Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots

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1 ISSN Acta Biochimica et Biophysica Sinica 2004, 36(10): CN /Q Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots Hui-Zhi WANG, Hai-Yan WANG, Ru-Qiang LIANG, and Kang-Cheng RUAN* Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai , China Abstract Semiconductor quantum dots (QDs) offer several advantages over organic dyes in fluorescence-imaging applications, such as higher quantum yield, exceptional photostability, and a narrow, tunable, and symmetric emission spectrum. To explore whether QDs could specifically and effectively label tumor markers and be used in immunohistochemistry as a novel type of fluorescent probe, we used quantum dots with maximum emission wavelength 605 nm (QD605) to detect the ovarian carcinoma marker CA125 in specimens of different types (fixed cells, tissue sections, and xenograft piece). Additionally, we compared the photostability of QD signals with that of a conventional organic dye, FITC. All labeling signals of QDs were found to be more specific and brighter than those of FITC. Moreover, the QDs exhibited exceptional photostability during continuous illumination for 1 h by a high-intensity laser (Ar laser power 100 mw) at 488 nm, while the FITC signals faded very quickly and became undetectable after 24 min of illumination. These results indicate that QD-based probes can offer substantial advantages over existing fluorophores in many applications, and can be used effectively in immunohistochemistry as a novel class of fluorescent probes. Key words quantum dots; detection of tumor marker CA125; ovarian carcinoma The fluorescent labeling of biological materials using small-molecule organic dyes is widely employed in biological imaging and clinical diagnosis. Organic fluorophores, however, have certain characteristics that limit their advantages in some applications. These limitations include narrow excitation bands and broad emission bands with red spectral tails, which make the simultaneous evaluation of several light-emitting probes difficult due to spectral overlap. Also, many organic dyes exhibit high photodegradation [1,2]. Quantum dots (QDs), also known as semiconductor nanocrystals, have recently emerged as promising alternatives to organic dyes in fluorescence-imaging applications. QDs are generally composed of atoms from elements in groups II-VI or III-V in the periodic table, and are defined Received: July 14, 2004 Accepted: August 15, 2004 This work was supported by the grants from the National Key Basic Research and Development Program of China (No. 2002CB713802), the National Natural Science Foundation of China (No ), and the National High Technology Research and Development Program of China (No ) *Corresponding author: Tel, ; Fax, ; , kcruan@sibs.ac.cn as particles with physical dimensions smaller than the exciton Bohr radius. This small size results in a quantum confinement effect, which endows nanocrystals with unique optical and electronic properties [3,4]. In the past 20 years, research into the applications of QDs has mainly focused on microelectronics and optoelectronics rather than biology and medicine, due to some unsolved technical problems [5]. However, developments in QD surfacecoating chemistry, especially key advances in the preparation of water-soluble and biocompatible nanocrystals, have led to the emergence of QD applications in biological labeling and cellular imaging [6,7]. QDs offer several advantages over conventional organic dyes that endow them with great potential as a novel class of fluorescent probes. Firstly, their emission spectra are narrow, symmetrical, and tunable according to their size and material composition, allowing closer spacing of different probes with less spectral overlap. Secondly, they exhibit excellent photostability and broad absorption spectra which make it possible to excite all QDs simultaneously with a single light source and minimize sample auto-fluorescence by choosing an appropriate excitation wavelength. Thirdly, they show higher luminescence and quantum yield

2 682 Acta Biochimica et Biophysica Sinica Vol. 36, No. 10 (potentially 100%) than conventional fluorophores under appropriate conditions [8 10]. For example, the emission spectra of ZnS-capped CdSe QDs are as narrow as 13 nm (full width at half maximum), one-third as wide as the spectral-line width of the rhodamine molecule (a type of organic dye), and 20 times as bright and 100 times as stable against photobleaching [11]. At room temperature, they are strongly luminescent (35% 50% quantum yield) and their emission wavelength can be tuned between blue and red wavelengths by changing the particle size. Another advantage of QDs is that they exhibit a larger cross-section to two-photon stimulation than organic dyes. Combined with multiphoton microscopy, QDs can be used in the imaging of thick specimens, angiography, and in vivo multiphoton imaging [12]. These advantages make QD an attractive alternative fluorescent probe that is superior to existing fluorophores in many cases. QDs have been used increasingly in cellular imaging and specific labeling in vivo in recent years [13 17]. Ovarian cancer is the second most-common malignancy of the female genital tract, and CA125 (carcinoma antigen 125) is an epithelial antigen that has been used as a useful tumor marker in the detection and therapy of ovarian cancer. CA125 is a mucinous glycoprotein that is overexpressed in more than 80% of nonmucinous ovarian carcinomas [18 20], and it has been shown that the production of CA125 is correlated with ovarian cancer activity [21]. The great value of CA125 in clinical diagnoses has led to its being used widely in the detection of ovarian cancer. Immunohistochemistry (IHC) and in situ hybridization are common detection methods in pathology and clinical diagnosis, in which small-molecule organic fluorescent probes are used to detect and localize proteins and nucleic acids. Here we describe the specific detection of CA125 in different types of specimen (fixed cells, tissue sections, and tissue pieces) using ZnS-capped CdSe QDs conjugated to streptavidin and a specific monoclonal antibody. We also compare the immunofluorescence images of CA125 obtained using QD conjugates and a conventional organic fluorescent probe, FITC. Additionally, we compare the photostability of QD and FITC signals during continuous illumination by a high-intensity laser (Ar laser power 100 mw) at 488 nm for 1 h. Materials and Methods Reagents and instruments ZnS-capped CdSe QDs (Qdot TM 605) conjugated with streptavidin (QD605) were purchased from Quantum Dot Corporation, USA. All reagents used in the experiments were of analytical grade. Monoclonal anti-ca125 antibody (Mab CA125) was purchased from Biodesign Corporation, USA. All solutions were prepared using deionized and autoclaved water. FITC dye was purchased from Molecular Probes Corporation, USA and DAPI dye and cover slides were purchased from Sigma, USA. All images of CA125 detection in cells and tissue sections were obtained on a confocal microscope (Leica TCS SP2), in which excitation was at 488 nm both for FITC and QD605, and emission filters from 505 nm to 530 nm and from 590 nm to 620 nm were used to detect FITC and QD605, respectively. Meanwhile, the images of CA125 in fixed tissue pieces of ovarian tumor were obtained on a conventional microscope using a commercial Olympus CCD camera (CAMEDIA C-4000). Cell line and tumor tissue Human ovarian carcinoma HO8910 cells were obtained from Cell Bank of the Chinese Academy of Sciences, and human ovarian carcinoma tissue was resected from patients diagnosed in Hospital of Obstetrics and Gynecology, Fudan University. To establish tumor xenografts in mice, exponentially growing HO8910 cells were injected subcutaneously into the chest and dorsal area of BALB/c nu/nu mice. After 9 weeks inoculation, the xenograft tumor tissue was resected and used in CA125 detection. Labeling of Mab CA125 with biotin and FITC Mab CA125 (mice anti-human ovarian CA125) was labeled with biotin or FITC according to the protocol of Molecular Probes Corporation. The ratios of Mab CA125 to biotin and FITC were 1:4 and 1:6, respectively, as determined by measuring the absorption. The QD605s could then be conjugated to biotin-labeled Mab CA125 by the specific binding of biotin and streptavidin. Preparation of samples and procedure of CA125 detection To detect of CA125 in cell, the human ovarian carcinoma HO8910 cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum, with no antibiotics added. The culture medium was changed every 2 days until the cells grew to exponentially growing period. CA125 was detected in the cytoplasm of HO8910 cells by collecting sufficient cells, washing them with 0.02 M phosphate-buffered saline (PBS) at 4 C, and then centrifuging them at 1000 g for 3 min at room temperature and transferring the supernatant. This process was repeated

3 Oct., 2004 Hui-Zhi WANG et al.: Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots 683 three times and then the cells were fixed with 4% paraformaldehyde for 10 min. The fixed solution was blocked for 30 min in PBS at 4 C containing 3% BSA (W/V) and 0.1% Triton X-100. Cells were incubated with Mab CA125 overnight at 4 C, washed three times with PBS containing 0.1% Triton X-100, and then incubated with QD605s with 100-fold dilution for 1 h at room temperature. The cells were washed five times at room temperature to remove unbound QDs, and then resuspended in 50% glycerol-pbs. A drop of cell suspension was placed on a glass slide, covered with a coverslip, and imaged under a laser-scanning confocal microscope. The cell nuclei were counterstained with DAPI. To detect the CA125 in human ovarian tumor tissues, the human ovarian tumor tissues obtained from Hospital of Obstetrics and Gynecology, Fudan University, were fixed with 4% paraformaldehyde, paraffin embedded, sectioned at 10 µm, and treated as described in a previous IHC protocol [22]. After being blocked in the solution containing 10% normal goat serum (V/V) and 3% BSA (W/V), these sections were incubated with 10 µg/ml biotinylated Mab CA125 overnight at 4 C, QD605 for 1 h at room temperature, and DAPI solution to stain nuclei. Full rinsing was performed between each step. The cells were covered with coverslips and imaged. The excitation and emission wavelengths were the same as for the fixed cells. Using conventional organic probes, targets in a tissue piece can be detected only the tissue is sectioned. QDs exhibit uniquely high light absorbance and quantum yield, and reports that the fluorescence of a single QD can be detected [9] indicate that it is possible to detect targets in a tissue piece directly. To explore the potential of QD in the direct imaging of tissue pieces, we labeled the tissue piece directly by the successive incubation with Mab CA125 and QD605. Excitation was provided by an ultraviolet lamp, and emissions were detected using a filter cutting below 490 nm light. The cultured cells were collected during the period of exponential growth, and cells were inoculated into subcutis of nude BALB/c nu/nu mice to establish the xenograft. The xenografts were resected from the nude mice 9 weeks after inoculation. After the careful removal of its outer membrane, the tumor tissue was rinsed with PBS at 4 C and then immediately fixed in 4% paraformaldehyde solution. The fixed tissues were incubated with biotinylated Mab CA125 overnight at 4 C and QD605 for 30 min successively, with five times washing with PBS containing 0.1% Triton X-100 between each step. Control tissues were incubated with Mab CA125 linked to FITC and QD605 under the same conditions. The treated tissues were excited with ultraviolet light and imaged using the commercial CCD camera with a filter cutting below 490 nm light. To further explore the penetrability of Mab CA125 conjugated with QD605s, we cut the sample labeled with QD605s to determine whether QD fluorescence was evident in the internal transects. Photostability comparison between QD605 and FITC To compare the photostability of QDs and FITC, the specimens were continuously illuminated with a 488 nm laser (Ar laser power 100 mw) for 1 h under a 60, N.A. 1.3, oil-immersion objective. Images were automatically captured with the confocal microscope at 3 min intervals for each color. Excitation and emission conditions were the same as for the fixed cells. The experimental conditions for FITC and QD605 were the same, and the mean fluorescence intensity was measured automatically with software provided with the confocal microscope. After normalizing the absolute fluorescence intensity, we analyzed changes in fluorescence intensities of QD605 and FITC during the entire illumination period. Results and Discussion Detection CA125 with quantum dots at the cellular level Fig. 1 shows images of CA125 detection in a fixed ovarian cancer HO8910 cell labeled with QDs (panel A) and FITC (panel C). Nuclei were counterstained with DAPI to localize CA125. These images clearly show that CA125 images with the QD605 probe are brighter than those using FITC. Moreover, in QD605 image, there was a lower background and the expression of the CA125 antigen on the tumor cell membrane was clear, whereas the FITC image had some nonspecific staining in the cytoplasm. When the fixed cells were incubated with QD605 and PBS as a control, no detectable QD signals were observed in intracellular spaces, and there were only DAPI signals evident in the nucleus [Fig. 1(E,F)]. This result indicates that the QD605 conjugates have a very low nonspecific binding and a high signal in a biotin-streptavidin labeling system. Additionally, a limitation of the light source of the instrument was that the QD605 was excited at 488 nm, which was not the maximum excitation wavelength for QD605 but was the maximum excitation wavelength for FITC. Therefore, it was reasonable to assume that the distinction between the QD and FITC results would be

4 684 Acta Biochimica et Biophysica Sinica Vol. 36, No. 10 Fig. 1 Confocal fluorescence images of CA125 in fixed human ovarian carcinoma HO8910 cells (A,C) The cells were incubated with biotinylated monoclonal anti-ca125 antibody and QD605 and FITC conjugated Mab CA125, respectively. (E) Negative control, in which the cells were incubated with PBS and QD605. (B,D,F) The overlap images in (A), (C), and (E), and their corresponding DAPI images, respectively. Excitation was at 488 nm both for FITC and QD605. Emission filters from 505 nm to 530 nm and from 590 nm to 620 nm were used to detect FITC and QD605, respectively. Images were captured with a confocal microscope. All scale bars in photo are 40 µm. greater if the excitation was at a shorter wavelength. Detection of CA125 with quantum dots on tissue sections Fig. 2 shows images of CA125 detection with fixed tissue sections of ovarian cancer patients obtained by labeling QD605 (panel A) and FITC (panel D), respectively. Nuclei were counterstained with DAPI to localize CA125 (panel B, D). Panel C and F are the overlap images of panel A and B, and panel D and E, respectively. Tumor Fig. 2 Confocal fluorescence images of human ovarian carcinoma tissue sections (A) The tissue sections were incubated with Mab CA125 and QD605. (D) The tissue sections were incubated with Mab CA125 conjugated FITC. (G) Negative control, in which the tissue sections were incubated with PBS and QD605. (B,E, H) The cell nuclei were counterstained with DAPI. (C,F,I) The overlap images in (A) and (B); (D) and (E); (G) and (H); respectively. Excitation and emission wavelength were the same as in Fig. 1. All scale bars are 40 µm. cells with the CA125 antigen were clearly detected and brighter images were obtained when using QDs than when using FITC. When the tissue was incubated with QD605 and PBS as a control, only very weak QD signals were observed, and there were only DAPI signals evident in the nucleus (panel G). These results indicate that QD-based probes can be adapted to applications involving tissue sections and have significant potential applications in IHC. Detection CA125 in the thick specimen (fixed tissue piece) with quantum dots Fig. 3 shows the detection of CA125 in xenograft tissue pieces from ovarian cancer specimens, indicating that after labeling the tissue pieces directly by successive incubation with Mab CA125 and QD605, the very strong red fluorescence was visible to the naked eye, and could Fig. 3 Scheme of the detection of specific labeling of the ovarian carcinoma tissue pieces (A) The picture of nude mice (BALB/c nu/nu) with ovarian cancer xenograft. (B,C) Images of ovarian carcinoma tissue pieces labeled with Mab CA125 conjugated with FITC (left) and Mab CA125 and QD605 successively (right). The images were obtained under natural light (B) and ultraviolet light (C) respectively. (D) The images of transected sample labeled by QDs. All the fluorescence images were captured by a commercial CCD camera with a 490 nm low-pass filter.

5 Oct., 2004 Hui-Zhi WANG et al.: Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots 685 be captured clearly by the commercial CCD camera [Fig. 3(C), right-hand object]. However, under the same experimental conditions, only weak fluorescence was evident on the surface of the tissue piece labeled with FITC [Fig. 3(C), left-hand object]. These results demonstrate that QD enables thick specimens such as tissue pieces to be labeled directly and effectively with a simple apparatus. Images of the transected samples labeled by QDs revealed no red fluorescence in the internal transects, indicating that QDs, like other organic dyes, have no special ability to penetrate in tissue pieces. Photostability of quantum dots Fig. 4 shows a time course of images for the use of QDs (top row) and FITC (bottom row) under continuous illumination with a 488 nm laser (Ar laser power 100 mw) for 1 h. It is obvious that the FITC signals faded quickly and became undetectable after 24 min, whereas QD605 signals showed no obvious change during the entire 1 h illumination period. This trend was expressed precisely by calculating the fluorescence intensity of QDs and FITC and the data were plotted in Fig. 5, indicating that QD605 Fig. 4 Photostability comparison between QD605 and FITC conjugated to HO8910 cells Top row is the fluorescence of QD605 and bottom row is that of FITC. Emission filters from 505 nm to 530 nm and from 590 nm to 620 nm were used to collect FITC and QD605 signals, respectively. The images from left shown were obtained at 0, 3, 6, 9, 12, 18, 24, and 30 min. Scale bars are 20 µm. Fig. 5 Photostability of QD605 and FITC conjugated to HO8910 cells exhibits much higher photostability than does FITC. Conclusion We have demonstrated that semiconductor QDs can be used for the detection of tumor marker CA125 in IHC as a new type of fluorescent probe that exhibits superior characteristics in many applications. Moreover, QDs have great potential in the imaging and labeling of thick specimens. Because of their unique optical properties and exceptional photostability, QDs may also be more effective than a conventional organic fluorescent probe in sensitive immunoassays. References 1 Hall M, Kazakova I, Yao YM. High sensitivity immunoassays using particulate fluorescent labels. Anal Biochem, 1999, 272(2): Hermanson GT. Bioconjugate Techniques. 2nd ed. London: Academic Press, 1996, Alivisatos AP. Semiconductor clusters, nanocrystals, and quantum dots. Science, 1996, 271: Nirmal M, Brus L. Luminescence photophysics in semiconductor nanocrystals. Acc Chem Res, 1999, 32: Henglein A. Small-particle research: Physiochemical properties of extremely small colloidal metal and semiconductor particles. Chem Rev, 1989, 89: Klarreich E. Biologists join the dots. Nature, 2001, 413: Mitchell P. Turning the spotlight on cellular imaging. Nat Biotechnol, 2001, 19: Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science, 1998, 281: Achermann M, Petruska MA, Kos S, Smith DL, Koleske DD, Klimov VI. Energy-transfer pumping of semiconductor nanocrystals using an epitaxial quantum well. Nature, 2004, 429: Pitsillides CM, Joe EK, Wei X, Anderson RR, Lin CP. Selective cells targeting with light-absorbing microparticles and nanoparticles. Biophys J, 2003, 84: Chan WC, Nie S. Quantum dot bioconjugates for untrasensitive nonisotopic detection. Science, 1998, 281: Larson DR, Zipfel WR, Williams RM, Clark SW, Bruchez MP, Wise FW, Webb WW. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science, 2003, 300: Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson PJ, Ge N et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol, 2003, 21: Kaul Z, Yaguchi T, Kaul SC, Hirano T, Wadhwa R, Taira K. Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates. Cell Res, 2003, 13(6): Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol, 2003, 21: Watson A, Wu X, Bruchez M. Lighting up cells with quantum dots. Biotechniques, 2003, 34:

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