Working solutions of vinblastine were made by diluting a 1 mM stock in distilled water with PBS. CEM and CEM/VLB cells in complete media were treated with indicated concentration of the drug as described above. Preparation of Nuclei. Cell nuclei were isolated using the Nuclei EZ Prep Nuclei Isolation Kit (Sigma, U.K.); 2 106 cells were suspended in Dulbecco’s phosphate-buffered saline (PBS, Gibco BRL) and centrifuged at 500  g for 5 min at 4 C. The cell pellet was resuspended and incubated with Nuclei EZ lysis buffer for 5 min at 4 C and then centrifuged at 500  g for 5 min at 4 C. Supernatant containing cytoplasmic components was removed. Nuclei in the pellet were further purified by repeating this procedure twice more. The pellet containing the final isolated nuclei was resuspended in 200 μL of PBS at 4 C. Nuclei were counted using the trypan blue dye according to the instructions of the manufacturer of the Nuclei EZ Prep Nuclei Isolation Kit. Briefly, 10 μL of nucleicontaining solution were mixed with 10 μL of trypan blue. Using this methodology, nuclei stain blue with a uniform circular or sausageshaped appearance, whereas cytoplasmic contamination and cell debris stain light blue with an irregular morphology, and will be clearly distinguishable, if present (SIGMA, U.K.). Nuclei isolated in PBS were immediately used for Raman data acquisition. 114

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Figure 1. Effect of doxorubicin on Raman spectra of cell nuclei Spectra showing the DNA and amide regions corresponding to the mean of 10 spectra of 50 different HL60 cell nuclei treated with (red line) and without (black line) doxorubicin at 80 nM for 16 hr. The positions of Raman bands at 723, 782 788, 1004, 1095, 1255, 1339, 1375, 1422, 1449, 1489, 1575, and 1660 cm 1 were marked.

Table 1. Raman Bands Observed in the Spectra of Isolated Nuclei and Their Tentative Assignment Raman shift (cm 1)

tentative assignment

728

nucleic acid (adenine)

782 788

nucleic acid (cytosine, thymine); DNA backbone O P O stretching

1004

symmetric ring breathing mode of phenylalanine

1095

DNA backbone: O P O stretching

1257

nucleic acid (adenine, cytosine); amide III: β-sheet

1339

nucleic acid (adenine, guanine); protein: CH2 deformation

1375

nucleic acid (adenine, guanine, tyrosine)

1422

nucleic acid (adenine, guanine)

1449 1489

lipid, protein, DNA CH2 deformation nucleic acid (adenine, guanine)

1578

nucleic acid (adenine, guanine)

1660

amide I α-helix

Figure 2. Doxorubicin caused significant decrease in individual Raman bands of nuclei isolated from HL60 cells but not from doxorubicin resistant cells HL60/ADR. (A) The intensity of individual Raman bands obtained from nuclei of HL60 cells, treated with varying concentrations of doxorubici overnight and the nuclei were isolated and analyzed. Statistical significance (P) was calculated by Student’s t test. Error bars show s.e.m.; *, p < 0.01, and #, p < 0.001. (B) Relative intensities of Raman peaks of isolated cell nuclei of untreated HL60 and doxorubicinresistant cells HL60/ADR treated with doxorubicin at 80 nM. All Raman intensity were taken with 514 nm laser excitation and were corrected by subtracting the spectra of HL60 or HL60/ADR cells treated with doxorubicin by the spectra of untreated cells.

’ RESULTS

when the cells were treated with doxorubicin as compared to control cells. Peaks at 728, 782, 1095, 1375, 1422, 1489, and 1578 cm 1 are from nucleic acids.20 22 The peak at 1004 cm 1 represents symmetric ring breathing mode of phenylalanine.21,22 Peaks at 1257, 1339, and 1449 cm 1 can be attributed to DNA, amide, proteins, and lipids21,22 (Table 1). These findings are consistent with the expected biochemical changes in nuclei induced by doxorubicin. Thus, Raman spectrometry is a useful tool to distinguish cells with and without drug treatment. Relationship between Nuclear Changes and the Concentration of Cytotoxic Agents. To determine the sensitivity of Raman spectroscopy for detecting cytotoxic changes, HL60 cells was treated with varying concentrations of doxorubicin overnight and the nuclei were isolated and analyzed. HL60/ADR, a cell line

Monitoring of Nuclear Changes by Raman Spectroscopy. To induce cytotoxicity, a promyelocytic leukemia cell line HL60 was incubated with the antineoplastic drug, doxorubicin, at 80 nM for 24 h, and nuclei were isolated from the cells to minimize the interfering signals from cytoplasm. Raman spectra of each nucleus were obtained from an average of 10 spots on a selected nucleus and spectra of 100 nuclei were collected. Figure 1 shows Raman spectra obtained from the nuclei of the HL60 cancer cells treated with or without doxorubicin. Peak assignments at different wavenumbers are given in table 1, and most of them were from nucleic acids. Only a few peaks are from proteins and lipids. It is clear that all peaks in Figure 1 were significantly decreased 115

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Figure 3. In vitro cytotoxicity of doxorubicin as determined by 3Hthymidine incorporation and Alamar Blue assay. The HL60 and doxorubicin-resistant cells HL60/ADR were treated with doxorubicin overnight and the cytotoxicity was determined by 3H-thymidine incorporation (A) and Alamar blue assay (B). The IC50 of doxorubicin was 240 nM for HL60 and >1000 nM for HL60/ADR cells by 3H-thymidine incorporation assay. The cytotoxicity of doxorubicin on either HL60 or HL60/ADR as measured by Alarmar Blue assay was negligible.

Figure 4. Treatment with vinblastine causes significant decrease in individual Raman bands of nuclei isolated from CEM cells but not from resistant cells CEM/VBL. (A) The intensity of individual Raman bands obtained from nuclei of CEM cells treated with varying concentrations of vinblastine overnight and the nuclei were isolated and analyzed. Statistical significance (P) was calculated by Student’s ttest. Error bars show s.e.m.; *, p < 0.01, and #, p < 0.001. (B) Relative intensities of Raman peaks of isolated nuclei of untreated CEM and vinblastineresistant cells CEM/VBL treated with vinblastine at 10 nM. All Raman intensity were taken with 514 nm laser excitation and were corrected b subtracting the spectra of CEM or CEM/VBL cells treated with vinblastine by the spectra of untreated cells.

resistant to adriamycin and derived from HL60 by stepwise selection, was also treated for comparison. Figure 2 showed significant differences in Raman spectra of HL60 and drugresistant cells HL60/ADR exposed to doxorubicin. The intensities of individual Raman peaks decreased steadily with increasing drug concentration of doxorubicin from 0.8, 3.2, and 16 80 nM in HL60 but not HL60/ADR cells. For Raman peaks at 728, 782, 1095, 1339, 1422, 1489, and 1578 cm 1, respectively, the corresponding intensities of each Raman peak in the cells treated with 80 nM doxorubicin decreased to the following percentages of control cells: 55.6 ( 2.2% (p < 0.001), 75.2 ( 2.4% (p < 0.001), 72.0 ( 3.5% (p < 0.001), 81.2 ( 1.9% (p < 0.001), 68.5 ( 2.8% (p < 0.001), 71.2 ( 1.7% (p < 0.001), 76.2 ( 3.0% (p < 0.001), respectively (Figure 2A). A decrease in the intensities of individual peaks was detectable in HL-60 at concentration as low as 3.2 nM doxorubicin. In contrast, there was no significant change of Raman peak intensities in the nuclei of HL60/ADR even after treatment with 80 nM doxorubicin (96.4 ( 3.1% at 782 cm 1 of control, Figure 2B). On the other hand, conventional cytotoxicity assay including 3H-thymidine incorporation and Alamar Blue assay showed less sensitivity than Raman spectroscopy in detecting cytotoxic changes of cells (Figure 3A, B). Upon incubation

with 80 nM doxorubicin for 24 h, DNA synthesis as measured by 3 H-thymidine uptake was 74.2 ( 1.9% and 94 ( 3.8% of control in HL60 and HL60/ADR cells, respectively (Figure 3A). Alamar Blue assay that assessed mitochondrial function as an index of cell viability was even less sensitive, with the value of 102.0 ( 1.8% and 101.7 ( 2.5% of control at 80 nM and 85.8 ( 0.9% and 101.3 ( 2.0% at 2000nM in HL60 and HL60/ADR, respectively. To verify the sensitivity and reproducibility of single cell Raman spectroscopy, the cytotoxicity of an antimicrotubule agent, vinblastine, was examined in CCRF-CEM, a T lymphoblastoid cell line, and its drug-resistant derivative, CEM/VBL cell line. As shown in Figure 4A, the intensities of Raman peaks from CEM cells decreased dramatically with increasing concentrations of 116

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Figure 5. In vitro cytotoxicity of vinblastine as determined by 3Hthymidine incorporation and Alamar Blue assay. The CEM and vinblastine-resistant cells CEM/VBL were treated with vinblastine overnight and the cytotoxicity was determined by 3H-thymidine incorporation (A) and Alamar blue assay (B). The IC50 of vinblastine was 1.18 nM for CEM, and >10 nM for CEM/VBL cells by 3H-thymidine incorporation assay. The cytotoxicity of vinblastine on either CEM or CEM/VBL as measured by Alarmar Blue assay was negligible.

vinblastine, from 0.016, 0.08, 0.4, and 2, to 10 nM. In this case, the reduction in the Raman signals of the CEM cell nuclei spectra was readily discernible at vinblastine concentration of 0.4 nM, and became overtly diminished at g2 nM vinblastine. Upon exposure to 10 nM vinblastine, the intensities of peaks at 782 and 1422 cm 1 representing nucleic acid dropped to 64.5 ( 3.0% (p < 0.001) and 49.1 ( 4.6% (p < 0.001), respectively, of control CEM cells. The peak at 1660 cm 1, which corresponds to protein amide I also dropped to 67.5 ( 5.1% (p < 0.001) of the control. On the other hand, CEM/VBL, a subline of CEM resistant to vinblastine, showed negligible intensity changes for the peak at 782 cm 1 (98.3 ( 3.7% of control) and other peaks after treatment with up to 10 nM vinblastine (Figure 4B). In comparison, when cytotoxicity was measured by 3H-thymidine incorporation, there was no significant changes in 3H-thymidine uptake in CEM cells after treatment with 0.4 nM vinblastine (94.8 ( 4.1% of control) while the intensity of Raman peak at 782 cm 1 decreased to 85.0 ( 2.1% of control (p < 0.001) (Figure 5A). A decline in 3H-thymidine became evident only at g2 nM vinblastine. For CEM/VBL cells, negligible changes in 3Hthymidine incorporation (102.5 ( 1.2% of control) or Raman peak intensities were observed even at 10 nM vinblastine. In addition,

Figure 6. Single nuclei Raman spectroscopy is useful for quantifying the time-dependent biochemical changes induced by cytotoxic drugs. HL60 and resistant cells HL60/ADR were incubated with or without 80 nM doxorubicin for various time periods; the nuclei of the cells were analyzed by Raman spectroscopy and 3H-thymidine incorporation assay. For 3H-thymidine incorporation assay, the cells with or without doxorubicin treatment were exposed to 3H-thymidine at zero hour. Comparison of time course of doxorubicin induced reduction of Raman shift intensity between HL60 cells and HL60/ADR cells (A) and cell cytotoxicity by 3H-thymidine incorporation assay (B).

Alamar Blue assay was also less sensitive than Raman spectroscopy, with the value of 96.1 ( 1.7% and 94.3 ( 5.0% of control in CEM and CEM/VBL, respectively, upon treatment with 0.4 nM vinblastine (Figure 5B). Thus, Raman spectroscopy of single nuclei has the ability to detect and identify nuclear changes linked to cytotoxicity at lower drug concentrations than conventional cell based assays. Time Kinetics of Doxorubicin Induced Reduction of Raman Peak Intensities in HL60 and HL60/ADR Cells. We next investigated whether single nuclei Raman spectroscopy could be used to detect and quantify the time-dependent biochemical 117

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Figure 7. Comparison of Raman peaks of nuclei isolated from HL60 cells at G0/G1 and those at G2/M phase. (A) G2/M Cell cycle analysis of synchronized HL60 cells. Representative DNA content histograms are shown for cells arrested in G0/G1 induced by double-thymidine block and for cells released into G2/M phase after 6 h. (B) Relative intensities of Raman peaks of nuclei isolated from HL60 cells at G0/G1 and G2/M phase.

changes induced by anticancer drug. HL60 and resistant cells HL60/ADR were incubated with or without doxorubicin at 80 nM for various time periods and the Raman peak intensities were analyzed (Figure 6A). As early as 8 h, a decline in the Raman intensity of doxorubicin-treated HL60 cells was detectable, which decreased further with time. At 8 h, the declines were noted in the Raman peaks at 782 and 1489 cm 1 (nucleic acids), 1660 (protein) and 1449 cm 1 (nucleic acids, protein, and lipid). At 12 h, obvious decreases were observed in the Raman peaks at 728, 1095, 1422, 1578 (nucleic acids), 1004, and 1257 cm 1 (protein). On the other hand, there were no significant changes in doxorubicin-treated HL60/ADR cells over 24 h. To validate the significance of the observed reduction of doxorubicininduced Raman intensities, the cytotoxicity of doxorubicin was evaluated by conventional assay, using incorporation of 3Hthymidine. As shown in Figure 6B, doxorubicin-treated HL60 cells displayed a similar time dependent decrease in the incorporation of 3H-thymidine, which is not evident until 12 h after doxorubicin exposure (p < 0.001). Therefore, single nuclei Raman spectroscopy appears to be more sensitive than the conventional cytotoxicity assays in detecting the doxorubicininduced cellular biochemical changes in response to cytotoxic agent at earlier time point. Correlation of Raman Peak Intensities to Cell Cycle. We further assessed the validity of single nuclei Raman spectroscopy in deciphering cellular biochemical changes by examining cells synchronized in various phases of cell cycles. To this purpose, cells were treated with excessive amount of thymidine twice (double-thymidine) to arrest the cells in G0/G1 phase. Upon

removal of excess thymidine, the synchronized cells entered S phase, followed by G2/M phase. DNA content histograms as determined by flow cytometry showed that double thymidine treatment resulted in accumulation of 60.0% of the cells in G0/G1 with only 2.4% in G2/M phase (Figure 7). Six hours after release from excess thymidine 46.8% of the cells entered into G2/M phase, with only 17.2% remaining in G0/G1 phase. Nuclei from cells enriched in G0/G1 and G2/M phases were analyzed by Raman spectroscopy. Consistent with the fact that G2/M cells contains twice the DNA content of the G0/G1 cells, G2/M cells displayed higher signals than G0/G1 cells in most Raman peaks representing nucleic acids (e.g., peaks at 728, 782, 1095, 1375, 1489, 1578 cm 1), and some Raman peaks standing for protein (e.g., protein, with peaks at 1660 cm 1). Such analysis of cell cycle dynamics with Raman spectroscopy further validate the potential of single nuclei Raman spectroscopy for monitoring cellular and biochemical changes in cells without radioactive labels or colorimetric reactions.

’ DISCUSSION In this study, we demonstrated that single nuclei Raman spectroscopy has the ability to detect early nuclear changes in response to cytotoxic agents, such as doxorubicin and vinblastine at lower concentrations and in shorter times than conventional cell-based assays. Doxorubicin and vinblastine are chemotherapeutic agents widely used for antitumor therapy and both induce apoptosis in tumor cells. Doxorubicin interacts with DNA by intercalation and inhibits topoisomerase II, which 118

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Analytical Chemistry catalyzes the unwinding of DNA of transcription and replication, leading to inhibition of macromolecular biosynthesis.23,24 Doxorubicin stabilizes the topoisomerase II after it has broken the DNA chain for replication, inhibiting the DNA double helix from being replicated, and resulting in an apoptotic response. Vinblastine depolymerizes microtubules and destroy mitotic spindles at high concentrations (e.g., 10 100 nM in HeLa cells), leaving the dividing cancer cells blocked in mitosis with condensed chromosomes.25 At low but clinically relevant concentrations (e.g., IC50 0.8 nM in HeLa cells), vinblastine blocks mitosis and induces apoptosis by suppressing microtubule polymerization rather than depolymerization.26 The cells undergoing apoptosis reveal a specific morphology, characterized by cell shrinkage, membrane blebbing, deep condensation and marginalization of nuclear chromatin,27 formation of apoptotic bodies, and fragmentation of DNA at internucleosomal linker sites giving rise to multiples of 180 200 base pairs.28 In this study, we also evaluated for the first time Raman spectrometry for the characterization of biochemical changes related to cell cycle dynamics within single cell nucleus. Cells enriched in G0/G1 obtained by synchronization with excessive thymidine, and those enriched in G2/M phases harvested after removal of thymidine were analyzed by Raman spectroscopy. Previously, Raman spectroscopy combined with algorithm principal component analysis was used to analyze the changes in whole cell spectra as a function of cell cycle.16 Such analyses revealed cell cycle dependent variations in Raman spectral signatures with a concurrent relative increase in signals of nucleic acids and proteins from G0/G1 to G2/M phase.16 However, our Raman spectra measurement of cell cycle changes was derived from an average of single nuclei within the whole population of cells, but not from algorithm analysis of Raman spectra of whole cells. Single nuclei Ramen spectroscopy during G0/G1 transition to G2/M phase in our study showed that most Raman peaks representing nucleic acids (e.g., peaks at 728, 782, 1095, 1375, 1489, 1578 cm 1), and some Raman peaks standing for protein (e.g., peaks at 1660 cm 1) were higher in G2/M phase than G0/ G1 phase. These findings attested to the potential application of single nuclei Raman spectroscopy for assessing cell-cycle related changes without adding exogenous probes or chemicals.

ARTICLE

induced by external stress or agents, such as growth retardation, apoptosis, as well as cell attachment differentiation. Moreover, with the addition of a robotics workstation and compatible software, this single-nuclei-Raman-spectroscopy could be automated and adapted for the high throughput screening of large compound libraries.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (A.L.Y.); [email protected]. edu.tw (C.-H.C).

’ ACKNOWLEDGMENT This work is supported by National Science Council and National Health Research Institute (C.-H.C.) and Academia Sinica (A. L.Y.). ’ REFERENCES (1) Crow, P.; Molckovsky, A.; Stone, N.; Uff, J.; Wilson, B.; WongKeeSong, L. M. Urology 2005, 65, 1126–1130. (2) Kanter, E. M.; Vargis, E.; Majumder, S.; Keller, M. D.; Woeste, E.; Rao, G. G.; Mahadevan-Jansen, A. J. Biophotonics 2009, 2, 81–90. (3) Huang, Y. S.; Karashima, T.; Yamamoto, M.; Ogura, T.; Hamaguchi, H. J. Raman Spectrosc. 2004, 35, 525–526. (4) Naito, Y.; Toh-e, A.; Hamaguchi, H. O. J. Raman Spectrosc. 2005, 36, 837–839. (5) Nijssen, A.; Koljenovic, S.; Bakker Schut, T. C.; Caspers, P. J.; Puppels, G. J. J.Biophotonics 2009, 2, 29–36. (6) Chan, J. W.; Taylor, D. S.; Lane, S. M.; Zwerdling, T.; Tuscano, J.; Huser, T. Anal. Chem. 2008, 80, 2180–2187. (7) Wachsmann-Hogiu, S.; Weeks, T.; Huser, T. Curr. Opin. Biotechnol. 2009, 20, 63–73. (8) Moritz, T. J.; Taylor, D. S.; Krol, D. M.; Fritch, J.; Chan, J. W. Biomed. Opt. Express 2010, 1, 1138–1147. (9) Notingher, I. Sensors 2007, 7, 1343–1358. (10) Buckmaster, R.; Asphahani, F.; Thein, M.; Xu, J.; Zhang, M. Analyst 2009, 134, 1440–1446. (11) Uzunbajakava, N.; Lenferink, A.; Kraan, Y.; Volokhina, E.; Vrensen, G.; Greve, J.; Otto, C. Biophys. J. 2003, 84, 3968–3981. (12) Yao, H. L.; Tao, Z. H.; Ai, M.; Peng, L. X.; Wang, G. W.; He, B. J.; Li, Y. Q. Vib. Spectrosc. 2009, 50, 193–197. (13) Draux, F.; Gobinet, C.; Sule-Suso, J.; Trussardi, A.; Manfait, M.; Jeannesson, P.; Sockalingum, G. D. Anal.Bioanal.Chem. 2010, 397, 2727–2737. (14) Pijanka, J. K.; Kohler, A.; Yang, Y.; Dumas, P.; Chio-Srichan, S.; Manfait, M.; Sockalingum, G. D.; Sule-Suso, J. Analyst 2009, 134, 1176–1181. (15) Zheng, F.; Qin, Y.; Chen, K. J. Biomed. Opt. 2007, 12, 034002. (16) Swain, R. J.; Jell, G.; Stevens, M. M. J. Cell Biochem. 2008, 104, 1427–1438. (17) Oshima, Y.; Shinzawa, H.; Takenaka, T.; Furihata, C.; Sato, H. J. Biomed. Opt. 2010, 15, 017009. (18) McGrath, T.; Latoud, C.; Arnold, S. T.; Safa, A. R.; Felsted, R. L.; Center, M. S. Biochem. Pharmacol. 1989, 38, 3611–3619. (19) McGrath, T.; Center, M. S. Biochem. Biophys. Res. Commun. 1987, 145, 1171–1176. (20) Neugebauer, U.; Schmid, U.; Baumann, K.; Ziebuhr, W.; Kozitskaya, S.; Deckert, V.; Schmitt, M.; Popp, J. ChemPhysChem 2007, 8, 124–137. (21) Notingher, I.; Hench, L. L. Expert Rev. Med. Devices 2006, 3, 215–234. (22) Schuster, K. C.; Reese, I.; Urlaub, E.; Gapes, J. R.; Lendl, B. Anal. Chem. 2000, 72, 5529–5534.

’ CONCLUSION Raman spectroscopy with laser tweezers allows single cells to be directly interrogated without need for any exogenous probes or chemicals, and thus is free of the artifacts introduced by the external agents. Single nuclei Raman spectroscopy collects the information about the entire biochemical composition of a cell nucleus and reports the contributions of various chemical functional groups in parallel. The rich information provided by singlenuclei-Raman-spectroscopy makes it a versatile technique, in which the decision is made on each cell nucleus, not the ensemble average of 1000 cells or more. This single-nuclei-Ramanspectroscopy has the ability to detect and identify nuclear changes related to cytotoxicity at lower concentrations and in earlier time points than conventional cell based assays. Thus, this new cytotoxicity detection strategy using Raman spectroscopy of single, isolated nuclei may be useful for rapid and sensitive detection of cellular changes in response to chemotherapeutic agents and thus facilitate the selection of appropriate chemotherapeutic agents for personalized cancer therapy. This technology will be valuable for monitoring alteration in cellular processes 119

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dx.doi.org/10.1021/ac201900h |Anal. Chem. 2012, 84, 113–120