New directions in quantum dot-based cytometry detection of cancer serum markers and tumor cells

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1 Critical Reviews in Oncology/Hematology 86 (2013) 1 14 New directions in quantum dot-based cytometry detection of cancer serum markers and tumor cells Olga Akinfieva a, Igor Nabiev a,b,, Alyona Sukhanova a,b, a Laboratory of Nano-Bioengineering, Moscow Engineering Physics Institute, 31 Kashirskoe Shosse, Moscow, Russian Federation b European Technological Platform Semiconductor Nanocrystals, Institute of Molecular Medicine, Trinity College Dublin, James s Street, Dublin 8, Ireland Accepted 5 September 2012 Contents 1. Introduction Quantum dots: advantages over organic fluorochromes; safety and toxicity issues Fluorophore-encoded microbeads for multiplexing Applications of quantum dots Cell labeling Cellular uptake of quantum dots Multicolor flow cytometry for T cell immunophenotyping Detection of multiple cancer markers Two-photon spectroscopy and imaging with the use of quantum dots Detection of multiple cancer cell populations: the use of two-photon cytometry Properties of quantum dots as FRET donors Use of quantum dot-tagged microbeads for multiplexed detection and diagnosis Conclusion: implications of quantum dot-encoded suspension arrays for diagnosis and therapy monitoring Reviewers Conflict of interest Acknowledgments References Biographies Abstract The use of fluorescent quantum dots (QDs) incorporated in or tagged with polymeric microbeads allows multiplexed coding of biomolecules. Compared to organic dyes, QDs are characterized by improved imaging capabilities, brightness, and photostability and may be used for simultaneous detection of multiple biomarkers. Development of QD conjugates and QD-encoded suspension arrays has given rise to new promising approaches to cell labeling, in vivo visualization, and diagnostic assay techniques. QDs have proved to be efficient donors for Förster resonance energy transfer (FRET) and are characterized by high multiphoton absorption coefficients. Implication of QD-based suspension arrays for identification of autoantibodies, tumor-specific T cells, and detection of circulating cancer cells by means of flow cytometry, holds considerable promise for earliest diagnosis of human abnormalities and effective monitoring of the therapeutic effects. This review summarizes recent advances in QD-based suspension arrays application to cancer diagnosis and attempts to predict their diagnostic potential in a future Elsevier Ireland Ltd. All rights reserved. Keywords: Flow cytometry; Colloidal nanocrystals; Quantum dots; Microbeads; Autoantibodies; Cancer cells; FRET; Multiphoton excitation Corresponding authors at: Institute of Molecular Medicine, Trinity College Dublin, James s Street, Dublin 8, Ireland. Tel.: ; fax: addresses: igor.nabiev@gmail.com (I. Nabiev), nanomedicine.mephi@gmail.com (A. Sukhanova) /$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.

2 2 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) Introduction 1.1. Quantum dots: advantages over organic fluorochromes; safety and toxicity issues One of the main goals of biology is to comprehend the complex spatiotemporal interplay of biomolecules at all levels from cell to body. In order to study this interplay, researchers generally use fluorescent labeling for both in vivo cellular visualization and in vitro assay [1 4]. Until recently, organic and genetically encoded fluorophores have been widely applied to bioimaging, however, the photophysical properties of these dyes broad absorption/emission profiles and low photobleaching thresholds, to name but a few have limited their effectiveness in long-term labeling and multiplexing [5 7]. Recent advances in nanotechnology have made it possible to obtain a new class of highly fluorescent homogeneous semiconductor nanocrystals 2 6 nm in size or quantum dots (QDs) [5 9]. Two unique properties of QDs that organic dyes lack are of particular interest to biologists: broad excitation spectra, which allow excitation of different QD populations at the same wavelength remote from their respective emission bands, and dependence of fluorescent emission on the QD composition and core size [5,6]. Therefore, QDs of various sizes may be excited with light of a single wavelength, resulting in many emissions of different colors that may be detected simultaneously [5,7]. In general, QDs used for biological applications are composed of a photoluminescent semiconductor core (consisting of, e.g., cadmium selenide, CdSe) coated with a shell of another semiconductor with a larger bandgap (e.g., zinc sulfide, ZnS). These QDs are characterized by a high quantum yield; high molar extinction coefficients (approximately times higher compared to organic dyes); broad absorption spectra and narrow, symmetric photoluminescence spectra ranging from 400 nm to 2 m; large Stokes shifts; and a high resistance to photobleaching and chemical degradation [5 7]. Phase transfer of QDs into an aqueous solution requires surface functionalization with hydrophilic ligands, which not only ensure colloid solubility, but also protect the QD surface from deterioration in biological media and promote the attachment of biomolecules [10 12]. Despite the evident advantages of QDs over the organic dyes, it should be noted that related safety and toxicity issues have been insufficiently addressed to date. Data on QD toxicity are quite contradictory. The use of QDs for in vivo targeting in mice revealed no toxic effect within 24 h [13] or even several days [14,15]. Jaiswal et al. report that nm QDs in the medium had no detectable effects on cell morphology or physiology [16]. Recent research on fibroblast activity demonstrated changes in cell function within 5 6 h; however, these were mainly caused by the toxic effect of free cadmium rather than the assembled QDs [17]. Beside these data the other results were published establishing the safety of QDs for in vivo studies [18]. However, QD degradation products (in particular, cadmium and selenium) may have a harmful effect [17]. The formation of these products depends on the degree of QD susceptibility to oxidation and photolysis, which, in turn, depend on the parameters of QDs, including the chemical composition, size, charge, and characteristics of the outer shell [19]. Therefore, estimation of QD toxicity requires further systematic risk assessment studies of potential health risks of QDs in relationship with their physicochemical properties Fluorophore-encoded microbeads for multiplexing Semiconductor nanocrystals have proved to be unique fluorescent labels offering outstanding possibilities for highthroughput detection and diagnosis [5,18,20]. Still, however informative QD-based biological labeling and optical encoding are, up-to-date research in genomics and proteomics, not to mention clinical diagnosis, require technologies for rapidly screening a large number of nucleic acids, proteins, and cell surface antigens simultaneously [21,22]. Polymeric optically encoded microbeads (3 10 m in size) have made it possible to perform mass-scale parallel analyses of biological molecules [7,22 25]. Multicolor optical coding for biological assays with the use of microbeads is based on the two strategies: on the encoding of microspheres by fluorescent organic dyes and on the incorporation of semiconductor QDs into beads at precisely controlled ratios [21,25]. Recent advances concerning the applications for the simultaneous detection of various molecules have resulted in the development of a microsphere-based flow cytometric assay (MFCA) by Luminex Company (Austin, TX, USA). The multiplex Luminex technology involves the covalent coupling of polysterene microspheres (5.6 m), encoded with three internal organic fluorophores, with capture antibodies or target molecules, which in their turn bind to reporter molecules labeled with a fluorescent marker, such as phycoerythrin (PE) or AlexaFluor 532. Variations of the ratio of the three organic dyes make it possible to distinguish up to 500 different bead sets coupled to different biological probes. In the Luminex technology the quantification of the multicolour fluorescence of each microsphere and of the intensity of the fluorescent reporter signal is carried out by a conventional flow cytometer. As the individual bead sets can be separated by a flow cytometer, many assays can be performed simultaneously, thus allowing the multiplexed quantitation of multiple analytes in a single sample. Reportedly, this type of technology was used for multiplexed assays of cytokines, antibodies, hormones, nucleic acids, viral antigens and other biomolecules. In addition to its multi-analyte capability, the MFCA developed by Luminex proved to be highly reproducible, sensitive and fast technique which up to date could be considered a primary particle-based flow cytometric assay [25].

3 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) In the recent years an alternative approach with the use of optically encoded microbeads has been developed for massively parallel and high-throughput analysis of biomolecules. This encoded-bead technology is based on the optical properties of semiconductor QDs which are incorporated into polymeric microbeads at precisely controlled ratios. Combination of the molecular recognition capacity of bioprobes, such as antibodies, conjugated to a bead surface, and an identification code inside the bead makes each bead a chemical laboratory that detects a unique molecule in a complex mixture [7,24]. The principle of multiplexed optical coding with the use of microbeads embedded with QDs is shown in Fig. 1. Using 10 intensity levels (0, 1, 2,..., 9) at a single wavelength (i.e., color), it is possible to obtain 9 unique codes (level 0 merges into the background). An increase in the wavelength and intensity ranges gives an exponential increase in the number of codes. In general, the theoretically available number of codes is determined by the number of QD colors and the number of intensity levels of QD emission: C = N m 1, where C is the number of theoretically available detectable codes, N is the number of intensity levels, and m is the number of colors. The actual number of codes, however, is lower due to spectral overlapping, variations of fluorescence intensity, and signal-to-noise requirements [21]. It is worth mentioning that microbeads developed by the Luminex and encoded with internal organic fluorophores, suffer from considerable disadvantages compared to beads embedded with semiconductor nanocrystals [7,21]. First of all, an increase in the number of organic dyes requires multiple excitation lasers, which increases the cost of the decoding instrument. On the contrary, QDs broad absorption spectra ensure excitation of various populations of QDs with light of a single wavelength, i.e. single laser. Secondly, organic dyes which are incorporated in microbeads used in Luminex technology are characterized by broad emission profiles and low photobleaching thresholds, whereas QDs, used as fluorescent tags, have narrow emission spectra and provide resistance to photobleaching. As a result, polymeric microbeads optically encoded with organic fluorophores allow for a limited number of unique codes, whereas beads embedded with QDs display multiplexing capacity, photostability, and sensitivity of antigen detection. Thus, due to the inherent properties of QDs listed above, they are generally considered to be the most suitable for microbead encoding [7,24]. 2. Applications of quantum dots 2.1. Cell labeling Since the creation and development of the QD technology, fluorescent nanoparticles have been used in various imaging techniques. The use of QDs for specific labeling of various targets (cell surface receptors, cytoskeleton components, and nuclear antigens) with different locations in the cell (the cell membrane, cytoplasm, or nucleus) in various types of specimens (cultured live cells, fixed cells, and tissue sections) has attracted much interest [18,26,27]. Generally, the strategies for QD labeling of live cells include endocytosis, direct microinjection, electroporation, mediated uptake, targeted uptake, and selective targeting of cell surface proteins (Table 1) [18,28 32]. Fig. 1. Optical coding based on wavelength and intensity multiplexing. Large spheres represent polymer microbeads in which small colored spheres (multicolor QDs) are embedded at predetermined intensity ratios. Molecular probes (A E) are attached to the bead surface for biological binding and recognition. The number of colored (red, green and blue) spheres shows the fluorescence intensity level, rather than the number of individual QDs. Both absolute intensities and intensity ratios at different wavelengths are used for coding purposes; e.g., (1:1:1) and (2:1:1) are distinguishable codes. Reproduced from Ref. [21] Cellular uptake of quantum dots Reportedly, peptide translocation domains or cationic lipids are efficient facilitators of endocytosis, allowing rapid labeling of whole cell populations with specific colors [33]. Higher specificity and efficiency can be obtained with functionalized QDs. Transferrin has been used to facilitate endocytosis by mammalian cells, a strategy also used successfully to label pathogenic bacteria cells [34]. According

4 4 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) 1 14 Table 1 Approaches to labeling of cells with quantum dots. Approach Mechanism Example Endocytosis Nonspecific uptake; QDs end up in endocytic compartments HeLa cells take up TAT peptide-conjugated QDs via macropinocytosis, a fluid-phase endocytosis process triggered by TAT QD binding to negatively charged cell membranes [30] Direct microinjection Injection of QDs into individual cells Delivery of QDs combined with peptide sequences: nuclear localized sequence (NLS) and mitochondrial localized sequence (MLS) for direct transport of quantum dots to the nucleus and mitochondria of HeLa cells [28] Electroporation Mediated uptake Targeted uptake Selective targeting of cell surface proteins Generation of hydrophilic pores in the plasma membrane by applying an electric field pulse. The pores allow passive transport of nanoparticles into the cell Encapsulation of QDs in lipid vesicles in order to facilitate their penetration into cells Based on the cell propensity to recognize and internalize QDs labeled with specific peptides and deliver them to specific cellular compartments Based on the specific interaction between antibodies linked to QDs and cell surface antigens Delivery of single QDs through pores in the plasma membrane of HeLa cells [28] Encapsulation of QDs in lipid vesicles with the use of Lipofectamine 2000, which facilitates the penetration of nanoparticles into cells [18] Internalization of engineered protein G conjugated with mitochondria-targeting antibodies and QDs into cervical cancer cells [32] Binding of QD Ab conjugates with the breast cancer marker Her2 on the surface of live cancer cells [31] to Ruan et al., HeLa cells take up QDs conjugated with the TAT peptide (a trans-activating transcriptional activator of HIV-1) via macropinocytosis [30]. Introduction of QDs into cells via direct microinjection or electroporation is more specific, however, these approaches have considerable disadvantages: reportedly, the former method is quite tedious and limits the number of labeled cells; in the latter technique, QDs stabilized with PEG loose electrostatically adsorbed surface ligands and form clusters (up to 500 nm in diameter) inside the cells [28]. Furthermore, the delivery of nanoparticles conjugated with antibodies into cells by electroporation or microinjection damages the cell membrane, decreases the cell viability, and affects the stability of the antibodies [32]. The transport of QDs into live cells via mediated and targeted uptake proved to be more effective and specific than methods based on invasive delivery of nanoparticles. For example, Lipofectamine 2000 proved to efficiently facilitate the entry of nanoparticles into cells through encapsulation of QDs in lipid vesicles [18]. According to Lim et al., an engineered protein G system containing affinity tags and cell penetration peptides can capture surface-modified QDs and targeting antibodies, and then noninvasively deliver them into cells for the targeting, visualizing, and manipulating mitochondria [32]. It has been found that matrix metalloproteinases can induce the transport of QD conjugates into cells [35]. With the use of fluorescent microscopy, it has been demonstrated that QDs linked to immunoglobulin G (IgG) and streptavidin specifically and efficiently label the breast cancer marker Her2 on the surface of fixed and live cancer cells, stain actin and microtubule fibers in the cytoplasm, and detect nuclear antigens inside the nucleus [31]. Reportedly, QDs can serve as markers for tracking cell movement, differentiation, and fate [36]. These results indicate that QDs are remarkably effective in cell labeling and have substantial advantages over organic dyes in multiplex target detection Multicolor flow cytometry for T cell immunophenotyping The crucial role of CD8 + T cells (cytotoxic T lymphocytes, CTLs) in elimination of malignant or virus-infected cells can hardly be overestimated. Indeed, CTLs may be regarded as the main inducers of apoptosis or programmed cell death (PCD) in transformed or damaged cells [37]. In addition, IFN- which is produced predominantly by CTLs and NK cells, inhibits proto-oncogene expression in transformed cells, potentiates the phagocytic activity of granulocytes and macrophages, which form an essential part of the innate immune system, and enhances the immune cytotoxicity of CTLs and NK cells (Fig. 2) [38]. CTLs are capable of recognizing and efficiently attacking a wide variety of tumor cells that display specific tumor-associated antigens (TAAs) bound with major histocompatibility complex class I (MHCI) proteins on their surfaces, including the cells of ovarian [39] and colon tumors [40], sarcomas [41], renal cell carcinomas [42], pancreatic tumors [43], adenocarcinomas, and squamous tumors of the head [44]. Reportedly, TAAs can stimulate adaptive immune response preventing metastatic disease progression and eliminating malignant cells in the body [45]. Although most TAAs are still unidentified, analysis of CTLs and MHC motifs binding TAAs have allowed researchers to detect a few TAAs that proved to be effective in triggering adaptive immune response in healthy individuals at risk of cancer [46]. The breakthrough advantages of QD use for both the disentangling of the intricacies of adaptive immune response pathways and the development of diagnosis and immunotherapy were convincingly demonstrated by Chattopadhyay et al. in a study on EBV-, CMV-, HIV Nef-, and HIV Gag-specific CD8 + T cell populations with the use of multicolor flow cytometry [26]. Unique spectral properties of QDs enabled researchers to use a 17-color staining panel consisting of

5 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) Macrophage Immature DC DC maturation Mature DC IFN-γ IL-12 NKT IL-12 NKT IFN-γ IFN-γ IFN-γ NK IFN-γ IFN- γ CD8 + IL-2, IFN- γ lysis NO, perforin, FasL, granzymes Tumor cells IL-12 Fig. 2. Elimination of tumor cells by CD8 + T cells and other major components of adaptive and innate immunity. CD8 + T cells, as well as macrophages and NK cells, stimulate the cytolysis of tumor cells through different pathways, such as production of NO, perforin, granzymes, and FasL. NKT cells can produce IFN-, which facilitates the maturation of dendritic cells and activates NK cells, CD8 + T cells, and macrophages. The mature dendritic cells produce IL-12, which enhances the IFN- and IL-2 production by NKT cells. DC, dendritic cells; FasL, Fas ligand; NO, nitritic oxide. seven QDs and ten organic dyes. QD reagents used for biolabeling were conjugates with antibodies against CD4, CD45RA, and CD57, as well as peptide MHC class I (pmhci) multimers designed to detect EBV-, CMV- and HIV-specific CTLs (Fig. 3) [26,47]. Configuration of the multi-detector, multi-laser flow cytometer used by Chattopadhyay et al. made it possible to excite all QDs with a 408-nm violet laser; an octagon detection system collected QD emissions. This study clearly demonstrated the potential of the multiplexed approach. Simultaneous identification of multiple phenotypically distinct subsets within each antigen-specific T cell population provides an insight into the complexity of T-cell immunity [26]. The development of Fig. 3. Quantum dot application to immunophenotyping of CTL cell populations. Schematic representation of a QD/peptide MHCI protein conjugate bound to the membrane of a CD8 + T cell. Multivalent interactions between the CD8 co-receptor and MHCI protein determine the stability of TCR/pMHC I/CD8 complexes and enhance the CD8 + T cell responses. Reproduced from Ref. [47]. QD suspension arrays for T cell phenotyping may prove to be a major step in the implementation of multicolor flowcytometry diagnostic technique Detection of multiple cancer markers Early detection and treatment of cancer is the ultimate goal of cancer research. This is an issue of much concern, especially in the cases of tumors that are typically diagnosed at late stages and, hence, have low therapeutic response rates (e.g., ovarian cancer [39]). Identification of proteins from cancer patients sera holds considerable promise for better understanding of the early-stage immune response to cancer. There is increasing evidence that the immune system produces a response to cancer-derived antigens. Circulating autoantibodies against TAAs have proved to be early, sensitive, and efficient diagnostic indicators of cancer. It has been shown that autoantibodies appear long before any pathology manifests itself [48]. Autoantigens represent a variety of intracellular and surface proteins that are overexpressed in tumors, such as p53, c-myc, HER2, NY-ESO-1, BRCA1, BRCA2, and MUC1 [49,50]. Autoantibodies against tumor antigens have been found in sera of patients first diagnosed with primary invasive breast cancer and ductal carcinoma in situ [51]. A mass spectrometry-based differential immunoproteomic method and ELISA proved to be highly effective in identification of autoantibodies in cancer patients sera. Alternatively, QD-based suspension arrays may have important implications for diagnosis, thus contributing to the prevention of metastasis and promoting efficient and complete tumor elimination. Moreover, antibodies in QD assays have been used to detect carcinoembryonic antigen (CEA) [52]. Recently, QDs were used as fluorescent labels for ultrasensitive detection of proteins after rolling circle amplification (RCA) of an oligonucleotide probe [53]. By using affinity

6 6 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) 1 14 capturing of the target protein, RCA of the bound DNA probe, and the oligonucleotide-functionalized CdTe QDs as complementary detection tags, Cheng et al. [53] dramatically improved the detection limit for proteins. They have achieved an impressive detection limit of 0.27 am (or 16 molecules in a 100- L sample) of human vascular endothelial growth factor (VEGF), a protein that plays an important role in tumor growth and metastasis. The DNA methylation status can be altered in cancer, and, hence, can be used as an epigenetic biomarker [54]. DNA hypermethylation may result in silencing of genes, when the gene products are no longer expressed [55]. A unique method termed methylation-specific QD fluorescence resonance energy transfer (MS-qFRET), has been developed to quantify the extent of DNA methylation and has been used to determine the methylation status of the PYCARD, CDKN2A, and CDKN2B tumor suppressor genes [56]. DNA was first extracted from sputum samples, and sodium isulfate was used to convert unmethylated cytosines into uracil without altering methylated cytosines. Depending on the gene to be detected, specific forward and reverse primers for both methylated and unmethylated states were designed. The forward primer was labeled with biotin, and the reverse primer, with the Cy5 fluorophore. PCR was used to amplify DNA; DNA was then captured by QD streptavidin conjugates through the binding of biotin. This binding enabled FRET to occur between the QD and the fluorophore. The signal was quantified as a q-score based on the efficiency of FRET, where methylated and unmethylated controls have q-scores of 1 and 0, respectively. The standard curve was linear, and the method was capable of detecting 15 pg of methylated DNA in 150 ng of unmethylated DNA [56] Two-photon spectroscopy and imaging with the use of quantum dots The method of two-photon spectroscopy proved to be one of the most promising approaches used for the investigation of the structure of living tissues, where signals can be obscured by scattering and competing intrinsic emissions [15]. Two-photon spectroscopy enables deep imaging of a variety of biological samples with less overall photobleaching than with wide-field or confocal microscopy, and it has now become the primary fluorescence imaging technique in thick specimens. The method of two-photon spectroscopy is based on the nonlinear two-photon excitation of fluorescent particles by femtosecond laser in the infrared (IR) range ( nm) and the registration of the fluorescence in the visible spectrum (Fig. 4). A focused pulsed femtosecond tunable IR laser provides a high photon flux which is required for the near simultaneous absorption of the two photons by fluorescent particles. Emission in the IR range is characterized by far more penetration power than emission in the visible spectrum, thus making it possible to visualize the structure of living tissues in vivo [15]. Energy Laser (400 nm) Single photon Excited state Excitation of electrons to higher energy levels Ground state Laser (800 nm) Two-photon Fluorescence emission Fig. 4. Energy level diagram of single photon and two-photon absorption. In one as well as two-(or multi-) photon fluorescence, a fluorescent particle emits light when an electron relaxes from an excited energy state to its ground level. The energy difference between the two levels (electronic and vibrational states) determines the emission spectrum. In single photon fluorescence, a particle absorbs one photon with a shorter wavelength than the emitted photon. In two-photon fluorescence two photons with about double the wavelength and, therefore, half the energy are absorbed. Before emitting a photon and relaxing to the ground level the electron dissipates energy. Since the probability of near simultaneous absorption of the required two photons is very low, a high photon flux is required and it is provided by the output of a focused pulsed (femtosecond) tunable infrared laser. Since tissues are characterized by autofluorescence in the blue and yellow green regions due to the presence of innate fluorophores, such as porphyrins, flavins, NADH, elastin, etc. [57], near-infrared (NIR) dyes are used for biomedical imaging in living tissues. However, this technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores [15]. It has been demonstrated that QDs can be effectively used for intravital tissue labeling [20,58,59]. Kim et al. applied NIR-emitting QDs ( nm) to mapping sentinel lymph nodes (SLNs) in cancer surgery of animals. Injection of 400 pmol of NIR QDs permitted SLNs located 1 cm deep from the skin surface of pigs to be imaged easily in real time [20]. Therefore, intraoperative NIR fluorescence imaging allows the optical identification of lymph flow and SLN without the use of radioactive tracers or organic dyes. This technique provides a surgeon with image guidance throughout the operation procedure. Moreover, unlike small molecules of organic fluorochromes, NIR QDs have an optimal size (and, hence, do not flow past the SLN) and do not photobleach [20]. NIR QDs have also been used for gastrointestinal and pulmonary SLN mapping in various studies, which show that they are promising for clinical lymphatic mapping [60 64]. Visualization with targeted NIR QDs results in a significant increase in tumor fluorescence. Cai et al. [65] successfully used a peptide-labeled QDs for in vivo tumor vasculature labeling. Tumor angiogenesis is an important target for diagnostic in vivo imaging, as well as for anticancer treatment strategies based on the inhibition of angiogenesis. v 3-Integrin is an important marker of angiogenic blood

7 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) vessels and is normally upregulated in cancer cells. It binds specifically to arginine glycine aspartic acid (RGD) peptide containing components of the interstitial matrix. The authors studied the binding of RGD-labeled QDs specifically to v 3-integrin in vitro, ex vivo, and in living mice with the U87MG tumor. The tumor fluorescence intensity reached a maximum within 6 h after injection to the mice. Tumors from euthanized mice were imaged to show the presence of the QD 705-RGD probe. These data show the potential of QDs as a universal tool for detection of many tumor types, labeling of tumors, and imaging-guided surgery [65]. Kobayashi et al. [66] reported on multiplexed in vivo imaging employing five types of QDs. The authors detected five different lymphatic basins using five NIR QDs, which were injected intracutaneously into the middle digits of the forelegs, the ears, and the median chin to simultaneously visualize lymph flow from the neck and upper trunk of mice. The route of cancer metastasizing into the lymph nodes could be predicted by simultaneous visualization of five separate lymphatic flows [66]. In addition to the properties described above, QDs have a high two-photon cross-sectional efficiency, which makes these nanoparticles suitable for in vivo deep-tissue imaging using two-photon excitation at low intensities. Using this technique to excite green-emitting QDs in the NIR region allowed the imaging of mouse capillaries hundreds of micrometers deep in skin and adipose tissues [15]. Since the QD cross sections of are three orders of magnitude greater than those of conventional organic dyes, the use of QDs enables imaging at greater depths than the techniques using standard fluorophores, which makes QDs especially useful for fluorescence imaging in living tissues [15] Detection of multiple cancer cell populations: the use of two-photon cytometry It has always been of particular interest to researchers to analyze circulating tumor cells (CTCs) in the bloodstream in cancer and other diseases. It is well known that the number of CTCs is an independent predictor of survival in patients with metastatic breast cancer [67]. The genotype of CTCs seems to be identical to that of tumor tissue cells; therefore, analysis of CTCs may promote better understanding of the genetic changes that occur in the course of the disease [68]. Investigation of molecular features that regulate the dynamics of CTCs in experimental animals and patients would potentiate broadening of the cancer biology horizons and contribute to diagnostic and therapeutic approaches. The numbers of CTCs estimated using singlecolor detection may vary in response to physiological changes vasoconstriction, fluctuations of the heart rate, and intrinsic changes in CTCs, to name but a few, which means that accurate assessments of CTCs could be conducted only through simultaneous detection of multiple cell populations. A two-photon flow cytometry system has the important advantage over a single-photon system, due to the fact that a single femtosecond-range NIR laser can be used to excite multiple fluorescent dyes or semiconductor nanocrystals via two-photon transitions [69]. The large difference between the NIR excitation and emission wavelengths attenuates scattered excitation light while collecting the entire fluorescence spectrum with a high efficiency, thereby decreasing the detection background [70]. The use of two-channel, two-photon flow cytometry in vivo with the application of QDs has made it possible to quantify and monitor several populations of malignant cells in the same animal [69]. The fate of cells of two breast cancer lines has been analyzed by Tkaczyk et al. in the mouse bloodstream: MDA-MB-435 cells, which are characterized by a high metastatic potential, and MCF-7 cells, which, in contrast, do not form metastases. It was found that MCF-7 cells, labeled with QDs that emitted at 585 nm (QD585), were almost completely cleared from the circulation 1000 min after injection, while MDA-MB-435 cells, labeled with QDs that emitted at 655 nm (QD655), remained detectable during the entire 1000 min of the experiment; in addition, greater numbers of MDA-MB-435 cells were initially retained in the lungs and liver and persisted in these organs as compared to MCF-7 cells [69]. The researchers obtained the data indicating that QDs do not influence the behavior of labeled cells, and that both cell lines remained labeled adequately for detection for over 5 6 days. These data convincingly demonstrate the effectiveness of the two-photon flow cytometry technique in revealing differences in the fates of different malignant cells in circulation. Unique photophysical properties of semiconductor nanocrystals and their advantages in brightness and photostability over organic dyes indicate the perspectives of QDs as cell labels in the application in two-photon flow cytometry method of cancer cell detection. Further studies might allow scientists to interrogate functions of specific molecules implicated in metastatic breast cancer, such as the chemokine CXCL12 and its receptor CXCR4 [71]. Itisevident that this approach may add to our knowledge of the characteristics of circulating cancer cells and the progress of the metastatic disease Properties of quantum dots as FRET donors It is generally known that the Förster (or fluorescent) resonance energy transfer (FRET) mechanism is used in biology for monitoring intracellular interactions and binding events [72]. The development of QDs and their application to biological labeling have demonstrated that these nanoparticles can be far more efficient FRET donors than conventional fluorophores [73]. First, the QD donor emission can be size-tuned in order to improve the spectral overlap with a particular acceptor dye. Second, several acceptor dyes can interact with a single QD donor, which improves the FRET efficiency. Different sensing schemes have been developed with QDs as FRET donors. A common method for FRET-based sensing of an analyte involves the displacement of a bound quencher [74]. It has been demonstrated that displacement of the QSY- 9 dye-labeled -cyclodextrin from maltose-binding protein

8 8 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) 1 14 Fig. 5. Quantum dot application to FRET-based sensing of maltose involving displacement of a bound quencher. CdSe/ZnS QD based competitive assay of maltose using the maltose binding protein (MB) as a sensing material and the -cyclodextrin QSY-9 conjugate as a FRET quencher. Reproduced from Ref. [73]. (MBP) linked to a CdSe/ZnS QD upon addition of maltose results in regeneration of the luminescence of the QDs [73]. Another method for FRET-based biosensing involves structural changes in proteins upon interaction with their substrates. This approach has been used for the assembly of a QD-based sensor for maltose [75]. QDs are functionalized with a mutant maltose-binding protein, MBP41C, which includes a cysteine at a peristeric site that is not involved in maltose binding. This residue is specifically labeled with a fluorescent dye, Cy3, which acts as a FRET acceptor from the QD. Upon binding maltose, MBP undergoes conformational changes that alter the environment surrounding the dye, thus changing its fluorescence in a concentration-dependent manner (Fig. 5). These conformational changes enhance both the FRET from the QDs to the dye and the nonradiative decay of the dye. FRET as a readout mechanism in QD-modified dyelabeled peptides may also be useful for analysis of the hydrolytic functions of a series of proteolytic enzymes. Shi et al. have recently used a QD FRET probe consisting of the donor QD and the acceptor rhodamine-labeled peptide to measure the enzymatic activity of tripsin [76]. Local excitation of the QDs is able to induce efficient energy transfer to the adjacent rhodamine dye. Approximately 48 molecules of the rhodamine-labeled collagenase substrate (RGDC) were conjugated on a single QD via the sulfhydryl group of the cysteine residue. In a test with trypsin (500 g/ml for 15 min), these probes exhibited a 60% increase in the photoluminescence of the QDs and the corresponding decrease in the emission of rhodamine due to the FRET signal changes [76]. QDs conjugated with nucleic acids have been used to monitor biocatalyzed replication of DNA [77]. Patolsky et al. functionalized QDs with the DNA primer that was complementary to a specific domain of M13 mp18 DNA. Hybridization of single-stranded DNA with the nucleic acidfunctionalized QDs was followed by the replication of the resulting duplex in the presence of polymerase and a mixture of dntps that included Texas Red-functionalized dutp [77]. This process resulted in the incorporation of the dye labels into the DNA replica. The FRET from the QDs to the incorporated dye units resulted in emission from the dye and quenching of the QD fluorescence. A similar method was used to follow the activity of telomerase, which is considered an important marker of cancer. Telomeres are sequences composed of G-rich repeats that protect the genetic information in chromosomes. During the cell cycle, telomeres are gradually shortened; at a certain length of the DNA strand, an intracellular signal that terminates cell proliferation is activated [78,79]. In some cells, telomerase is accumulated and elongates the telomere strands parallel to their natural shortening, which results in immortal cell lines. In over 95% of various cancer cells, elevated amounts of telomerase have been observed, and the enzyme is considered to be a universal marker for tumor cells [80,81]. To analyze the telomerase activity, QDs were modified with a nucleic acid primer recognized by telomerase (Fig. 6). In the presence of telomerase and a dntp mixture containing Texas Red-functionalized dutp, telomerization of the QD-associated primer occurred, and the Texas-Red-labeled nucleotide was incorporated into the telomeres (Fig. 6). The FRET from the QDs to the dye enabled the time course of telomerization to be followed [77]. Fig. 6. Optical analysis of the telomerase activity by means of incorporation of Texas Red dutp into telomeres associated with CdSe/ZnS quantum dots. The telomerization of the nucleic acid associated with QDs occurs in the presence of telomerase and a dntp mixture containing Texas Red-functionalized dutp. D, dye unit. Reproduced from Ref. [77].

9 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) The energy transfer from QDs was also used to monitor hybridization of DNA in QD molecular beacons (MBs) [82,83]. Kim et al. used MBs consisting of a QD and a quencher molecule linked to the opposite termini of a hairpin structure of a single-stranded oligonucleotide. In the absence of the target DNA, the oligonucleotides were in the hairpin form, hence, the QDs and quenchers were close to each other, and FRET was quenched. Hybridization of the target DNA with the single-stranded loop of the oligonucleotide hairpin opened the stem duplex region, and the MB assumed an extended configuration. The increased spatial separation of the QD from the quencher restored the fluorescence of the QD [82]. Whereas the original FRET-based biosensing systems used QDs as energy donors, there are several recent developments with QDs as energy acceptors. Conventional organic dyes cannot serve as donors for any significant FRET because of the long exciton lifetime of the QD acceptor compared to that of the dye, combined with substantial direct excitation of the QD at the dye excitation wavelength [84]. Ithas been found, however, that lanthanide complexes exhibit long luminescence lifetimes. In a model biotin avidin biosensing system, in which compounds were labeled with terbium or europium complexes as donors and QDs as acceptors, FRET was found to occur [85,86]. Recently, So et al. have demonstrated the feasibility of using QDs as energy acceptors in a bioluminescence resonance energy transfer (BRET) system [87]. The effect of self-illuminating quantum dots was demonstrated with Luc8, a variant of R. reniformis luciferase, linked to carboxylic acid-capped QDs. Upon addition of coelenterazine, which is a luminescent substrate of the luciferase enzyme, emission peaks at both 480 nm (coelenterazine emission) and 655 nm (QD emission) appeared. This method also provides highly sensitive detection of proteases [88]. In addition to FRET and BRET, electron transfer (ET) quenching may be used as an alternative way to monitor biorecognition events and biocatalytic transformations. The activities of different enzymes was probed by electrontransfer quenching of semiconductor QDs. Gill et al. used QDs capped with a monolayer of methyl ester tyrosine in a study on tyrosinase activity [89]. Tyrosinase-induced oxidation of tyrosine units into the corresponding dopaquinone units yielded active quencher units that suppressed the fluorescence of the QDs. Depletion of QD fluorescence upon interaction with different concentrations of tyrosinase was used to monitor the tyrosinase activity by the time course of oxidation of L-DOPA residues. Apart from demonstrating that QDs can be used to monitor biocatalytic processes, analysis of the tyrosinase activity using QDs has practical implications. Elevated amounts of tyrosinase have been found in melanoma cells; therefore, rapid optical detection of this biocatalytic biomarker by means of QDs could be a useful diagnostic technique. Several studies have demonstrated that QDs conjugated to target-binding molecules, which may be either proteins [73,75], antibody fragments [90], or DNA aptamers [91], can serve as efficient FRET donors in detecting small analytes. In addition, QD aptamer (QD Apt) conjugates have proved to be efficient in imaging and delivering anticancer drugs to prostate cancer (PCa) cells and sense the delivery of drugs to the targeted tumor cells based on the FRET mechanism [92]. QD Apt conjugates used by Bagalkot et al. comprise three components: QDs, which function as fluorescent imaging vehicles [2,93,94], RNA aptamers covalently attached to the QD surface, which fulfill a dual function as targeting molecules and drug-carrying vehicles [95], and doxorubicin (Dox), which is a widely used anthracycline drug with known fluorescent properties interacting with RNA and DNA by intercalation and inhibition of their biosynthesis, thus acting as a therapeutic agent [96]. The assembly of these components results in the formation of a bi-fret complex: the QD Dox donor acceptor FRET pair, where the fluorescence of QD is quenched as a result of Dox absorbance, and the Dox aptamer donor quencher FRET pair, where Dox is quenched by a double-stranded RNA aptamer. Therefore, both QD and Dox of the conjugate are in the off fluorescence state when the QD Apt is loaded with Dox [QD Apt(Dox)]. After the particle is taken up by targeted cancer cells, Dox is gradually released from the conjugate, which induces the transition of QD and Dox fluorescence to the on state. This multifunctional nanoparticle system could be used to deliver Dox to the targeted cells and sense the delivery of Dox by activating the fluorescence of the QD, which concurrently images the cancer cells. Bagalkot et al. used conjugates of QDs with the A10 RNA aptamer, which recognizes the extracellular domain of prostate-specific membrane antigen (PSMA), to develop a QD Apt system that is specifically taken up by prostate cancer cells expressing the PSMA protein and can be used to image these cells [92]. 3. Use of quantum dot-tagged microbeads for multiplexed detection and diagnosis The spectral coding technology with the use of QD-embedded microspheres is expected to offer new opportunities in gene expression studies, high-throughput screening, and medical diagnosis. Reportedly, QD-encoded microbeads could be efficiently used for multiplexed detection of oligonucleotide probe hybridization [21]. Han et al. used triple-color-encoded microbeads conjugated with oligonucleotide probes complementary to the target oligonucleotides labeled with the fluorescent dye Cascade Blue. DNA hybridization studies demonstrated that the coding and the target signals can be simultaneously read at the single-bead level [21]. NC-tagged microbeads proved to be a sensitive tool for high-throughput genotyping of ten single nucleotide polymorphisms of PCR-amplified genomic DNA [97]. Eastman et al. used magnetic microbeads encoded with four colors and twelve intensity levels of QDs for gene expression analysis with accuracy and sensitivity comparable to those of

10 10 O. Akinfieva et al. / Critical Reviews in Oncology/Hematology 86 (2013) 1 14 microarrays [98]. Gao et al. demonstrated the efficiency of QD-encoded microbeads for multiplexed genetic detection of non-amplified DNA samples [24]. Early screening and detection of tumor markers in sera of cancer patients could hardly be overestimated; it is at the early stage of protein secretion, before the manifestation of obvious symptoms, that most cancer patients have the greatest chance to be cured. The development of flow cytometric assays based on the use of QD-encoded microbeads in analysis of tumor serum markers may considerably contribute to the prevention of metastatic disease and complete tumor elimination. Alternative technologies for the detection of cancer serum markers, such as ELISA or MFCA, developed by Luminex Company, provide effective quantification of tumor markers, however, these techniques have considerable disadvantages compared to the multiplexed detection with the use of QD-encoded microbeads. ELISA can detect only a single protein in one sample, which makes analysis of multiple cancer markers expensive and time-consuming. ELISA has also other limitations, such as the need for a large sample volume, a narrow dynamic range, and complicated dilution procedures [99,100]. MFCA are characterized by greater dynamic range of measurement and considerably less preparation time and labor over the conventional gold standard, which was ELISA. Sun et al. have demonstrated that MFCA used for quantification of human AFP, CEA, cancer antigens (CA) 19-9, CA24-2 and CA72-4 has considerable advantages over ELISA, including the capacity for detecting large numbers of analytes simultaneously, thereby providing a tool for multiple tumor marker profiling [25]. However, polymeric beads optically encoded with organic dyes which are used in the Luminex technology allow for a limited number of unique codes, whereas semiconductor nanocrystals as fluorescent tags improve the multiplexing capacity, photostability, and antigen-sensitivity of microbeads [7,21,24]. Identification of autoantibodies in cancer patients sera is important for a better understanding of the early-stage immune response to cancer, and for the finding the antigens that may be suitable for immunotherapy. There is evidence that assay of autoantibodies against a panel of antigens could be used as an aid to mammography in the detection and diagnosis of the early primary breast cancer [51]. A mass spectrometry-based differential immunoproteomic method uses native tumor autoantigens against immunoprecipitate autoantibodies from cancer patients sera to identify autoantibody signatures that are common in sera of diseased individuals and are not found in control sera. In this connection, QD-based microbead suspension arrays may also have important implications for further development of diagnosis and immunotherapy. QD-encoded microbeads have proved to be highly informative in the FRET detection of autoantibodies. The scheme of the autoantibody profiling in the energy transfer mode, included several steps: conjugation of nanoparticle-encoded fluorescent beads with antigens, incubation of microspheres with autoantibodies against bead-tagged antigens, and staining of the beads with AlexaFluor 633-labeled secondary antibodies [7]. It was demonstrated, that a sufficient spectral overlap of the QD emission with the absorption of the dye potentiated excitation energy transfer from QDs to the neighboring dye labels on secondary antibodies, which confirmed the binding effect (Fig. 7). The large spectral distance between the emissions of QDs and the dye allowed the two emission peaks to be easily differentiated, providing multiplexed detection. It was found out that when a QD-selective Fig. 7. Flow cytometry assay for immunodetection of autoantibodies with the use of quantum dot-encoded, antigen-coated microbeads. Microbeads optically encoded with orange-emitting QDs and coated with the human topoisomerase I antigen are incubated with anti-topoisomerase I monoclonal antibodies or with the serum samples of patients and stained with the AlexaFluor 633-labeled secondary antibodies. Excitation is provided by the nm broad band from a fluorescence microscope light source. Adapted from Ref. [7].