Peptide-Based Method for Detection of Metastatic Transformation in

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Peptide-Based Method for Detection of Metastatic Transformation in Primary Tumors of Breast Cancer Hao Li,† Yue Huang,† Yue Yu,‡ Weiwei Li,† Yongmei Yin,§ and Genxi Li*,†,∥ †

State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, Nanjing University, Nanjing 210093, China Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China § Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ∥ Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China ‡

Downloaded by RUTGERS UNIV on August 29, 2015 | http://pubs.acs.org Publication Date (Web): August 28, 2015 | doi: 10.1021/acs.analchem.5b01750

S Supporting Information *

ABSTRACT: Detection of metastatic activity before the onset of the actual metastasis can be a promising method to combat metastasis, the foremost cause of death in cancer. Therefore, in this work, we have developed an assay method for the detection of metastatic tumor cells in primary tumor, by using a protein of the metastatic cell signaling as the biomarker. In this assay, a peptide-based probe targeting the marker protein and a sensitive nanoparticle doped graphene nanolabel are combined to enable the detection of metastatic cells. Consequently, the metastatic cells can be specifically detected and discriminated from primary tumor cells. By applying this assay method to clinical samples of primary tumor, a low amount of metastatic activity can be detected in the tumor sites, which may suggest the activity of local metastatic transformation. So, these results may point to the prospect of using the proposed method for controlling metastatic cancer.

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discriminating metastatic cells from the primary tumor cells; instead, metastatic markers, such as Smo used in this work, are able to provide early warning of metastasis in primary tumors. Due to its intimate connection with metastasis, Smo has become a therapeutic target for antimetastatic drugs;10 thus, a probe has been successfully constructed in this work by using a Smo specific drug in combination with peptides and other synthetic agents. Moreover, since peptide-based probes have been well studied in this lab, specific modifications can be employed to give them more diverse biosensing functions,11−14 for the development of a method to detect metastatic tumor cells in primary tumor. On the other hand, to obtain a satisfactory performance of the cell assay, another governing factor has also been considered, namely, the low amount of metastatic cells in the primary tumor sites. Accordingly, we have equipped our cell assay with a nanolabel loaded with large numbers of sensitive nanoparticles, enabling a sensitive method to detect metastatic cells. To our knowledge, this is the first time that novel biosensing elements such as peptides and nanolabel are applied in Smo bioassay. The ability of this method in specifically and quantitatively detecting metastatic cells is first verified using various types of primary tumor cells as the control. Then, in the examination of clinical samples using

n cancer, up to 90% of death is caused by metastasis, or the uncontrollable migration and proliferation of malignantly transformed tumor cells. In order to stop metastasis at an early stage, great efforts have been devoted to improve the understanding of it. Nowadays, it is found that metastasis originates from the metastatic transformation of a few tumor cells in the primary tumor sites.1,2 This discovery helps to strengthen the notion that detection of metastatic transformation, even before the onset of clinically observable metastasis, is essential for the effective control of metastasis. So, it is urgently needed to find a method that can specifically target metastatic activity in cells of the primary tumor sites.3,4 In this work, such a method is developed by using a cell surface metastatic protein as the biomarker. This protein, Smo, is known as an effector protein of the Hedgehog signaling pathway, a pathway that is frequently up-regulated in tumor cells for initiating and sustaining the metastatic transformation. In the previous reports, the employed biomarkers are epithelial markers overexpressed by the primary tumor cells. Conventionally, metastatic cells are treated simply as the direct descendants of the primary tumor cells. Nevertheless, recent researches have yielded results quite different from the conventional view.5,6 According to these latest findings, metastatic cells have undergone a transformation termed epithelial-mesenchymal transition (ECM), in which the parental epithelial characteristics are lost and replaced by mesenchymal characteristics such as high proliferative and invasive power.7−9 So, epithelial markers cannot be effective in © XXXX American Chemical Society

Received: May 8, 2015 Accepted: August 24, 2015

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DOI: 10.1021/acs.analchem.5b01750 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

nanolabel, UV−vis measurements were taken at 25 °C using a Shimaztu UV-2450 spectrophotometer. Dynamic light scattering (DLS) measurements were conducted on a particle size analyzer (Brookhaven 90Plus, USA) under the spreadsheet mode (scattering angle, 15°) and at 25 °C. Transmission electron microscope (TEM) images were taken using Hitachi H7650 with accelerating voltages of 80 kV. The Atomic force microscopy (AFM) images are recorded using Agilent 5500 AFM system. Samples were imaged at a scan rate of 0.5−1 Hz in a tapping mode. AFM tips with resonant frequency in a range 160−260 kHz were used. Images were acquired at a resolution of 512 × 512 pixels. ITC study of probe/Smo interaction. Isothermal titration calorimetry (ITC) measurements were conducted using a MicroCal ITC200 System (GE healthcare life sciences) at 25 °C. The titration schedule consisted of 38 consecutive injections of 1 μL with at least a 120 s interval between injections. All solutions were degassed prior to titration. Electrode treatment and modification. These steps were essentially the same as previously reported.14 Briefly, the electrode was reacted with 5 μM peptide in 10 mM PBS (pH 7.4) at 4 °C for 16 h, followed by being dipped in 1 μM 9mercaptononanol for 3 h. Cell assay. The modified electrode, after incubation with cell sample at 37 °C for 60 min, was thoroughly rinsed first with 5% tween-20, to lyze the surface-bound cells, and then with ddH2O, to reduce nonspecific absorption. Thermolysin cleavage (7 nU/ml thermolysin in 50 mM Tris-HCl, 0.5 mM CaCl2 and trace amount of ZnCl2, pH 8.0) was then applied for 80 min at 50 °C, followed by rinsing and 6 min incubation with the nanolabel. After a further rinsing step, the electrode was ready for signal readout. Electrochemical measurements. These steps were essentially the same as previously reported.14 Briefly, electrochemical measurements were carried out on a CHI660D Potentiostat (CH Instruments) with a conventional threeelectrode system: the electrode immobilized with peptide as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. Square wave voltammograms (SWVs) were recorded in 10 mM PBS, pH 7.4, which was deoxygenated by purging with nitrogen gas and maintained under this inert atmosphere during the electrochemical measurements.

our method, a low amount of Smo overexpressed metastatic cells is detected in primary tumors, pointing to the effective use of this method in the diagnosis of metastasis.

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EXPERIMENTAL SECTION Chemicals and reagents. Graphene oxide (GO), chemically exfoliated, was from Nanjing XFNANO Materials Tech Co., Ltd. Peptide probe and clickable peptide, their sequence as shown in Figure 1, were synthesized by Shanghai Science

Figure 1. (a) Structure of the probe. (b) Structure of the nanolabel. (c) Detection procedure. All Figures are not drawn to scale.

Peptide Biological Technology Co., Ltd., as lyophilized powder, purity >95%. Human recombinant Smo was from Abnova. Other reagents were of analytical-grade. Powder of the peptide probe was dissolved to 5 μM in 10 mM phosphate buffer solution (PBS) (pH 7.4). The same solution was used to dissolve Smo. For all solutions, double-distilled water (ddH2O) was prepared in Milli-Q purification system to 18 MΩ·cm. T47D, from Type Collection of Chinese Academy of Science, was cultured in Dulbecoo’s Modified Eagle’s Medium (DMEM, from Gibco co.) containing 10% fetal cattle serum (FCS, from Hyclone co.) and maintained in a humidified atmosphere with 5% CO2 at 37 °C. Biopsy samples from breast cancer patients were obtained from the Department of General Surgery of the Affiliated Hospital of Nanjing University, after elected consent by the local ethical committee. The retrieved samples were immediately sliced on ice to 1 mm3, followed by digestion with type II collagenase at 37 °C for 30 min. The resulted supernatant was centrifugated at 800 rpm for 5 min, the pellet was resuspended with 10 mM PBS, counted and diluted to 1 × 105 cell/mL, ready for cell assay. Synthesis and characterization of the nanolabel. First, ddH2O suspension of GO was probe-sonicated (Scientz-IID) for 300 min, followed by 1000 rpm centrifugation for 30 min. The supernatant was then subject to several rounds of centrifugation filtration (500 kDa filter) and dried in vacuum at room temperature. After that, 100 μg/mL resuspended GO was conjugated with 50 μM clickable peptide using 5 mM NHS and 10 mM EDC. After overnight-stirring at ambient condition, the resulted modified GO was repeatedly washed, filtered and resuspended to 1 mL. Then 100 μL modified GO was mixed with 150 μL ddH2O, 750 μL ο-phenylenediamine (OPD) (0.437 mg/mL) and trace amount of CuCl2, water-bathed at 60 °C for 30 min. The obtained nanolabel was washed, filtered and reconstituted with 10 mM PBS to 1 mL. To characterize the

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RESULTS AND DISCUSSION Figure 1 may illustrate the principle of the proposed method for cell assay. We have first designed a peptide-based probe for targeting Smo (Figure 1a), by attaching cyclopamine, a wellstudied anti-Smo drug,15,16 to a peptide. Since the conjugation is realized through a nonessential group of cyclopamine, its affinity toward Smo can be preserved. To convert target binding to signal readout, the peptide-based probe is also designed to be able to not only conjugate with cyclopamine but also have a binding site (clickable azide group) of the nanolabel at one end, at the other end, the probe has a thiol-group for surface immobilization, separated by a cleavage site of thermolysin in between. Since target binding can shield the probe from thermolysin cleavage, the subsequent attaching of nanolabel, the structure of which is shown by Figure 1b, can then lead to signal readout. Moreover, since each nanolabel is loaded with a large number of nanoparticles consisting of electroactive organic molecules, high sensitivity of the detection can be achieved. B


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Analytical Chemistry The nanolabel is also designed to be modified with Glycine10 (Gly10) peptides bearing the clickable dibenzylcyclooctyne groups at the ends. The biorthogonal group is for attaching the nanolabel to the probe via copper-free click chemistry, while the Gly10 linker is necessary for the nanolabel to negotiate the steric-hindrance in its binding with the probe. In the detection procedure, as shown in Figure 1c, the probes, immobilized on the surface of the working electrode, can capture target cells via multiple specific interactions with the cell surface Smo. The captured cells are then lyzed, thus only the Smo molecules are left, being bound by the probes. Next, thermolysin cleavage is employed to remove Smo-free probes from the electrode surface, while Smo-bound probes can be shielded from cleavage by the captured Smo molecules. Subsequently, the nanolabels are attached to the Smo-bound probes. The great numbers of nanoparticles loaded in the nanolabel can then give rise to prominent signal readout, due to the electroactivity of 2,3diaminophenazine (DAP) in the nanoparticles. Previously, we have employed the cupric ion-catalyzed rapid oxidation of OPD to DAP, as the means to amplify signal.14 In re-examining this reaction, the product is found to be a colloidal suspension of nanoparticles (Figure S1). Furthermore, centrifugation-separated and resuspended nanoparticles still show evident electrochemical response (Figure S2). For biosensing application, this electroactive nanoparticle can be synthesized in the presence of GO, to form a nanolabel. In fabricating the nanolabel, GO is first modified with the clickable peptides. After conjugation, the original GO (Figure S3, left column) is densely modified by the peptide (Figure S3, right column). The electroactive nanoparticles are then synthesized in the presence of the modified GO, as is shown in Figure 2a. The amount of OPD used, as well as the reaction time have been optimized to avoid excessive formation of nanoparticles (Figure S4 ∼ S5). Under the optimized condition, the nanolabel shows characteristic surface morphology on AFM images (Figure 2b): the GO is densely occupied by the nanoparticles. This feature is then confirmed by TEM images (Figure 2c), in which GO, similar in size to AFM result, is shown densely decorated with nanoparticles. Judging from the size of nanoparticle shown with TEM, and the thickness of the nanolabel, as well as that of unmodified GO, on AFM height map, the label is confirmed to have a single layer of nanoparticle. After click-attached to the surface-immobilized probes, the label can give rise to evident electrochemical response (Figure 2d). Function of the designed probe has been evaluated by various methods. First, the proper function of the Smotargeting motif is verified by investigating probe/Smo interaction using ITC. Probe/Smo binding may result in a typical sigmoid binding curve (Figure S6), with a Kd of 50 nM, comparable to previous report.16 So the Smo-targeting motif can efficiently recognize Smo in the presence of the other functional motifs. Second, the effective inhibition of protease cleavage by Smo binding is examined by applying thermolysin cleavage to surface immobilized probes in the presence/absence of surface-bound Smo. To directly report the effect, azide group of the probe is replaced by ferrocene. As shown by the resulted CVs of ferrocene signal (Figure S7), Smo-binding can effectively block the otherwise thermolysin cleavage. To verify the detection procedure depicted in Figure 1c, electrochemical impedance spectra (EIS) is recorded step by step. As is shown in Figure 3, both a straight line (curve a) representing the bare electrode and a small semicircle

Figure 2. Characterization of the nanolabel. (a) UV absorption recorded under various conditions. A/A′, UV spectra of the reaction mixture before/after nanoparticle formation in the absence of GO; B/ B′, the same reaction recorded in the presence of GO; C/C′, UV of GO before/after reaction. The inset is the photoimage corresponding to the UV curves. Left to right: upper row, A → C; bottom row: A′ → C′. (b) AFM topographic image of the nanolabel; the inset is the height image for a selected piece of nanolabel, and the height distribution along the marked line is also shown below. (c) TEM image of the nanolabel; the inset is an enlarged view of the nanoparticle. (d) Cyclic voltammogram (CV) of the nanolabel. . The arrow marks the scan direction; scan rate: 0.1 V/s.

Figure 3. Stepwise electrochemical impedance spectra (EIS) of the electrode in the process of the cell assay. (Target T47D cell concentration: 1 × 105 cell/mL.)

manifesting the surface monolayer of probe (curve b) can be obtained, while a drastic augment of resistance to electron transfer is observed after the capturing of target cells (curve c). After cell lysis and thoroughly rinsing of the electrode surface, the resistance drops evidently (curve d), and a subsequent thermolysin cleavage further reduces the resistance (curve e). Finally, binding of nanolabel increases resistance, due to its size and charge (curve f). Meanwhile, AFM images taken at major steps of the detection procedure (Figure S8) also agree with the EIS data: the probe modified electrode shows moderately dense spots representing peptide probes (a); the electrode after cell lysis shows, on the contrary, prominent patches of great height (b); while protease digestion leaves only residual elevated spots (c); and finally, the nanolabel binding can also be observed (d). C


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Smo expression. As is shown in Figure 5, even at very high cell concentration, these cells can only have background signal. This

Next, several aspects of the experimental procedure have been examined. First, surface density of probe needs optimization because only probes of sufficient density can provide multiple probe/Smo interactions strongly enough for capturing target cells. Besides, since the cells were paved on the probes, the interface between the probe and a cell may take up less than half of the cell membrane. So, probe with ferrocene instead of azide group is employed to study probe density (Figure S9a, b), and it is observed that the response of ferrocene levels off as probe concentration for electrode modification grows high. At 5 μM, maximum surface density is achieved. Second, probes with different concentrations are employed to modify the electrode, and assays of a given amount of the target cells are conducted (Figure S9c, d). The obtained signal response of the nanolabel approaches plateau beyond 1 μM of probe, so 1 μM is enough for surface modification to capture the target cells. Third, the influence of nanolabel size and size distribution on the reproducibility has also been investigated, thus nanolabels with varied size and size distribution (Figure S10) are separately applied to assay a given amount of target cells. In all assays, signal response is very similar (Figure S11). So morphology of the nanolabel has no evident influence on the reproducibility, owing to the small size of the label in relative to target cells. Other conditions of the detection have also been optimized. First, 60 min incubation of the cell sample leads to nearly saturated response (Figure S12), so this incubation time is selected. Second, sufficient amount of thermolysin is applied to the electrode for gradually longer time, and 80 min for effective digestion has been known (Figure S13). Third, the time required for click-attaching of nanolabel has been studied (Figure S14), and 6 min is known to be sufficient for this labeling process to approach saturation. Under the optimal conditions, the assay method is then employed to detect cancer cell lines. T47D is a Smo overexpressed cell line derived from metastatic breast cancer. Us-ing buffer diluted T47D suspension as the standard sample, signal response of the assay increases in proportion to cell concentration. A linear relation to the logarithm of cell concentration can be established from 1 × 102 cell/mL to 3.16 × 105 cell/mL, with the limit of detection calculated as 0.65 × 102 cell/ml, at the signal-to-noise ratio of 3:1 (Figure 4). The standard deviation of all repetitive measurements is within 5%. Specificity of the assay method has also been studied by detecting other breast cancer cell lines which have much lower

Figure 5. SWVs showing the specificity of the assay; the inset is the enlarged view of the peak response in detecting control cell lines. All the cells are diluted to 1 × 105 cell/mL.

confirms the ability of our assay method in discriminate metastatic cell from other types of cells in the primary tumor samples. In addition, the ability of this method to detect serum sample has been verified as well by quantitative analysis of T47D diluted with serum of healthy volunteer, and the obtained results are also satisfactory (Figure S15). In detecting these complicated samples, the stability of probe and nanolabel has been studied (Figure S16, S17), the results suggest an acceptable stability of these sensing elements. Our cell assay method is then extended to clinical samples of breast cancer. In the meantime, since the conventionally employed markers of breast cancer, such as human epidermal growth factor receptor, have no explicit connection with metastasis, while Smo has been known to have intimate connection with metastasis,17,18 and Smo has been employed in our method, it may also evaluate the metastatic potential of primary breast cancer. First, effectiveness of our proposed method in analyzing clinical sample is examined by detecting cells retrieved via mastectomy from a stage II primary case, as well as by detecting cells obtained in the liver lesion of metastatic breast cancer (Figure 6). The retrieved cells are counted and diluted, and it can be known that the well-diluted metastatic cell sample can still have evident signal (Figure 6a, 6b). On the contrary, only slightly diluted or even undiluted stage II sample (Figure 6c, 6d) can have signal readout, indicating early metastatic events in the primary stage II tumor, consis-tent with the recently advanced understandings of metastatic progression. Second, the assay is extended to more samples from patients of different stages of the primary tumor, as well as the metastatic stage. Cell samples from different sampling sites such as the primary tumor sites, sentinel lymph nodes surrounding the primary tumor sites and the distant metastatic sites are counted and diluted to 1 × 105 cell/mL. The signal response of different stages is then compared (Figure 7): readout of stage II is just discernible, suggesting the early sign of metastatic transformation among the primary tumor cells. Stage III, or the locally advanced cancer, shows larger signal, indicating the formation of metastatic cell clusters in the primary tumor. Finally, as a control, the signal of stage IV, the distant metastatic cancer, is much more prominent. In all the primary stages, the low or moderate level of metastatic signal response, especially that detected in the sentinel lymph nodes, is an unequivocal sign of invasion. These results are also

Figure 4. (a) Square wave voltammograms (SWVs) of the nanolabel for quantifying cultured T47D cell. (b) Calibration curve of SWV peak response in (a) as a function of cell concentration; the inset is the linear relationship between the logarithm of cell concentration and signal response, with error bars representing the standard deviation (n = 3). D



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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01750. Various experimental conditions, as well as validation of the detection principle (PDF)

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AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant Nos. 21235003, 21327902), and the National Science Fund for Distinguished Young Scholars (Grant No. 20925520).

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Figure 6. (a) SWVs obtained in detecting cell samples from a metastatic case. (b) Relationship between the signal response and cell concentration in serial dilution of the cell sample, with error bars representing standard deviation (n = 3). (c) and (d) have the same meaning as (a) and (b), respectively, except that the cell samples are collected from a stage II case.

REFERENCES

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Figure 7. Distribution of signal response in detecting cell samples from breast cancer pathological samples of the primary tumor sites, sentinel lymph nodes, as well as the distant metastatic sites, in different stages of cancer. Cell samples are counted and diluted to the same concentration, 1 × 105 cell/mL.

comparable with that obtained with conventional methods (Figure S18), validating the feasibility of this method.

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CONCLUSION We have developed a cell assay method to evaluate the metastatic potential of the primary tumors. This method may also offer precaution for controlling metastasis. For the development of this method, a peptide-based probe is designed to realize cell capturing and signal generation via its multiple functional motifs. In the meantime, a nanolabel is also designed for amplified signal readout by using an electroactive nanoparticle doped graphene. The proposed method can detect a low amount of early metastatic activity in the primary tumor sites, therefore serving as a precaution for the metastatic potential in the early stages of cancer. The experimental results obtained in this work may also suggest the prospective application of the proposed cell assay method in combating metastatic cancer in the future. E


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(18) Souzaki, M.; Kubo, M.; Kai, M.; Kameda, C.; Tanaka, H.; Taguchi, T.; Tanaka, M.; Onishi, H.; Katano, M. Cancer Sci. 2011, 102 (2), 373−381.

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Peptide-Based Method for Detection of Metastatic Transformation in

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