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A two-photon tracer for human epidermal growth factor receptor-2: Detection of breast cancer in a live tissue Ji-Woo Choi, Seung Taek Hong, Dong Eun Kang, Kyu Cheol Paik, Man So Han, Chang Su Lim, and Bong Rae Cho Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00912 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016
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Analytical Chemistry
A two-photon tracer for human epidermal growth factor receptor-2: Detection of breast cancer in a live tissue Ji-Woo Choi†, Seung Taek Hong†, Dong Eun Kang‡, Kyu Cheol Paik§, Man So Han§, Chang Su Lim*,∥, and Bong Rae Cho*,†, ‡,§ †KU-KIST
Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea ‡Department
of Chemistry, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul, 02841, Republic of Korea
§ Department
of Chemistry, Daejin University, 1007 Hoguk-ro, Pochun-si, Gyeonggi-do, 11159, Republic of Korea
∥Department
of Chemistry, Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 16499, Republic of Korea
ABSTRACT: We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 10-50cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90130 m in live mouse tissue by TPM.
Receptor-targeted imaging is emerging as a promising strategy for cancer diagnosis.1 An attractive target for such applications is the human epidermal growth factor receptor-2 (EGFR-2, HER-2, Her-2/neu, ErbB-2), which controls cell growth and differentiation.2 Whereas HER-2 exists at a low level on the healthy epidermal cell surface, it is over-expressed in 2030% of breast and lung cancers, and in other cancers such as ovarian carcinomas and B-cell acute lymphoblastic leukemia.3 Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the most common methods for assessing HER-2 expression levels.4,5 IHC examines immunostaining patterns in cells and tissues labeled with chromogens such as diaminobenzidine, using field microscopy,6,7 whereas FISH utilizes immunofluorescent tracer detection in cells and tissues hybridized with fluorescent molecules such as fluorescein and Texas Red, using fluorescence microscopy.8,9 However, neither of these methods can detect cancers deep inside living tissue. Furthermore, both methods use formalin fixed and paraffin-embedded cells and tissues that require time-consuming pre-treatment,10 during which cells and tissues may be deformed. Therefore, there is a great need to develop a faster and more reliable method of detecting cancer, without pre-treatment, deep inside live tissue. An attractive approach for such applications is the use of two-photon microscopy (TPM). TPM, which utilizes two photons of low energy during excitation, has now become an indispensable tool in biomedical research due to the capability it affords of imaging deep inside live tissue for a long period of time with high spatial resolution.11,12
Figure 1. Structures of Pyr, PLT-yellow, PMT-yellow Pyr-CT, Pyr-SIM, and Pyr-affibody; and synthesis of Pyr-affibody. (a) EDCI, DMP/DMF; (b) Affibody/PBS.
Combined with an efficient two-photon (TP) tracer for HER-2, it is possible to detect HER-2 in a live tissue by TPM. Therefore, we have developed a TP tracer for HER-2, named ‘Pyr-affibody’, comprising Pyr as the fluorophore and a His-affibody oligopeptide (Affibody) that targets HER-2 (Figure 1). Affibodies are oligopeptide antibodymimics engineered to bind to a large number of target proteins or peptides with high affinity.13 We have adopted Pyr from our earlier work because Pyr-CT has an emission maximum at 561 nm with very large effective TP action cross-section (eff = 1555 GM) in the cell.14 We utilized His-affibody oligopeptide as the affibody, because it has a very high affinity for HER-2 (KD ~22 pM), low molecular weight (~10 kDa) compared to that of antibodies (~185
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Analytical Chemistry
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kDa),15 and it has been applied to various carriers, including liposomes, nanoparticles, biopolymers, and adenovirus vectors.13 Herein, we report that Pyr-affibody is an efficient TP tracer for HER-2, which can be used to detect breast cancer on both the cellular and organism levels. ■ EXPERIMENTAL SECTION Synthesis of Pyr-SIM. A mixture of Pyr-CT (0.30 g, 0.56 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide· HCl (0.13 g, 0.67 mmol), and 4-dimethylaminopyridine (6.8 mg, 0.056 mmol) in DMF (10 mL) was stirred for 30 min. Then, N-hydroxysuccinimide (77 mg, 0.67 mmol) was added to this mixture and stirred at RT for 12 hr under argon. The mixture was extracted with CH2Cl2 and dried over MgSO4. After removing the solvent by evaporation, the mixture was separated by silica gel column chromatography, using CH2Cl2/EA (1/1) as the eluent to obtain the product as an orange solid. Yield: 0.20 g (58%); 1H NMR (500 MHz, CDCl ): δ 9.05 (2 H, s), 7.53 (2 H, m), 3 7.44 (1 H, s), 7.43 (1 H, s), 7.15 (1 H, d, J = 2.2 Hz), 6.96 (1 H, dd, J = 8.8, 2.2 Hz), 6.92 (1 H, d, J = 2.2 Hz), 6.81 (1 H, dd, J = 8.8, 2.2 Hz), 4.25–4.21 (2 H, m), 3.95–3.88 (4 H, m), 3.78– 3.75 (2 H, m), 3.63–3.59 (2 H, m), 3.42 (3 H, s), 3.09 (3 H, s), 2.94 (2 H, t, J = 7.0 Hz), 2.87 (4 H, br); 13C NMR (100 MHz, CDCl3): δ 170.0, 167.2, 158.2, 157.7, 156.5, 152.0, 150.8, 147.3, 142.9, 142.2, 140.3, 140.1, 122.2, 121.9, 121.8, 119.3, 113.4, 110.7, 106.9, 106.3, 96.6, 95.0, 71.9, 70.7, 69.6, 67.9, 59.1, 48.8, 39.5, 39.2, 28.6, 25.5 ppm; HRMS(ESI): m/z calcd. for [C33H32N4O9+Na+]: 651.2067, found: 651.2063. Preparation of HER-2 specific affibody. The modified HER-2-specific affibody (ZHER-2:342)17 DNA construct (5’ATGGTTGATAATAAATTTAATAAAGAAATGCGTAATGC TTATTGGGAAATTGCCCTTCTTCCAAATCTTAATAATC AACAAAAACGTGCTTTTATTCGTTCTCTTTATTGATCCT TCCCAATCCGCTAATCTTCTTGCCGAAGCTAAAAAACT CAATGATGCCAAGCTCCTAAATAGCTCGAG-3’), designed for the prokaryotic vector, pET15b, which appends an N-terminal His6 tag and encodes a T7 RNA polymerase site, was purchased from Bioneer, (Daejeon, Korea). His-affibody oligopeptide was produced in Escherichia coli on a pET15b vector. The amino acid sequence: (NH2-MGSSHHHHHHSSGLVPRGSHMVDNKF NKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSAN LLAEAKKLNDAQAPK-COOH). The pET15b vector encoding the HER-2-specific affibody gene was transformed into BL21 (DE3) competent cells (Novagen, Germany). Bacterial cell cultures were initially grown overnight at 37 °C in Luria-Bertani (LB) medium (3 mL), while shaking in an air shaker set at 200 rpm. Cultures were scaled up by growing overnight in LB media (50 mL) under the same conditions, before inoculating 1 L volumes of LB media containing ampicillin (50 mg/L). When the optical density readings (O.D.600 nm) reached a value of 0.6, isopropyl-1-thio-β-D-galactopyranoside (IPTG) (0.5 mL, 1.0 M) was added to cultures to activate the T7 RNA polymerase based expression system. The induced cultures were maintained at 37 °C for 4 hr, before being pelleted by
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centrifugation at 6,000 rpm for 30 min. Cell pellets were resuspended in PBS buffer (1X PBS, pH 8.5, 50 mL) containing phenylmethanesulfonyl fluoride (PMSF) (1 mM), lysed by pulse sonication on ice, and then centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was collected, incubated for 1 hr in a Ni2+nitrilotriacetic acid (Ni-NTA) (5 mL) beads (Qiagen, USA), and allowed to pass through a manually operated gravity column (Bio-Rad, USA). The Ni-NTA beads were washed with PBS buffer (50 mL), and the His-affibody was purified by immobilized metal ion chromatography (IMAC) using imidazole (00.5 M)/PBS as the eluent. The eluted protein was concentrated to 5 mL using a centrifugal filter (Centricon, Millipore, Billerica, MA) with a molecular weight cut-off value (MWCO) of 3,000 Da, while adding small increments of PBS buffer. The product was separated by size exclusion chromatography (SEC) and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of HER-2-affibody in PBS as measured by UV absorption at 280 nm was 50 mg/mL. Conjugation of Pyr-affibody. A solution containing Pyr-SIM (10 mM in DMSO) and affibody (1 mM in PBS) in a NaHCO3 buffer (0.1 M, pH 9.5) was incubated for 4 hr at RT while shaking. Unreacted Pyr-SIM was removed by dialysis (PBS, pH 8.5, 2 days) and passed through a PD-10 desalting column (GE Healthcare, USA). Pyr-affibody was separated by gel filtration, concentrated while washing with PBS in a Centricon filter (MWCO = 3,000 Da), and purified by eluting with distilled water through disposable PD-10 desalting columns. The product was identified by MALDI TOF mass spectrometry and SDS-PAGE. Fluorescence signals were detected in gels upon excitation at 470 nm with an LED Gel Illuminator. The ratio of Pyr to Affibody (number of Pyr molecules coupled to each Affibody molecule) was estimated by counting the number of peaks with successive increments of 515 Da (molecular weight of Pyr) above the molecular ion peak of uncoupled Affibody in the MALDI-TOF mass spectrum. Spectroscopic measurements. Absorption and fluorescence spectra were recorded on an Agilent 8453 diode array UV-Vis spectrophotometer and a Shimadzu RF-5301PC spectrofluorophotometer using a 1-cm standard quartz cell, respectively. The fluorescence quantum yield () was determined by using Coumarin 307 as reference, according to established methods.25 Solubility. The solubility of Pyr-affibody was determined by the fluorescence method as reported.21 Circular dichroism (CD) spectra. The CD spectra were collected under nitrogen gas on a Jasco J-715 spectropolarimeter equipped with a thermostated cell holder and a Peltier bath, using a quartz cell with 1 mm path length. The stock solutions of affibody and Pyraffibody were prepared in nitrogen-purged PBS buffer, and the concentrations were determined by UV-vis spectroscopy. The CD spectra of the affibody and Pyraffibody were recorded in the wavelength range of 200–260 nm at 20 °C. The thermal denaturation experiments were
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Analytical Chemistry
performed over 20 °C and 100 °C, with 1 °C increments and 1.5 min equilibration times between temperature changes. The following parameters were used during acquisition: 2nm scale, continuous scanning, 100 nm/min velocity, and accumulation of 5 runs. Molar residue ellipticities (MRE, deg cm2 dmol−1) were calculated using the following equation:
where θobs is the observed ellipticity in millidegrees, C is the molar concentration, l is the path length of the cell in cm, ‘residue’ refers to the number of residues in the sequence and 10 is a conversion factor to dmol.19 The melting temperature (Tm) was determined by an established method.20 Measurements of the TP action cross-section (δФ). The TP action cross-section (Фδ) value for the Pyr-affibody was determined by a fluorescence method as reported.23 The Фδ value of Pyr-affibody was calculated by assuming that three molecules of Pyr are attached to each molecule of Affibody. Measurements of effective TP action cross-section (Фδeff). The effective value of the TP action cross-section (Фδ)eff was calculated using the equation (Фδ)eff = Φr δr(Ip’/Ir’): where Ip’ and Ir’ are the TPEF intensities from the probe-labeled cells and 5.0 M of Rhodamine 6G in MeOH in delta T-dishes measured under the TPM setup, respectively.14 However, the TPEF intensity emitted from 5.0 M of Rhodamine 6G in MeOH in delta T-dishes was much weaker than that from the probe-labeled cells. Since the TPEF intensity of Rhodamine 6G in MeOH was linearly proportional to the concentration up to 50 M, we have measured the TPEF from 50 M of Rhodamine 6G in MeOH and divided it by 10 to obtain Ir. The intracellular concentration of the probe was estimated using the equation cp = cr(Φδ)eff/Φpδp by assuming that the TPEF intensity of the probe in the cell would be the same as that in the PBS buffer. Cell cultures. SK-BR-3, MCF-7, MDA-MB-231, HT-29, NCI-H460, and HeLa cell lines were all obtained from the American Type Culture Collection (Rockville, MD) and cultured according to their specifications. SK-BR-3 (human breast carcinoma cell line), HT-29 (human colorectal adenocarcinoma cell line), and NCI-H460 (human large cell lung carcinoma cell line) were cultured in RPMI 1640 (WelGene Inc, Seoul, Korea); HeLa (human cervical carcinoma cell line), MCF-7 (human gland/breast carcinoma cell line), and MDA-MB-231 (human gland/breast carcinoma cell line) were cultured in DMEM (WelGene); and all cells were supplemented with 10% FBS (WelGene), penicillin (100 units/ml), and streptomycin (100 μg/ml). The cells were kept under the humidified atmosphere in an incubator containing 5/95 (v/v) of CO2/air at 37°C. Two days before imaging, the cells were detached and re-placed on glass-bottomed dishes (MatTek). For labeling, the growth medium was removed
and replaced with RPMI without FBS. The cells were incubated with Pyr-affibody (3 μM) for 20 min at 37°C, then washed three times with RPMI without FBS, and imaged. Western blot analysis of HER-2 protein in cells and tissues. Samples (cells and ex vivo slices) were homogenized in a RIPA buffer (50 mM Tris, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.4) containing a protease inhibitor cocktail tablet (Roche, Palo Alto, CA, USA). The mixture was centrifuged at 14,000 rpm (10 min, 4C) to isolate the supernatant. The Bradford assay (Bio-Rad, USA) was used to measure the total protein concentration present in the lysate supernatant. An equal amount of lysate protein from each of the 6 samples was loaded onto a 10% polyacrylamide gel and separated by SDS-PAGE. The gel was transferred to a polyvinylidene fluoride (PVDF) membrane with a semi-dry blotting system. The PVDF membrane was blocked using 5% skim milk in Tris buffered saline containing 0.1% Tween 20 (TBST) at RT for 1 hr. The membrane was incubated overnight with a rabbit monoclonal anti-human HER2/ErbB-2 antibody (1:1000; Cell Signal Technology, USA), using a -actin antibody (1:5000; Santa Cruz biotechnology, USA) as a loading control. The membrane was washed three times (15 min, each) and then incubated with horseradish peroxidase conjugated to anti-rabbit antibody (1:5000) in TBST for 1 hr. The chemiluminescence intensities of the PVDF membranes treated with lysates obtained from healthy and malignant ex vivo slices were compared using an ECL kit (Pierce, MA USA). Selectivity. The selectivity of Pyr-affibody for HER-2 receptors was tested using the SK-BR-3 cells and tissue slices from xenograft models. MCF-7 cells and healthy tissue were also used as the control. The cells were incubated with 300 μM of His6-affibody for 1 hr, labeled with 3 μM of Pyr-affibody, and then washed 3 times with PBS buffer16. The TPEF intensities of the images obtained with and without treatment with His6-affibody were compared. The selectivity study for the tissue sample was conducted by the same method except that the concentrations of Pyr-affibody and His6-affibody were increased by 10-fold to facilitate staining. TPM imaging. TPM images of tracer-labeled SK-BR-3 human breast carcinoma cells and ex vivo slices were obtained over the 450–650 nm range using a multiphoton microscopy as described. 22 Cytotoxicity. The cytotoxicity of Pyr-affibody was measured by using a Cell Counting Kit-8 (CCK-8 kit, Dojindo, Japan), following the manufacturer’s protocol. The results are shown in the Supporting Information (Figure S5).
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Analytical Chemistry Photostability. Photostability was determined by monitoring the TPEF intensity in SK-BR-3 human breast carcinoma cells and ex vivo slices labeled with Pyr-affibody, as described (Supporting Information, Figure S4). 22
■ RESULTS AND DISCUSSION Preparation of Pyr-affibody. Pyr-SIM was obtained at a yield of 58% by the coupling of Pyr with Nhydroxysuccinimide. His-affibody oligopeptide (NH2MGSSHHHHHHSSGLVPRGSHMVDNKFNKEMRNAYWE IALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDA QAPK-COOH, Affibody) was produced recombinantly in Escherichia coli as reported by Orlova et al. (2006).17 PyrSIM was then reacted with Affibody to obtain Pyr-affibody (see the ‘Experimental section’ for details). The molecular weight of Pyr-affibody was almost the identical to Affibody (10 kDa), as indicated by the SDS-PAGE analysis (Figure 2a). When the gel was excited at 470 nm, it also showed a strong emission band at 10 kDa and a weaker one at 20 kDa, the latter of which can be attributed to the dimer, which exists in equilibrium with the monomer (Figure 2a).18 In addition, the MALDI-TOF mass spectrum of Pyr-affibody showed a base peak corresponding to the molecular weight of the Affibody and three smaller peaks in 515 Da increments above it (Figure 2b). These results confirmed that Pyr is indeed conjugated to the Affibody, and that 3 molecules of Pyr are attached to each molecule of Affibody.
Conformation of Pyr-affibody. To assess whether the conformation of the Affibody changes upon conjugation to Pyr, we obtained the CD spectra of both the Affibody and Pyr-affibody (Figure 3a).19 They were nearly identical. In addition, the thermal denaturation curves of the two molecules overlapped well with identical Tm values of 66. 2 ± 0.2C (Figure 3b).20 These results confirm that the 3dimensional structures of the Affibody and Pyr-Affibody are almost identical. (a)
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Preparation of xenograft mice tissue. Ex vivo slices were obtained from the epidermal tissues of 12-week-old cBALB nu/nu mice. We acquired 8 slices each from healthy and tumor-bearing mice. The tissues were placed in sterile specimen bottles containing PBS buffer. Subsequently, 30 µM Pyr-affibody in PBS was added and the mixture was incubated at 37°C for 1 hr, in an atmosphere containing 95% O2 and 5% CO2. Following incubation, slices were washed three times with PBS, transferred to glass-bottomed dishes (MatTek), and observed in a spectral confocal multiphoton microscope.
Figure 2. (a) SDS-PAGE analysis of Affibody and Pyr-affibody by molecular weight (left) and fluorescence (right). The fluorescence signal was obtained following excitation at 470 nm using an LED Gel Illuminator. (b) MALDI-TOF mass spectrometry results for Affibody and Pyr-affibody.
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Xenograft models. Xenograft models were grown and tissues were obtained in accordance with the approved protocol of the institutional review board of Korea University Medical Center, Anam Hospital. Institutionally bred, 6-week-old female c-BALB nu/nu mice were used (Orient Bio Inc., Korea), with body weights in the range 1825 g at the start of the experiment.24 The animals were maintained under pathogen-free conditions at the Korea University College of Medicine, Animal Research Center laboratory. Food and water were supplied ad libitum. The mice were reared for 2 weeks and then injected subcutaneously with a mixture of 1 × 107 SK-BR-3 cells (~100 µL each) and Matrigel (~100 µL each, BD Bioscience, USA). Each mouse was ear-tagged and monitored throughout the study. Tumor growth was monitored twice a week using calipers. Tissue slices were taken for the imaging experiments 4 weeks after inoculation, when the diameter of the tumors attained ~5 mm (equivalent to 100 mg).
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Figure 3. Circular dichroism measurements of Affibody (●) and Pyr-affibody (○). (a) Circular dichroism (CD) wavelength spectra at 20°C. (b) Thermal denaturation monitored by CD at 222 nm. A two-state folding model was used to determine the melting temperature. Tm = 66.2 ± 0.2°C.
Photophysical properties. The solubility of Pyraffibody in PBS buffer, as determined by the fluorescence method,21 was 3.0 µM (Figure S1), which was sufficient to stain the cells. Pyr-affibody showed an absorption maximum (max) at 439 nm ( = 93,100), with an emission maximum (fl) at 574 nm ( = 0.040) in PBS buffer (pH 7.4) (Figure S2). The max of Pyr-affibody was similar to that of Pyr-derived TP tracers such as Pyr-CT (437 nm), PLTyellow (444 nm), and PMT-yellow (440 nm).14,22 However, these other tracers emitted only scant fluorescence in PBS buffer, which increased with concomitant decrease in the fl values in a less polar solvent. 14,22 Hence, the shorter fl and much brighter fluorescence of Pyr-affibody may be attributed to the hydrophobic pocket in the Affibody helical bundle structure within which Pyr can be located. Two-photon brightness. Pyr-affibody emitted broad TP excited fluorescence (TPEF) centered at 555 nm in the
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cell, and not in the other cell lines. Moreover, the TPM image of the SK-BR-3 cells pre-incubated with a 100-fold excess His6-affibody was much dimmer than that of untreated cells, indicating almost complete binding inhibition of the HER-2 receptor (Figure S6b,d). This outcome indicates that Pyr-affibody competes with His6affibody for the HER-2 receptor.16 A similar result was observed in the tumor tissues labeled with Pyr-affibody (Figure S6f,h). In contrast, there was little change in the TPM images of MCF-7 cells and healthy tissues upon treatment with His6-affibody (Figure S6a,c and 6e,g). These results established that Pyr-affibody can selectively detect HER-2 over-expressing cells and tissues by TPM.
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Figure 4. (a) TPEF spectra of Pyr-affibody in PBS buffer and HeLa cells. The excitation wavelength was 740 nm. (b) TP excitation spectra of Pyr-affibody in PBS buffer and HeLa cells.
We also determined the effective Фδ values (Фδ)eff by comparing the TP excited fluorescence (TPEF) intensity of the tracer-labeled cells with that of the 5.0 M Rhodamine 6G in MeOH, typical concentration to stain the cells, under the imaging condition (see the ‘Experimental section’ for details).14 The TPEF emitted from the Pyr-affibody labeled The TP action cross-section (Фδ) of Pyr-affibody in PBS buffer was determined by the fluorescence method.23 The Фδ value was calculated by assuming that three molecules of Pyr are attached to each molecule of Affibody. The Фδmax value was 40 10-50cm4s/photon (GM) at 740 nm (Figure 4b). The value for the Pyr-affibody, which is much larger than the near-zero values determined for other Pyr derivatives, can be attributed to the much larger Ф value (see above). We also determined the effective Фδ values (Фδ)eff by comparing the TP excited fluorescence (TPEF) intensity of the tracer-labeled cells with that of the 5.0 M Rhodamine 6G in MeOH, typical concentration to stain the cells, under the imaging condition (see the ‘Experimental section’ for details).14 The TPEF emitted from the Pyr-affibody labeled cells was brighter than that of the reference by 10.6-fold. The (Фδ)eff value calculated from this ratio was 600 GM at 730 nm (see the ‘Experimental section’ for details), a value sufficient to obtain bright TPM images (Figure 4b). A comparison of Фδmax and (Фδ)eff values indicates that the intracellular concentration of Pyr-affibody is approximately 75 M. This value is undoubtedly overestimated because (Фδ)eff value was measured in the bright regions in the TPM images and the Фδmax value measured in the PBS buffer should be smaller than that in the cells due to the more hydrophilic microenvironment around Pyr-affibody in the aqueous buffer (see above). Nevertheless, the much higher concentration of Pyr-affibody than in the staining medium (3 M) can be attributed to the more favorable interactions that makes in the intracellular environment than it does in the staining medium. The TPM image of the SK-BR-3 cells was 47-fold brighter than that of all others (Figure 5g). This is a result consistent with the western blot experiments, since it demonstrates that HER-2 is over-expressed in the SK-BR-3
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Figure 5. (a-f) TPM images of various cell lines labeled with 3 µM Pyr-affibody. Images were obtained by collecting the emission at 450650 nm upon excitation at 750 nm. Scale bar = 30 µm. (g) Average TPEF intensity in each cell line. Cells shown are representative images from replicate experiments (n = 15). (h) Results of western blot experiments.
Pyr-affibody has the additional benefits of pH insensitivity at biologically relevant pH (Figure S3), high photostability as revealed by the negligible changes in the TPEF intensity in the Pyr-affibody-labeled SK-BR-3 cells and mouse tissues over 60 min (Figure S4), and a negligible decrease in cell viability as measured by a Cell Counting Kit (CCK)-8 assay (Figure S5). Detection of HER-2 in live tissue. We also evaluated the capability of detecting HER-2 over-expressing cells in live tissues. Institutionally bred, 6-week-old female c-BALB nu/nu mice were used (Orient Bio Inc., Korea), with body weights in the range 1825 g at the start of the experiment.24 Mice were grown in accordance with the protocol approved by the institutional review board of Korea University Medical Center, Anam Hospital. The tissues were obtained 4 weeks after inoculation, when the diameter of the tumors attained ~5 mm (see the ‘Experimental section’ for details).
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Analytical Chemistry (a)
■ CONCLUSIONS
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We have developed a TP tracer conjugated to a HER-2specific affibody (Pyr-affibody) that shows a large effective TP action cross-section and has high selectivity for HER-2 protein. We have demonstrated that it can detect breast cancer in live cells and deep inside live tissues by TPM with minimum interference from cytotoxicity, photodamage, or pH within the biologically relevant range. This tracer may find useful application in the broad-based diagnosis of cancers associated with the HER-2 protein. Further, our approach may serve as a new design strategy for generating TP biomarkers that target proteins-of-interest with high selectivity and specificity.
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Supporting Information. Figures S1−S7 and NMR data (Figures S8-10). This material is available free-of-charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Normal
Tumor
Corresponding Authors * E-mail:
[email protected];
[email protected] Figure 6. (a,b) Sectional and (c) Three-dimensional TPM images of tumor tissue samples labeled with Pyr-affibody (30 μM) at a depth of 90−130 μm with magnification at (a) 20× and (b, c) 100×. Images were obtained by collecting the emissions at 450650 nm upon excitation at 750 nm. The white boxes in Panel (a) indicate the 3 regions where 150 sectional images were collected at a magnification of 100×. TPM images of the healthy (d, e) and tumor (f, g) tissues labeled with Pyraffibody. The images were obtained at a depth of 110 μm. Magnification at 20× (d, f) and 100× (e, g). (h) The results of western blotting (top) and TPEF intensity in healthy tissue and tumor tissue (bottom). Much higher levels of HER-2 protein are found in xenograft samples than in healthy samples. Tissues shown are representative images from replicate experiments (a,d,f: n = 12; b,e,g: n = 36). Scale bars: (d, f) 150 µm, and (e, g) 30 µm.
We obtained 8 ex vivo slices from the tumor mice and control mice. The slices were labeled with Pyr-affibody and 450 TPM images were obtained for each slice; i.e., 3 TPM images for every 150 sections at a depth of 90130 m along the z-direction (Figure 6a and Figure S7). The TPM images of Pyr-affibody-labeled tissues taken at the middle section were much brighter in the tumor tissue than in the regular tissue from healthy mice (Figure 6d-h). Moreover, the average TPEF intensity calculated from 3,600 TPM images was more than 7-fold higher in the tumor tissue than in the regular tissue (Figure 6h, bottom). Furthermore, western blot analysis of the samples used in the imaging study indicated much higher levels of HER-2 protein expression in the tumor tissue than in the regular tissue (Figure 6h, top). These results demonstrated the capability of Pyraffibody for detection of HER-2 over-expressing cells in ex vivo slices by TPM.
Author Contributions The manuscript was written through the contributions of all the authors. All authors have given their approval to the final version of this manuscript.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This research was supported by the National Research Foundation of the Korea (NRF) grant funded by the Korean government (NRF-2015R1A2A2A01004052, NRF2013R1A6A3A04058351).
ABBREVIATIONS IHC: Immunohistochemistry; FISH: Fluorescence in situ hybridization; TPM: Two-photon microscopy; TP: Twophoton; PBS: Phosphate-Buffered Saline; TPEF: Two-photon excited fluorescence
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Analytical Chemistry
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Xenograft model
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