Supplementary Data. Detection of Circulating Tumor Cells in Human Peripheral Blood using Surface- Enhanced Raman Scattering Nanoparticles

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1 Supplementary Data Detection of Circulating Tumor Cells in Human Peripheral Blood using Surface- Enhanced Raman Scattering Nanoparticles Xu Wang 1 *, Ximei Qian 2 *, Jonathan J Beitler 1,3, Zhuo (Georgia) Chen 1, Fadlo R Khuri 1, Melinda M. Lewis 4, Hyung Ju C. Shin 5, Shuming Nie 2, Dong M. Shin 1 1 Department of Hematology and Medical Oncology, 2 Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, 101 Woodruff Circle Suite 2001, Atlanta, GA 30322, USA. 3 Departments of Radiation Oncology and Otolaryngology, 4 Department of Pathology, Winship Cancer Institute, Emory University School of Medicine, 1365 Clifton Road, Atlanta, GA 30322, USA. 5 Quest Diagnostics, Atlanta, GA. *These two authors contributed equally to this study. Materials and Methods Materials Ultrapure water (18 MΩ cm -1 ) was used throughout the work. The following chemicals were obtained from commercial sources and were used without further purification: 60-nm citratestabilized gold particles at a concentration of particles per milliliter (Ted Pella Inc., Redding, CA); QSY21 quencher dye (Invitrogen Corporation, Carlsbad, CA). mpeg-sh (MW ~ 5,000 Da) (Nektar Therapeutics), HS-PEG-COOH (MW ~ 3,000 Da) (Rapp Polymere, Germany), EGF peptide (AbCam, Cat. No. ab55483, CA). Tu212 cell line is established from a hypopharyngeal tumor, and kindly provided by Dr. Gary L. Clayman (University of Texas M.D. Anderson Cancer Center, Houston, TX). H292, MDA-MB-231 and H460 cell lines were obtained from the American Type Culture Collection (ATCC). Cell culture media, fetal bovine serum, hemacytometer, and cell culture supplies were purchased from Fisher Scientific. All other reagents were obtained from Sigma-Aldrich (St Louis, MO) at the highest purity available. The sequence for EGF peptide is NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR. Preparation of SERS nanoparticles A freshly prepared reporter QSY dye solution (5 µm) was added dropwise to a rapidly mixing 3 ml gold colloid which facilitated even distribution of the reporter molecules on the gold particle surface. The reporter dye molecules were adsorbed to the negatively charged gold nanoparticle surface through electrostatic interaction. The ratio of reporter molecules to Au particle was optimized and carefully titrated to achieve a near-maximal SERS signal and minimal colloid aggregation. Carboxylation of SERS nanoparticle 1.35 ml bi-functional HS-PEG-COOH (1 µm) was added dropwise to 3.3 ml Au-QSY solution (7.8x10 10 total Au particles) in a polypropylene tube under rapid mixing until the ratio of carboxy 1

2 group per Au nanoparticle was 5000:1. The Au-QSY complex was thoroughly mixed with HS- PEG-COOH overnight, then with ~1 ml HS-PEG (10 µm) for 3 hrs to ensure that any exposed regions of the Au nanoparticle surface were filled. The closely-packed PEG layer not only stabilizes the nanoparticle, but also helps the COOH function group extend to the outer layer for efficient conjugation. The carboxylated SERS tag was purified by 3 rounds of centrifugation at 2000g and resuspended in PBS. Freshly prepared 5µl EDC solution (40mg/ml) and 5 µl sulfo- NHS (110mg/ml) were added to the SERS tag solution, and then vigorously mixed for 15min at room temperature to activate the COOH group of carboxylated SERS tags. Excess EDC and sulfo-nhs were separated from the NHS-activated SERS tags by centrifugation for 5min at 3000g with Nanosep 10K MWCO OMEGA membrane (Pall Life Sciences, MI) and resuspended in PBS. Conjugation of EGF peptide to the carboxylated SERS nanoparticle EGF peptide (AbCam, Cat. No. ab55483, CA) (1.6 nmole) was immediately added to the NHSactivated SERS tag solution and mixed for 2 hours at room temperature. The N-terminal amine function group of EGF peptide reacted with NHS-activated carboxylated SERS tags. The mixture was stored at 4 o C overnight for continued reaction. Excess EGF peptide was then removed by 3 rounds of centrifugation and re-suspension in PBS. UV-Vis absorption spectra of protein at 280nm were measured to determine the amount of unbound EGF in the supernatant fractions collected from the synthesis and PBS rinsing steps. Given the initial amount of EGF added, the balance indirectly yields the conjugated EGF amount. Extinction coefficient of 18,500 cm -1 M -1 was used for EGF peptide. Based on gold nanoparticle concentration measured by plasmon resonance peak at 530nm, we estimated the conjugation efficiency to be ~500 EGF ligands per Au nanoparticle. Characterization of EGF-SERS nanoparticles. UV-Vis absorption spectra were recorded on a Shimadzu (UV-2401) spectrometer using disposable polyacryl cuvettes. Transmission electron micrographs (TEM) were taken by using a Hitachi H7500 high-magnification electron microscope. The EGF-SERS nanoparticle sample (5 µl) was dropped onto copper 200 mesh grids that were pre-treated with UV light to reduce static electricity. After 30 min, the solvent was drained with a filter paper and a phosphotungstic acid stain solution (1% by weight, adjusted to ph =6) was applied for 30 s. Dynamic light scattering (DLS) data were obtained using a Brookhaven 90Plus particle size analyzer instrument. Each sample was measured three times consecutively. SERS spectra were recorded on a hand-held Raman system using 785 nm (40 mw) excitation (Inspector Raman Series, DeltaNu, Laramie, WO). SERS intensities were normalized to the Raman spectra of cyclohexane and polystyrene to correct for variations in optical alignment and instrument response. The spectral resolution was approximately 5 cm -1 for the Inspector Raman systems. Mouse blood sample preparation All animal experiments were approved by the IACUC of Emory University. Blood was collected into BD Vacutainer PST tubes (containing gel and lithium heparin, BD Franklin Lakes, NJ) immediately after animal sacrificing. The blood was transferred to 15 ml centrifuge tubes and cultured cancer cells, as appropriate, were added at this time. Blood was mixed with equal volumes of balanced salt solution (0.01% D-glucose, 5.0 µm CaCl 2, 98 µm MgCl 2, 0.54 mm KCl 2, Tris 14.5 mm, 0.12 M NaCl, ph 7.6). 3 mls of Ficoll-Paque premium (GE Healthcare Bio- Sciences Corp, Piscataway, NJ) was added and then the specimens were centrifuged at 400 x g for 30 minutes at 20ºC. The low density cell layer containing lymphocytes and monocytes (WBCs) was transferred into a new tube, washed with PBS two times and fixed with 2% PFA for 10 min for the application. 2

3 Cultured cell labeling and characterization Tu212 cell line is established from a hypopharyngeal tumor, and kindly provided by Dr. Gary L. Clayman (University of Texas M.D. Anderson Cancer Center, Houston, TX). This cell line has been tested (genetic test using genotyping method) and authenticated by Research Animal Diagnostic Laboratory (4011 Discovery Drive, Columbia MO 65201). H292, MDA-MB-231 and H460 cell lines were obtained from the American Type Culture Collection (ATCC). Those cell lines were not further authenticated.the Tu212 human SCCHN and MDA-MB-231 breast cancer cell lines were maintained in DMEM medium supplemented with 10% FBS. Lung cancer cell lines H292 and H460 (ATCC) were maintained in RPMI medium supplemented with 10% FBS. To determine the cell labeling and detection sensitivity, 10, 100, 500, and 1000 Tu212 cells were spiked into 2 ml mouse blood samples. With constant mixing for 30 minutes at room temperature, these samples were incubated with 10 pm EGF-conjugated SERS nanoparticles. The SERS spectra from each sample were then measured and analyzed using 785 nm laser excitation on a handheld Raman system. To compare the labeling specificity of EGF-conjugated SERS nanoparticles, 10,000 cells from cell lines with different EGFR expression levels were added into separate mouse blood samples. These samples were incubated with 10 pm EGF-conjugated SERS nanoparticles for 30 min at room temperature, followed by three PBS washes. The SERS spectra from each cell line were then measured and analyzed. To examine the cell membrane EGFR expression of each cell line, the cells were incubated with primary antibody anti-egfr (BioGenex, San Ramon, CA) for 1 h, followed by three PBS washes. The cells were incubated with FITC-conjugated secondary antibodies for 30 min, then washed with PBS and analyzed using FACS (1). Patient blood sample preparation and circulating tumor cell (CTC) detection With IRB approval, blood ( ml) was collected from patients with different stages of head and neck cancer and from healthy donors. Blood samples were collected using BD Vacutainer CPT cell preparation tubes (BD Franklin Lakes, NJ) containing sodium heparin and polyester gel, and were centrifuged within 2 hrs of collection. After gentle mixing, the fresh blood was centrifuged at room temperature for 30 min at 1700 RCF. After centrifugation, the low density cell layer containing lymphocytes, monocytes and CTCs was transferred into a new tube, washed with PBS and fixed with 2% PFA for 10 min. The cells were washed with PBS, counted and split into 3 samples. One sample was incubated with 10 pm EGF-conjugated SERS nanoparticles, with constant mixing for 30 min at room temperature. Then, the cells were washed with ice-cold PBS three times and resuspended in 50 µl PBS before SERS measurement. A portion of the cells was incubated with 10 pm pegylated control SERS nanoparticles to assess nonspecific binding. The third sample received neither control SERS nanoparticles nor EGF-SERS nanoparticles, and was used as a control to better assess background cell scattering. The SERS spectra were then measured and analyzed. After the SERS signal measurement, the cells were prepared for H&E staining and immunostaining analyses. Immunohistochemistry staining of CTCs and primary tumor section Immunohistochemical analysis for cytokeratin and EGFR expression on slides was performed using Dako two steps kit (Dako, Carpinteria, CA). The slides were incubated with anti- 3

4 cytokeratin and anti-egfr antibody (1:100 dilution, Dako, Carpinteria, CA) overnight at 4 C, respectively. The slides were then stained with 3,3 -diaminobenzidine and counterstained with hematoxylin. Supporting Figures: Suppl. Figure 1. Flow cytometry analysis of EGFR expression on the surface of normal white blood cells from three healthy donors. The EGFR expression of healthy WBCs is at least two orders of magnitude lower than that of head and neck cancer (Tu212) cells. The negative control sample was set when no antibody staining was applied. 4

5 EGFR Cytokeratin 10µm Suppl. Figure 2. EGFR and cytokeratin expression in hematologic cells from healthy donor s blood sample. Control blood samples from healthy donors were evaluated for EGFR and cytokeratin by IHC staining. Representative bright field images show that hematologic cells were all stained negative (blue color of hematoxylin counterstaining of cell nuclei). No cytokeratin- or EGFR-positive cells were detected. References: 1. Xi L, Nicastri DG, El-Hefnawy T, Hughes SJ, Luketich JD, Godfrey TE. Optimal markers for real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma, breast, colon, esophageal, head and neck, and lung cancers. Clin Chem 2007;53(7):