Detection of specific biomarkers in a cancer lesion is one of the most important factors that affects the choice of cancer therapy. Developing drugs targeting specific tumor receptors such as Monoclonal AntiBodies (MAB) has opened an exciting opportunity to selectively target the cancer-causing biomarkers, inactivate specific molecular mechanisms responsible for cell malignancy, and deliver the toxin only to the malignant cells (1
). Recent advances in MABs show that their efficacy depends strongly on the expression of tumor-specific biomarkers (4
). For this reason, development of non-invasive in-vivo
imaging techniques for detection of cancer biomarkers and monitoring the efficacy of the treatment, especially, at the early stages of therapy is one of the major tasks in cancer diagnostics and treatment.
In clinical studies, the current diagnostic gold standards for specific cancer biomarkers are all based on ex-vivo
methods, such as immunohistochemistry (IHC), gene amplification based fluorescent in situ
hybridization (FISH), and enzyme-linked immunosorbent assay (ELISA)(6
). These methods are invasive and require biopsies from the patients. Inherently, biopsies have a risk of missing the malignant lesion and, during the therapeutic cycle, the number of times that the biopsy can be taken is limited. The current goal is to replace these invasive methods with non-invasive imaging, reduce the time between imaging and diagnosis, and facilitate analysis of therapy progression in the clinic with portable and accessible systems.
In cancer, understanding the pathophysiological status of the tumor is likely to be more important than structural imaging. Considering the different imaging modalities that are available now, it should be noted that MRI, CT and ultrasound (US) are optimal for structural imaging, while PET and optical imaging are better for functional and molecular imaging. In many cases, tumor and normal tissues are similar in appearance and structure, making it hard to discriminate them. Targeted molecular probes can be used to differentiate these regions based on their molecular specifications. They can be useful in finding the tumor margin in clinical surgery or diagnosing the metastatic tumors.
Incorporating advances in high quantum yield Near-InfraRed (NIR) fluorescence dyes (9
) and the excellent specificity of molecular probes, combined with significant improvements in fluorescence microscopy and macroscopic imaging systems (11
) make fluorescence imaging a promising candidate for cancer research.
In histopathology and cell biology, labeling the cell surface biomarkers with fluorescent probes helps to identify their role in the origin and progression of diseases (17
). Analysis of the affinity of a specific probe or drug molecule targeted to a cancer biomarker is one of the main goals of in-vitro
fluorescence imaging. These studies play an important role in the early stages of probe and drug development.
In contrast to in-vitro and ex-vivo experiments that deal with cell cultures and tissue samples, in-vivo preclinical studies facilitate investigation of different phases of a disease in a more realistic setting, i.e., in a live animal. Common methodologies in preclinical studies require sacrificing the animals at different stages of disease or treatment to study the lesion after excision of the organ. These methods require sacrificing many animals to obtain sufficient and reliable statistical results. Fluorescence imaging can be used as an in-vivo imaging technique to study the same phenomenon without removing the tumor or sacrificing the animal. In general, fluorescence imaging, compared to other imaging techniques, does not need ionizing radiation probes, and thus its cost is much lower than CT and MRI and can be implemented in a portable device.
In this paper, we review the fluorescence imaging methods including those that have been developed and used in our group to detect and monitor specific cancer biomarker expression in-vitro
for diagnostics and therapy. Here we focus our study on the HER2 receptor, a cancer biomarker that is highly expressed in about 30% of the breast cancer cases (18
). Overexpression of this receptor is correlated with poor prognosis and resistance to specific chemotherapy (21
). To optimize the treatment procedure, it is important to identify the level of expression of the HER2 receptors during the diagnostic process and to monitor it over the course of treatment.
In order to image the HER2 receptors, we used HER2 specific affibody molecules as a targeting agent (22
). Affibody molecules are highly water soluble and about 20 times smaller than antibodies and 4 times smaller than antibody fragments (24
). Due to their small size, they have better conjugation to HER2 receptors and shorter washout time from the body and normal tissues. To track these probes, affibody molecules were conjugated to NIR fluorescent dyes.
Currently, most of the in-vivo
fluorescence studies are based on mapping the fluorescence intensity. The drawback of this approach is the sensitivity of the fluorescence intensity to the fluctuations of the excitation light, distance of the probe from the tumor, and other parameters of the system. To overcome this problem and quantify the specific receptors of the tumor before and during the therapy, we introduced an algorithm based on the compartmental ligand-receptor model. This algorithm uses the dynamic of the normalized fluorescence intensity (uptake) in the tumor compared to the normal tissues at the contralateral site (28
). The results were compared with ELISA, a standard ex-vivo
method that is commonly used to quantify cancer biomarkers.
The other measurable parameter in fluorescence imaging is the fluorescence lifetime. It can provide useful clinical information, because fluorescence lifetime is potentially sensitive to local biochemical environment, e.g., temperature and pH, or molecular interactions (29
). On the other hand, its value does not depend on the concentration of the fluorophores or the intensity of the excitation light (31
). Potential applications of in-vivo
fluorescence lifetime in cancer diagnosis and investigation of the early-phase treatment response in the clinic are as follows: first, in-vivo
monitoring of the environmental differences (e.g. pH) in the tumor compared to normal tissues (32
); second, in-vivo
monitoring of the internalization of a specific drug into malignant and disease cells by using a fluorescent probe with a pH sensitive lifetime; third, developing a fluorescent probe that is sensitive to molecular interactions and capable of revealing the binding of a specific drug molecule to a specific disease/cancer receptor.