One of the most promising and rapidly developing areas of application of QDs is their usage as fluorescent labels during in vitro study of tumor cells: for imaging tumor cells and localizing the individual molecules expressed in them. In vitro diagnostics, is the only application of QDs out of all other alternatives available, which can be quickly implemented in clinical practice (as opposed to the in vivo use of QDs, which requires long investigations of QD toxicity and further consequences of their introduction into the body of research organism). The major utilities of QDs as an in vitro diagnostic tool are: imaging of tumor cells overexpressing certain oncomarkers, staining of tissues and their sections, and observation of individual molecules and cells in real time.

The imaging of tumor cells and identification of the individual oncomarkers and their quantitative assessment is very important for the early diagnosis, accurate classification, and optimal therapy of tumors. QDs that conjugated to various targeting agents (antibodies, ligands, peptides) have been known. They are intended for visualizing the cells of clinically significant human tumors: prostate carcinoma, breast adenocarcinoma and ductal carcinoma, pancreatic carcinoma, glioblastoma and squamous cell carcinoma of the tongue. Over a short period of time, appreciably simple, inexpensive, and well reproducible QD-based methods for the imaging of cancer cells were designed to aid in the diagnosis and predicting prognosis of cancers, especially breast cancer (Table 1) [48].

The surface area of nanocrystal QDs is appreciably large and is accessible for binding of several biological molecules. A range of 2 to 5 protein molecules and more than 50 small molecules (oligonucleotides or peptides) can be bound to one nanoparticle 4 nm in diameter. Targeting molecules (antigens, oligonucleotides, peptides, aptamers, etc.) that selectively bind to the surface oncomarker provide a high specificity for labeling the corresponding tumor cells. At the same time, such a feature of tumor cells as their extreme variability during the development of cancer and response to the action of therapeutic agents raises for researchers the problem of simultaneous imaging of several surface markers. The fundamental possibility of using QDs for simultaneous multiplex detection was demonstrated on five tumor markers from a human breast tumor cell culture. The simultaneous detection of the receptors estrogen receptor, progesterone receptor, EGF receptor, mTOP, and HER2/neu, using QDs fluorescence in different spectrum regions correlates positively with the results of conventional methods; including immunohistochemistry, Western blotting, and fluorescence in situ hybridization (FISH), while it considerably increases the rate of analysis and reduces cost [49]. Simultaneous imaging of two hypothesized cancer markers-integrin αvβ3 and nucleolin-using QDs conjugated to the RGD peptide and aptamer AS1411, respectively, enables to compare the localization of these markers within the cell [50]. Internalization of nucleolin and the surface distribution of integrin were confirmed using confocal microscopy, which will probably allow better understanding of how they participate in the processes occurring in tumor cells.

The results of these studies demonstrate that QDs conjugated to targeting molecules have a powerful potential as components of novel systems for assessing tumor types, their progression stage, and the metastatic potential on the basis of multiplex imaging. The combination of conventional IHC procedures with QD-based fluorescent dyes allows considerable improvement in the resolution and sensitivity of the method and provides possibility of simultaneous imaging of several markers. The feature of multiplexing is becoming increasingly important for cancer diagnosis since more and more studies are showing that a panel of biomarkers rather than a single one is needed to accurately determine the stage of the disease. Multiplexing has the advantage of reducing variability between tissue slices and is especially appealing in the case of precious specimens. Moreover the conjugation of QDs with the IHC method makes it much more illustrative. The biomarkers used to detect breast cancer micrometastases in lymph node like: anti-CK antibodies, anti-HER2 antibodies, anti-EpCAM, and anti-MUC1 can be detected by the combined QD-IHC approach, with results much superior to conventional IHC, and sensitivity nearly equal to that of FISH. As for example, HER2 status is usually assessed by standard IHC, with additional FISH in ambiguous cases. However, FISH is expensive, labor intensive and time consuming. Streptavidin coated QDs were used to visualize HER2 positivity after immunohistochemical staining with primary and secondary anti-HER2 antibodies and fluorescence image analysis used to identify and quantify positive signals. The results of QD-IHC were compared to both FISH analysis and conventional IHC in the same tissue samples. Good concordance between QD-IHC and FISH results was seen. The results indicate the feasibility and utility of quantitative IHC using QDs, this approach may be particularly useful in developing countries, where access to molecular techniques, such as FISH, is low [51]. Similarly, other biomarkers of micrometastases can also be detected with a combined QD-IHC approach.

Cell dynamics, molecular signaling mechanisms, conformational changes of proteins etc. are crucial in understanding the processes like chemotaxis and intercellular and intracellular signal transduction. In order to use QDs to gather information about specific cellular processes in real time, a specific cellular target must be considered QDs conjugated with the corresponding targeting ligands have been successfully used for such imaging modalities. Since a vast number of oncomarkers are represented by proteins that also have some regulatory functions in the normal cells and participate in various inter- and intracellular signal transductions.

Studying in detail the functioning of these oncomarkers/proteins is important to understand the multiple steps and mechanisms leading to malignant transformation of a normal cell. QDs conjugated with an EGF were used to study the mechanisms of EGF internalization and signal transduction pathways with the participation of proteins from the erbB-1/2/3 family of transmembrane tyrosine kinase receptors [52]. A study in breast cancer used QDs linked to streptavidin, which allowed them to bind with biotin conjugated targeting agents.

Streptavidin-conjugated QDs were first used for imaging of the tumor marker HER2/neu on the surface of human breast tumor SKBR-3 cells through biotin-conjugated antihuman secondary antibodies and humanized anti-HER2/neu antibodies. For all intended targets, specific labeling signals were brighter and considerably more photo-stable in comparison to the organic dye methods [53]. Multiplexed QDs diagnostics for the marker-molecules may be applicable for high antigen expression pattern contents analysis in the primary tumor tissue radically increasing the diagnostic value of the assays, elucidating differences in patterns and reflecting individual molecular signatures of events like microinvasions, etc. [54]. Since QDs were first introduced, their use in biologic experiments has become common, and important insights in cell biology have been made. For instance, QDs were used for the first time to document the dynamics of receptor transport at the cell membrane level. QDs have also been used to deliver and monitor siRNA in cells [55].

For the molecular genomic diagnosis, QDs conjugated oligonucleotide probes to visualize and map out the genetic material by FISH could identify the genes expressed even at very low levels [56]. In breast cancer, the QD based fluorescent probes were found to be more sensitive than traditional tags for FISH detections of HER2/neu oncogene, single-nucleotide polymorphism of cytochrome p450, and p53 tumor suppressor gene [57,58]. Recently, the HER had been under research focus as a likely dependable cancer marker, including breast cancer. QDs tagged with the EGF staining for the HER2 membrane receptor in cultured human cancer cells presented astonishing sensitive results [59]. QDs genomic analysis was also applicable in the in vitro studies of human metaphase chromosomes in transformed lymphocyte culture and breast cancer cell lines. In addition to tissue diagnostics, there are also emerging applications of QDs for in vitro detection assays and many of them are fluorescence resonance energy transfer based DNA hybridization applications [60]. For the targeted gene expression silencing therapy, the QDs conjugated silencing RNA (HER2-siRNA) may be tracked for delivery of the therapeutic siRNA and monitored for effectiveness of the siRNA-mediated down regulation of concerned receptor in the breast cancer cell lines [61].

Molecular imaging in combination with anatomical imaging, such as CT or positron emission tomography (PET)/CT, enables characterization of the molecular status of deep seated tumors. Their dependence on radioactivity-based methods has certain drawbacks, like short isotope half-lives, lack of multiplexing capability and low spatial resolution. Deep-tissue multiphoton microscopy, can image cells in three dimensions with high sensitivity and high spatial and temporal resolution. However, tissue penetrance of light is low, even for near infrared light, whilst conventional fluorophores are of insufficient brightness or stability for efficient visualization. QDs overcome these problems and have therefore been used extensively in live animal imaging, enabling detection of tumors at the deep sites. Large amounts of QDs can be transferred into live mammalian cells, either by nonspecific pinocytosis, microinjection or peptide-induced transport and up to two billion QDs have been delivered to the nucleus of a single cell without altering the cellular function or viability. Such labeled cells have been used to study embryogenesis [62], cancer metastases [63], stem cell therapy, and lymphocyte homing. They are a particularly powerful tool for embryogenesis and stem cell research, where multiplexing is extremely advantageous given the scarcity of tissue in such situations, whilst stem cells are rare and often require multiple markers for their correct identification. More important use of QDs is for lymph node mapping in cancer. QDs were used to perform simultaneous multicolor imaging of five different lymphatic basins as a tool for mapping lymphatic flow. Near infrared QDs were also used for sentinel node mapping in cancer surgery in animals. QDs were injected intradermally in the distal extremities and imaging methods were used to track their movement along the lymphatic channels, with identification of the sentinel lymph node. Furthermore, these experiments demonstrated high contrast between autofluorescence and emission signal, allowing minimal surgical incision for removal of the positive sentinel node [64]. Later, fluorescent tracking of solubilized NIR QDs injected subcutaneously in the anterior pawn mice demonstrated accumulation in the regional lymph nodes within 5 minutes of injection, and with a maximum concentration after 4 hours of injection, which then gradually falls down in the next 10 days, with resultant low-level uptake in other organs. Tracking, using fluorescent imaging, was compared with inductively coupled plasma-mass spectroscopy, demonstrating advantages of fluorescent imaging. These experiments were performed in mice and pigs, demonstrating their applicability for larger mammals, raising the possibility of intraoperative use in humans [65]. Such an approach would be particularly useful in breast cancer surgery, in which the sentinel node mapping is very common. QDs have also been used to image blood vessels in live mice, which demonstrated higher contrast and imaging depth than previously achieved with organic fluorophores. QDs have also been used to track cancer metastases, either by labeling antibodies reactive against cancer cells, or by direct labeling of cancer cells. Noh et al. [66] used QD-labeled dendritic cells to track their migration, following injection into mice foot pads, demonstrating that their final destination was popliteal or inguinal lymph nodes. This study demonstrated the ability of QD labeling to track immunotherapeutic cells, of importance for understanding the novel dendritic cell-based vaccination. These not only allow elegant imaging, but also enable real-time studies to be performed without frequent animal sacrifice, with advantages of improved experimental control and reduced cost and suffering, these factors could be vital for experimental feasibility. QDs with appropriate ligands as effecter molecules provide means for 'prolonged real-time visualizations' of the multistep signaling mechanisms in the living cell, and affording detailed movies of the processes involved. Cancer cell targeting by drug/Herceptin and its tracking by single QD conjugation with monoclonal anti-HER2 Ab was also a success in mice [67]. Various stages, which were visualized included: circulation in blood vessel, extravasation from blood vessel, presence in extracellular region, binding to HER2 on the cell membrane, moving from cell membrane to perinuclear region and finally presence in perinuclear region real-time imaging, using QDs, has an inherent limitation of autofluorescence (background signal) from intrinsic fluorophores in the normal tissue, lowering the target-to-background ratio. Several new technologies have been developed to reduce the autofluorescence. For instance, upconverting nanoparticles (UCNPs) are unique nanocrystals that emit light at shorter wavelengths than the excitation light in the NIR. This is achieved by converting energy of two photons serially absorbed by the UCNP. Conventional fluorophores, including QDs, typically emit light at longer wavelengths than the excitation light. Although, the shorter wavelength excitation light also excites autofluorescence, but in UCNPs, the longer wavelength excitation light (in the NIR) avoids exciting autofluorescence, and therefore, the shorter wavelength emission light is easily detectable. Optical lymphangiography in mice was successfully performed; superficial neck lymph nodes of the mice were depicted with no background signal in the visible range or NIR range. Furthermore, with serial injections of both the longer wavelength NIR and visible light, UCNPs lymphatic imaging was achieved in two colors simultaneously without a background signal.

In another approach to reduce the autofluorescence, QDs have been conjugated with luciferase, enabling self-illumination based on the principle of bioluminescence resonance energy transfer (BRET). The fluorescence emitted by these QDs can be illuminated by the bioluminescence produced by the reaction between luciferase and coelenterazine around the QD surface. Therefore, self-illuminating QDs can emit bright fluorescence with no requirement for external excitation, which exhibits extremely high sensitivity for use in vivo imaging. Such a bioluminescent approach enables QDs to be visualized at the deep sites where incident excitation light is low. Experiments on mice were performed to look for the feasibility of self-illuminating QDs. In all experimental mice, the SLN in each lymphatic basin were clearly visualized with no background signals. Sufficient signal for imaging was present for at least 30 minutes after coelenterazine injection. Moreover, by changing the QDs within the BRET-QD conjugate, it is possible to alter the emission wavelength, enabling multicolor in vivo lymphatic imaging with BRET-QDs [68]. QDs have a short half-life in circulation, due to hepatic uptake and efforts are going on to increase half-life by attachment of passivating molecules, such as polyethylene glycol, though this introduces further toxicity issues. Gao et al. [69] generated mercaptopropionic acid coated InAs/InP/ZnSe QDs with enhanced permeability and retention in vivo. They have an emission wavelength of approximately 800 nm and a very small hydrodynamic diameter (less than 10 nm) enhancing cellular uptake. They were highly accumulated in tumor xenografts in living mice, whilst additional coating with human serum albumin reduced localization in macrophages, and therefore, in the reticuloendothelial system, thus increasing their relative accumulation in tumors. There is an increasing body of work detailing the generation of multimodal QDs capable of both in vivo tumor cell tracking and of drug delivery. Weng et al. [70] conjugated liposomes to QDs together with anti-HER2 antibody, using the liposomes for doxorubicin loading, showing efficient anticancer activity in HER2 overexpressing breast cancer cells, and enabling tumor cell imaging. Recently, Harbeck and Thomssen [71] reported that node-negative breast cancer does not automatically suggest a good prognosis, or preclusion of chemotherapy benefits. Tumor-grade, Adjuvant! online algorithm, urokinase-type plasminogen activator could not be standard or dependable parameters to decide about the inclusion/exclusion of chemotherapy, hence, additional biomarkers are needed to help identify those patients who will actually benefit from chemotherapy. The dilemma that clinicians now face is about 30% of those node-negative patients who need chemotherapy because of their real chance of cancer recurrence, but there are no tools currently available to identify this subset of patients. In such cases that are declared as node negative by histopathology and other traditional IHC staining methods, QDs based applications may have a role to screen the SLNs, and have potential of elaborating any micrometastases >0.2 mm size as an authentic evidence to the determine need of adjuvant therapy for prevention of recurrence, and better cure. With the results obtained by QDs approach, a multiparametric tool for tumor cells detection is likely to be developed and implemented for early cancer and detection of micrometastases for monitored targeted cancer therapy.

The major limitation shared by all micrometastases detection techniques is a relatively poor tumor cell yield and the low detection limit of labeled cells. Thus, quantitative micrometastases diagnosis remains difficult to be resolved with present traditional means. A more effective approach, based on nanoparticles for biomarkers detection, would increase the diagnostic accuracy. As mentioned earlier, advantages of semiconductor fluorescent QDs over organic dyes include large absorption coefficients across a wide spectral range, size and composition tunable fluorescence emission, multiplexing characteristics (allowing simultaneous detection of several biomarkers), and very high levels of brightness and photostability, etc. Owing to these properties, QDs allow detection at the single particle level, making it possible to carry out an analysis of low-abundant biomolecules and rare cells, such as that of micrometastases. Indeed, a single nanoparticle is large enough for conjugation with multiple ligands, leading to enhanced binding affinity and exquisite specificity through the multivalency effect. These features are especially important in the detection of micrometastases cancer biomarkers, which are expressed in low concentration by few cells only. The possibility of intravital labeling of tumors with QDs was first demonstrated on mouse models. It was demonstrated that after intravenous administration, QDs conjugated to peptides specific to various type of tumors and their vessels are selectively accumulated in the tumor vasculature. This progress in the in vivo application of QDs has stimulated a large number of studies devoted to the intravital imaging of human model tumors in animals using QDs targeted at different tumor markers. Full-size antibodies and their fragments, specific peptides, and natural ligands were used as targeting ligands with equal success. The use of targeted QDs as fluorophores, in combination with modern optical imaging methods, allows to perform imaging of not only solid tumors, but also metastases in other organs, bone tissue and to reveal micrometastases at the early stages of the disease [47]. Direct covalent binding and streptavidin-biotin linkage may be used to conjugate the QDs with a panel of mAbs raised against various cancer markers present on the micrometastatic cells and may potentially be used for in vivo and in vitro serum and tissue diagnosis of the micrometastases [23]. Recently, micrometastases from lung cancer have been successfully detected with the help of magnetic nanoparticles (MNP) and QDs. Pan-cytokeratin (pan-ck) antibody was coupled to the surface of the MNPs, lunx (a novel human lung specific gene), and surfactant protein-A (SP-A) Abs were attached to QDs. MNPs were initially used for cell enrichment, and QDs were later utilized for micrometastatic cell detection [72]. This successful research of micrometastases detection in lung cancer by QDs has provided a solid scientific evidence, raising the hope that soon in near future, QDs can be similarly used to detect micrometestases in breast cancer. Therefore, the morbidity causing ALND and highly toxic adjuvant chemotherapy would be given to those patients only who are positive for micrometastases. An early detection and adequate removal of the micrometastatic cells will lead to prevention of recurrence later.

QDs toxicity depends on multiple factors derived from both the inherent physicochemical properties of QDs and environmental conditions. QDs size, charge, concentration, outer coating bioactivity (capping material and functional groups), and oxidative, photolytic, and mechanical stability are factors that, collectively and individually, determine the QDs toxicity [73].