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Determination of DNA and RNA Epigenetic Modifications in Circulating Tumor Cells by Mass Spectrometry Wei Huang, Chu-Bo Qi, Song-Wei Lv, Min Xie, Yu-Qi Feng, Wei-Hua Huang, and Bifeng Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03962 • Publication Date (Web): 27 Dec 2015 Downloaded from http://pubs.acs.org on December 30, 2015

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Analytical Chemistry

1

Determination of DNA and RNA Methylation in Circulating

2

Tumor Cells by Mass Spectrometry

3

Wei Huang,1,† Chu-Bo Qi,1,2,† Song-Wei Lv,1,† Min Xie,1 Yu-Qi Feng,1,* Wei-Hua

4

Huang,1,* Bi-Feng Yuan1,*

5 6 7 8

1

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China 2

Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P.R.

China

9 10

†

These authors contributed equally to this work.

11 12

*To whom correspondence should be addressed. Tel.:+86-27-68755595; fax:

13

+86-27-68755595. E-mail address: [email protected]; [email protected];

14

[email protected]

15 16 17

1

ACS Paragon Plus Environment


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ABSTRACT

19

DNA methylation (5-methylcytosine, 5-mC) is the best characterized epigenetic

20

mark that has regulatory roles in diverse biological processes. Recent investigation of

21

RNA modifications also raises the possible functions of RNA adenine and cytosine

22

methylations on gene regulation in the form of “RNA epigenetics”. Previous studies

23

demonstrated global DNA hypomethylation in tumor tissues compared to healthy

24

controls. However, DNA and RNA methylation in circulating tumor cells (CTCs) that

25

are derived from tumors are still a mystery due to the lack of proper analytical

26

methods. In this respect, here we established an effective CTCs capture system

27

conjugated with a combined strategy of sample preparation for the captured CTCs

28

lysis, nucleic acids digestion, and nucleosides extraction in one-tube. The resulting

29

nucleosides were then further analyzed by liquid chromatography-electrospray

30

ionization-tandem mass spectrometry (LC-ESI-MS/MS). With the developed method,

31

we are able to detect DNA and RNA methylation (5-methyl-2’-deoxycytidine,

32

5-methylcytidine, and N6-methyladenosine) in a single cell. We then further

33

successfully determined DNA and RNA methylation in CTCs from lung cancer

34

patients. Our results demonstrated, for the first time, significant decrease of DNA

35

methylation (5-methyl-2’-deoxycytidine) and increase of RNA adenine and cytosine

36

methylations (5-methylcytidine and N6-methyladenosine) in CTCs compared with

37

whole

38

hypermethylation in CTCs in the current study together with previous reports of

39

global DNA hypomethylation in tumor tissues suggest that nucleic acids

blood cells.

The

discovery

of

DNA

hypomethylation

2


and

RNA

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modifications play important roles on the formation and development of cancer cells.

41

This work constitutes the first step for the investigation of DNA and RNA methylation

42

in CTCs, which may facilitate uncovering the metastasis mechanism of cancers in the

43

future.

44 45

Keywords: DNA methylation, RNA methylation, circulating tumor cells, sample

46

preparation, mass spectrometry.

47

3



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INTRODUCTION

49

DNA cytosine methylation (5-methyl-2’-deoxycytidine, 5-mdC) is one of the

50

most important epigenetic marks that play critical roles in a variety of cellular

51

processes,1 including cell differentiation,2 genome imprinting,3 and X-chromosome

52

inactivation.4 Maintaining proper DNA methylation status is critical for the normal

53

functions of cells.5,6 Abnormal DNA methylation can cause many human diseases,

54

such as diabetes,7 neurological disorders 8 and cancers.9,10

55

Given the rich layers of epigenetic regulation from DNA methylation, reversible

56

RNA modification has been proposed to represent another realm for biological

57

regulation in the form of “RNA epigenetics”.11 Up to date, more than 100 modified

58

nucleosides have been identified in RNA in all three kingdoms of life,12 among which,

59

5-methylcytidine (5-mrC) and N6-methyladenosine (m6A) are the two most important

60

modifications on RNA molecules with potential functions on the control and

61

regulation of gene transcription and protein translation.13-16

62

Previous studies demonstrated that DNA typically exhibited hypomethylation in

63

tumor tissues compared to healthy controls, which therefore could be used as an

64

indicator for tumorigenesis.17-19 Whereas, tissue-based analysis normally requires

65

surgery, which is inconvenient and may cause excessive pain to patients. In this

66

respect, blood-based specimen analysis is a non-invasive strategy and frequently

67

employed in clinical investigation. However, recent studies suggested no obvious

68

genomic DNA methylation change was observed from human whole blood cells

69

between cancer patients and healthy controls.20-22 The reason could be due to that 4


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human blood is a complex system and includes various blood cells, which may not be

71

proper analytical target. In this vein, circulating tumor cells (CTCs) that are derived

72

from tumors in peripheral blood may truly reflect tumor status and progression instead

73

of whole blood cells.

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CTCs are highly related to the metastasis of cancer. CTCs appear at an estimated

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level of one against the background of millions (106~107) of surrounding normal

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peripheral mononuclear blood cells in peripheral blood.23 To date, no study of DNA

77

and RNA methylation on pure isolated CTCs has been reported, which could be

78

attributed to the technical limitations associated with studying DNA and RNA

79

methylation in a few or single cell.24 In this respect, development of effective strategy

80

for analysis of DNA and RNA methylation in a few or single cell, selective

81

enrichment of CTCs and/or systematic removal of peripheral mononuclear blood cells

82

and red blood cells are required in CTCs-based nucleic acids modification study.25,26

83

In the current study, we established a simple capture system for the isolation and

84

enrichment of CTCs from blood using prepared biotinylated antibody-streptavidin

85

(SA) modified magnetic beads (MBs). In addition, we further developed a combined

86

strategy of sample preparation for cell lysis, nucleic acids digestion, and nucleosides

87

extraction in one-tube. In this respect, the enriched CTCs were processed with the

88

one-tube strategy followed by liquid chromatography-electrospray ionization-tandem

89

mass spectrometry (LC-ESI-MS/MS) analysis. With the developed method, we

90

successfully determined DNA and RNA methylation in CTCs from lung cancer

91

patients. Our results demonstrated, for the first time, significant changes of DNA and 5



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92

RNA methylation in CTCs compared with whole blood cells. This work constitutes

93

the first step for the study of DNA and RNA methylation in CTCs.

94 95

EXPERIMENTAL SECTION

96

Chemicals and reagents

97

Streptavidin

(SA),

5-fluorescein

and

conjugate

carbodiimide

(FITC-biotin),

98

1-ethyl-3-(3-(dimethylamino)

99

N-hydroxysuccinimide (NHS), 4’,6-diamidino-2-phenylindole (DAPI), FITC-labeled

100

goat anti-mouse secondary antibody, calcein-acetoxymethyl ester (calcein-AM), and

101

propidium iodide (PI) were purchased from Sigma-Aldrich (Beijing, China).

102

Biotin-labeled mouse anti-human anti-EpCAM monoclonal antibody was obtained

103

from eBioscience (San Diego, CA). FITC-labeled mouse anti-human anticytokeratin

104

(FITC-CK) and phycoerythrin-labeled mouse anti-human CD45 (PE-CD45) were

105

purchased from BD Biosciences (San Jose, CA). Cytidine (rC), guanosine (rG),

106

adenosine (rA), uridine (rU), 5-methylcytidine (5-mrC), 2’-deoxycytidine (dC),

107

2’-deoxyguanosine

108

5-methyl-2’-deoxycytidine (5-mdC) and phosphodiesterase I were purchased from

109

Sigma-Aldrich (Beijing, China). N6-methyladenosine (m6A) was from Hanhong

110

Chemical Co., Ltd. (Shanghai, China). Proteinase K, S1 nuclease and calf intestinal

111

alkaline phosphatase (CIAP) were from Takara Biotechnology (Dalian, China).

112

Chromatographic grade methanol was purchased from Merck (Darmstadt, Germany).

113

The water used throughout the study was purified by a Milli-Q apparatus (Millipore,

(dG),

propyl)

biotin

2’-deoxyadenosine

hydrochloride

(dA),

6


thymidine

(EDC),

(T),

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114

Bedford, MA). Stock solutions of all the nucleosides were prepared in ultrapure water

115

at a concentration of 1 mM.

116

Oligonucleotides

117

The 16-mer DNA (5’-GTAGGTCGTGATGAGG-3’), 16-mer 5-mdC-containing

118

DNA

(5’-CCTCATCA

(5-mdC)

GACCTAC-3’),

10-mer

RNA

119

(5’-AUCUAUAUGC-3’) and 12-mer 5-mrC-containing RNA (5’-GACUAG (5-mrC)

120

GAGUC-3’) were purchased from Takara Biotechnology Co, Ltd. The 15-mer

121

m6A-containing oligonucleotide synthesized according to previously described

122

method 27 was kindly provided by Prof. Guifang Jia in Peking University.

123

Biological samples

124

MCF-7 (human breast cancer) cells were obtained from the China Center for

125

Type Culture Collection and maintained in Dulbecco’s Modified Eagle medium

126

supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL

127

streptomycin at 37°C in a 5% CO2 atmosphere.

128

A total of 18 human blood samples from lung cancer patients as well as 20

129

human blood samples from healthy controls were collected in Hubei Cancer Hospital

130

(Wuhan, China) and stored at 4oC in the presence of EDTA as an anticoagulant. This

131

study was approved by the ethics committee of Hubei Cancer Hospital.

132

Combined strategy of sample preparation

133

To realize DNA and RNA methylation analysis from small numbers of cells, here

134

we developed a combined strategy for cell lysis, nucleic acids digestion, and

135

nucleosides extraction in one-tube (Figure 1). Briefly, small amounts of cells in 7 µL 7



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136

PBS buffer were firstly incubated at 90°C for 10 min to break the cells, then

137

proteinase K (1 µL, 20 mg/mL) was added followed with incubating at 58°C for 1 h.

138

The resulting sample was then incubated at 95°C for 15 min to denature DNA as well

139

as inactivate proteinase K. After adding 1 µL of S1 nuclease buffer (30 mM

140

CH3COONa, pH 4.6, 280 mM NaCl, 1 mM ZnSO4) and 100 units of S1 nuclease, the

141

mixture (10 µL) was incubated at 37ºC for 4 h. To the solution was subsequently

142

added 4 µL of alkaline phosphatase buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 9.0),

143

0.0008 units of venom phosphodiesterase I, 6 units of alkaline phosphatase and 24.7

144

µL H2O. And then the incubation was continued at 37ºC for an additional 1 h

145

followed by adding 160 µL sterilized water and extraction with equal volume of

146

phenol/chloroform (v/v, 1:1) and chloroform once. The resulting aqueous layer was

147

collected and lyophilized to dryness and then reconstituted in 100 µL water for

148

subsequent LC-ESI-MS/MS analysis.

149

Fabrication and characterization of CTCs capture system

150

The schematic procedure for the fabrication of CTCs capture system is shown in

151

Figure S1 in Supporting Information. SA was firstly immobilized on the surface of

152

magnetic beads (MBs) by the reaction of amines of the SA with carboxylic acid of

153

MBs. Briefly, approximate 2 mg of MBs was activated in 50 mM EDC and 50 mM

154

NHS in 1 mL of ultrapure water at room temperature with gentle shaking for 30 min.

155

Subsequently, the activated MBs were collected by a magnetic scaffold and washed

156

with ultrapure water for three times. Then, activated MBs were resuspended in 1 mL

157

of ultrapure water to react with 400 µg of SA for 2 h with continuous shaking at room 8


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158

temperature. The resultant SA-immobilized MBs (SA-MBs) were washed with

159

ultrapure water to remove surplus SA. Finally, 0.2 mg SA-MBs were resuspended in 1

160

mL of ultrapure water to react with 10 µL biotinylated anti-EpCAM (100 µg/ml in

161

PBS) and incubated for 45 min. The obtained anti-EpCAM functionalized magnetic

162

beads (anti-EpCAM-Biotin-SA-MBs) were then magnetically separated and used for

163

following experiments.

164

Capture and identification of spiked cancer cells from human blood

165

Mimic CTCs-containing human blood samples were prepared by spiking human

166

breast cancer MCF-7 cells into healthy human blood at 102 cells/mL. Then 0.2 mg

167

anti-EpCAM-Biotin-SA-MBs were added to the blood samples and incubated at 37°C

168

for 30 min in dark. The captured cells were used for common three-color

169

immunocytochemistry (ICC) analysis. Briefly, cells were first fixed with 4%

170

paraformaldehyde for 30 min and then permeabilized with 0.2% Triton-X 100 for 30

171

min. Subsequently, the cells were stained with 10 µg/mL DAPI, FITC-labelled

172

anti-CK monoclonal antibody, and PE-labelled anti-CD45 monoclonal antibody at

173

4°C for 2 h according to the manufacture’s recommended protocol. After washing, the

174

captured cells were put into a 96-well plate for fluorescent microscopy imaging.

175

Capture of CTCs from human blood of lung cancer patients

176

The schematic procedure for CTCs enrichment is shown in Figure S2. Briefly,

177

human blood cells were collected by centrifugation at 2,000 rpm from 1 mL blood.

178

The cell pellets were washed with 1 mL PBS for three times. Then the blood cells

179

were resuspended in 1 mL of PBS followed by incubating with 0.2 mg 9



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for

30

min.

The

Page 10 of 28

180

anti-EpCAM-Biotin-SA-MBs

captured

cells

on

the

181

anti-EpCAM-Biotin-SA-MBs were magnetically separated and reconstituted in 7 µL

182

of sterilized water. Subsequently, the captured cells were subjected to our developed

183

combined strategy of sample preparation for cell lysis, nucleic acids digestion, and

184

nucleosides extraction in one-tube. The resulting nucleosides were then analyzed by

185

LC-ESI-MS/MS.

186

LC-ESI-MS/MS analysis of DNA and RNA methylation

187

Analysis of DNA and RNA methylation from CTCs was performed on

188

LC-ESI-MS/MS system consisting of an AB 3200 QTRAP mass spectrometer

189

(Applied Biosystems, Foster City, CA, USA) with an electrospray ionization source

190

(Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two

191

LC-20AD pumps, a SIL-20A autosampler, a CTO-20AC thermostatted column

192

compartment, and a DGU-20A3 degasser. Data acquisition and processing were

193

performed using AB SCIEX Analyst 1.5 Software (Applied Biosystems, Foster City,

194

CA). LC separation was performed on a Hisep C18-T column (150 mm × 2.1 mm i.d.,

195

5 µm, Weltech Co., Ltd., Wuhan, China) with a flow rate of 0.2 mL/min at 35oC.

196

Water (solvent A) and methanol (solvent B) were employed as mobile phase. A

197

gradient of 5 min 5% B, 10 min 5% -30% B, 5 min 30% -50% B, 3 min 50% -5% B

198

and 17 min 5% B was used.

199

Positive electrospray ionization mode was used to perform the mass

200

spectrometry detection. The target analytes were monitored by multiple reaction

201

monitoring (MRM) mode using the mass transitions (precursor ions product ions) 10


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202

of dC (228.4 112.2), T (243.3 127.2), dA (252.4 136.2), dG (268.4 152.4),

203

5-mdC (242.3 126.1), rC (244.4 112.2), rU (245.4 113.1), rA (268.4 136.2),

204

rG (284.5 152.2), 5-mrC (258.0 126.1), m6A (282.1 150.2). The MRM

205

parameters of all nucleosides were optimized to achieve maximal detection sensitivity.

206

(Table S1)

207 208

The modified nucleoside contents were calculated using the following expressions: M × 100% M M 5 − mrC % = × 100% M M m A % = × 100% M

5 − mdC % =

209

where M5-mdC, M5-mrC and Mm6A are the molar quantities of 5-mdC, 5-mrC and m6A,

210

while MdG, MrG and MrA are the molar quantities of dG, rG and rA determined in

211

samples.

212

Statistical analysis

213

Statistical analyses were performed using SPSS 19.0 software (SPSS Inc.,

214

Chicago, USA). Student’s paired t-test was used to assess the differences of DNA and

215

RNA methylation between captured CTCs and the corresponding whole blood

216

samples. Student’s unpaired t-test was used to assess the differences of DNA and

217

RNA methylation between the samples from lung cancer patients and healthy controls.

218

All p values were two-sided, and p ＜ 0.05 were considered to be statistically

219

significant.

220 11



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221

RESULTS AND DISCUSSION

222

Combined strategy of sample preparation

223

The typical procedure to analyze global DNA and RNA methylation contains

224

multiple steps, including cell lysis, nucleic acids extraction, enzymatic digestion,

225

extraction of nucleosides and analysis by instruments.28-32 However, this protocol is

226

not suitable to deal with small amounts of cells since the tiny amounts of nucleic acids

227

in a few cells may significantly lose with the traditional extraction and enzyme

228

digestion strategy. To realize the quantitative determination of global DNA and RNA

229

methylation from CTCs, here we developed a combined strategy for cell lysis, nucleic

230

acids digestion, and nucleosides extraction in one-tube (Figure 1).

231

To evaluate the performance of the combined strategy of sample preparation, 1, 5

232

and 10 cells were picked up from diluted cultured cells and transferred into PBS

233

buffer for further process with the developed combined sample preparation strategy.

234

The results showed that no signals of modified nucleosides were observed in the

235

control sample (without adding cells) (Figure 2A); while the signal intensities of

236

modified nucleosides, including 5-mdC in DNA and 5-mrC and m6A in RNA, were

237

clearly detected in the samples with 1, 5, and 10 cells (Figure 2B-D), indicating that

238

the method was effective to analyze DNA and RNA methylation from a few cells.

239

Characterization of CTCs capture system

240

In this work, a simple CTCs capture system was fabricated using biotinylated

241

anti-EpCAM and SA-modified MBs (anti-EpCAM-Biotin-SA-MBs) for the isolation

242

and enrichment of CTCs from human blood samples. SA-immobilized MBs (SA-MBs) 12


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243

were firstly examined using FITC-biotin (Figure S3). The results showed that

244

SA-MBs exhibited strong FITC fluorescence (Figure S3B) compared with MBs alone

245

(Figure S3D), indicating the successful immobilization of SA on MBs. Likewise,

246

FITC-labelled secondary antibody was used to examine the immobilization of

247

biotinylated anti-EpCAM on SA-MBs (anti-EpCAM-Biotin-SA-MBs, Figure S4).

248

Compared to MBs (Figure S4B), anti-EpCAM-Biotin-SA-MBs showed strong FITC

249

fluorescence

250

anti-EpCAM-Biotin-SA-MBs.

251

Capture of cancer cells using anti-EpCAM-Biotin-SA-MBs

(Figure

S4D),

suggesting

the

successful

fabrication

of

252

An EpCAM-positive cancer cell line (MCF-7) was used as the target cell line to

253

explore the performance of anti-EpCAM-Biotin-SA-MBs on the selective capture of

254

cancer cells. EpCAM is frequently over-expressed in many kinds of solid cancer cells

255

and is absent from hematologic cells; therefore, EpCAM has been typically used as

256

the target antigen to isolate cancer cells.33 In the current study, EpCAM-positive

257

MCF-7 cells were spiked into healthy human blood followed by enrichment with

258

anti-EpCAM-Biotin-SA-MBs. The captured MCF-7 cells were then used for

259

three-color immunocytochemistry (ICC) identification by FITC-labelled anti-CK

260

(marker for epithelial cells, such as MCF-7 cells) monoclonal antibody, PE-labelled

261

anti-CD45 (marker for white blood cells), and DAPI nuclear staining. As shown in

262

Figure 3, the enriched cells were DAPI+/CK+/CD45－, and white blood cells were

263

DAPI+/CK－/CD45+. These results demonstrated that EpCAM-positive cancer cells

264

can be selectively isolated and enriched by anti-EpCAM-Biotin-SA-MBs from whole 13



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265

human blood.

266

Analysis of DNA and RNA methylation by LC-ESI-MS/MS

267

MRM mode was used for the determination of DNA modification of 5-mdC and

268

RNA modifications of 5-mrC and m6A. The calibration curves were constructed by

269

plotting the mean peak area ratios of 5-mdC/dG, 5-mrC/rG, m6A/rA versus the mean

270

molar ratio of 5-mdC/dG, 5-mrC/rG, m6A/rA based on data obtained from triplicate

271

measurements. The results showed good linearities for 5-mdC/dG, 5-mrC/rG and

272

m6A/rA were achieved within the corresponding range of 0.5-20%, 0.05-2% and

273

0.02-1% (molar ratio) with the coefficient of determination (R2) higher than 0.9977

274

(Table S2).

275

Accuracy of the method was evaluated using the synthesized modified

276

nucleoside-containing oligonucleotides by comparing the measured contents to the

277

theoretical contents (Table S3). The results showed that the modified nucleosides

278

were determined from oligonucleotide hydrolysis products with relative standard

279

deviations (RSDs) being 1.8-10.9% and relative errors (REs) being 2.1-15.0% (Table

280

S4), indicating that the LC-ESI-MS/MS method was reliable for the determination of

281

5-mdC, 5-mrC and m6A. In addition, the precision of the LC-ESI-MS/MS method

282

were evaluated with the RSDs and REs being less than 14.9% and 16.0%, respectively

283

(Table S5-S7).

284

Validation of the developed method for analysis of DNA and RNA methylation in

285

CTCs

286

We used the mimic CTCs-containing blood sample prepared by spiking MCF-7 14


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287

cells to healthy human blood to evaluate the developed analytical strategy for the

288

determination of DNA and RNA methylation in CTCs. Since the contents of 5-mrC

289

and m6A between MCF-7 cells and human blood cells are distinctly different, MCF-7

290

cells were used to evaluate the specificity of the developed method. As shown in

291

Figure 4, the measured contents of 5-mdC, 5-mrC and m6A from enriched MCF-7

292

cells spiked in human blood samples were similar to those from the same amounts of

293

MCF-7 cells in PBS buffer; while the measured contents of 5-mrC and m6A from

294

enriched MCF-7 cells spiked in human blood samples were not similar to those from

295

the human blood cells. These results demonstrated that the developed CTCs capture

296

system with the combined strategy of sample preparation were reliable in the

297

quantitative determination of DNA and RNA methylation in CTCs from human blood

298

samples.

299

In our study, alkaline phosphatase was used to remove the phosphate group

300

during enzymatic digestion of nuclei acids. Alkaline phosphatase acts on

301

monophosphonucleotides, i.e., dNMP and NMP. Here we measured 5-mdC/dG,

302

5-mrC/rG and m6A/rA. Therefore, the cellular endogenous dGMP, GMP and AMP

303

could potentially generate dG, rG and rA by alkaline phosphatase. However, since the

304

amounts of cellular endogenous dGMP, GMP and AMP are more than two orders of

305

magnitude lower than dG, rG and rA from the enzymatic digestion of DNA and

306

RNA,34-38 the cellular endogenous dGMP, GMP and AMP will not affect the

307

quantitative analysis of DNA and RNA methylation.

308

Contents change of 5-mdC, 5-mrC and m6A in CTCs from lung cancer patients 15



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Lung cancer is one of the most common cancers in the world. It is a leading

310

cause of cancer death in men and women in the United States.39 Previous studies

311

demonstrated the global DNA hypomethylation of lung tumor tissues compared to

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tumor adjacent healthy controls.17,18 However, obtaining tissue samples needs

313

invasive procedures. In this regard, easily accessible human blood is a good

314

alternative for caner prognosis and study of metastasis. But recent studies

315

demonstrated no detectable genomic DNA methylation change from human whole

316

blood cells between cancer patients and healthy controls.20-22

317

In this respect, CTCs that derived from tumor cells may reflect the true changes

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of DNA and RNA methylation in cancer patients. As lung tumor cells express

319

EpCAM,40 we used the developed CTCs capture system and combined strategy of

320

sample preparation to analyze DNA and RNA methylation in CTCs captured from 18

321

blood samples of lung cancer patients. Shown in Figure 5A are the MRM

322

chromatograms of modified nucleosides from CTCs sample. The results demonstrated

323

that 5-mdC, 5-mrC and m6A can be distinctly detected. And the measured contents of

324

5-mdC in CTCs are much lower than that of whole blood cells (Figure 5B, Table S8).

325

The results suggested DNA hypomethylation in tumor cells, which is consistent with

326

previous reports that 5-mdC contents of lung tumor tissues significantly

327

decreased.17-19 As for RNA methylation, we observed the contents of both 5-mrC and

328

m6A increased in CTCs (Figure 5C and 5D, Table S8).

329

We then further analyzed the DNA and RNA methylation in the whole blood

330

cells in 20 healthy individuals whose genders and ages are matched to the lung cancer 16


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patients. The results showed that there are no significant differences of 5-mdC, 5-mrC,

332

and m6A levels in the whole blood cells between lung cancer patients and healthy

333

individuals (p = 0.36 for 5-mdC, 0.83 for 5-mrC and 0.85 for m6A) (Figure S5).

334

While the differences of 5-mdC, 5-mrC, and m6A levels between CTCs of lung cancer

335

patients and the whole blood cells of healthy individuals are significant (p = 7.6×10-13

336

for 5-mdC, 2.5×10-17 for 5-mrC, and 1.8×10-6 for m6A) (Figure S5), suggesting the

337

distinct statuses of DNA and RNA methylation between CTCs of lung cancer patients

338

and healthy individuals. The mechanism of the observed global changes of DNA and

339

RNA methylation in CTCs, however, need further investigation.

340

Early detection and characterization of CTCs is important as a general strategy to

341

monitor and prevent the development of overt metastatic diseases.41 Detection of

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DNA and RNA methylation in CTCs may be of great significance for the assessing of

343

tumor metastasis. Our results revealed, for the first time, the significant changes on

344

DNA and RNA methylation between CTCs and whole blood cells. The quantitative

345

evaluation of DNA and RNA methylation in CTCs can provide additional evidence

346

for the confirmation and characterization of CTCs in the scenario where CTCs are

347

used as cancer diagnosis.

348 349

CONCLUSIONS

350

In the current study, we established a combined strategy for cell lysis, nucleic

351

acids digestion, and nucleosides extraction in one-tube. With the developed sample

352

preparation method, DNA and RNA methylation can be successfully determined from 17



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one cell in combination with LC-ESI-MS/MS analysis. In addition, we developed a

354

simple system for the isolation and enrichment of CTCs from blood with high

355

specificity. Through the enrichment of CTCs from lung cancer patients and the

356

subsequent process with the combined strategy of sample preparation, we discovered

357

the significant decrease of 5-mdC and increases of 5-mrC and m6A in CTCs of lung

358

cancer patients. This study demonstrated DNA and RNA methylation may play

359

important roles on the formation and development of cancer cells and they may

360

facilitate the future investigation of tumor metastasis.

361 362

18


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363 364

Notes The authors declare no competing financial interest.

365 366

Acknowledgments

367

The authors thank the financial support from the National Basic Research

368

Program of China (973 Program) (2012CB720601), the National Natural Science

369

Foundation of China (21522507, 21205091, 21475098).

370 371 372 373

Supporting Information Additional information as noted in text. This information is available free of charge via the Internet at http://pubs.acs.org/.

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441

Figure legends

442

Figure 1. Schematic diagram of the combined strategy of sample preparation for cell

443

lysis, nucleic acids digestion, and nucleosides extraction in one-tube.

444 445

Figure 2. MRM chromatograms of modified nucleosides from cells prepared with the

446

combined strategy of sample preparation. (A) No cell; (B) 1 cell; (C) 5 cells; (D) 10

447

cells.

448 449

Figure 3. Characterization of the CTCs capture system using MCF-7 cells spiked in

450

healthy human blood. (A) Microscopic images of white blood cells (WBCs) stained

451

by DAPI, FITC-CK, and PE-CD45. (B) Microscopic images of cells captured from

452

mimic blood sample stained by DAPI, FITC-CK, and PE-CD45.

453 454

Figure 4. Validation of the developed method for analysis of DNA and RNA

455

methylation in CTCs.

456 457

Figure 5. Determination of the contents of DNA and RNA methylation in CTCs from

458

human blood of lung cancer patients. (A) MRM chromatograms of 5-mdC, 5-mrC and

459

m6A from CTCs. (B-D) The measured contents of 5-mdC, 5-mrC and m6A from

460

captured CTCs and the corresponding whole blood of 18 lung cancer patients.

461

22


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462

Figure 1.

463 464

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465

Figure 2.

466 467

24


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Figure 3.

469 470

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471

Figure 4.

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Figure 5.

475 476

27



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For “TOC” only

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Determination of DNA and RNA Methylation in ... - ACS Publications

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