Optical contrast can arise from endogenous fluorophores or exogenously administered contrast agents. In autofluorescence imaging (AFI), tissue excitation with light of a short wavelength results in emission of a longer wavelength. Alterations in the autofluorescence pattern of neoplastic tissue have been attributed to altered metabolic activity, such as FAD, NADH, and porphyrins as well as hemoglobin content and a breakdown of collagen fiber cross-links. This results in a shift towards the red spectrum when such tissue is excited with blue light. In addition, typical morphologic signs of malignancy such as increased nuclear-to-cytoplasmic ratio influence the propagation of light. The altered autofluorescence signal is translated into false colored images, usually depicting neoplasia in purple against a green background of healthy mucosa. Many of these tissue alterations are not specific for neoplasia, and the resultant AFI image is a combination of multiple molecular alterations. Therefore, AFI suffers from a low specificity and a high false-positive rate but benefits from the fact that no contrast agent has to be applied during endoscopy. On the other hand, the intrinsic signal can be enhanced by the application of precursor molecules that are metabolized to photodynamically active substances. 5-aminolevulinic acid (5-ALA) is the most widely used agent. Similar to AFI, inflammation negatively impacts on the specificity.
Induced fluorescence is several orders of magnitudes more intense than autofluorescence. Exogenous molecular probes usually target a disease-specific biomarker(1
). Such probes include antibodies, antibody fragments, peptides, nanoparticles and “smart” activatable probes (). Several studies have used fluorescently labeled antibodies against epitopes that are commonly overexpressed in most gastrointestinal cancers, such as vascular endothelial growth factor (VEGF) or epidermal growth factor receptor (EGFR) (2
). Antibodies bind to their target structure in a highly selective manner, thereby optimizing the signal-to-background ratio. In addition, the biologic relevance of their targets is often well established and exploited therapeutically even today, such as by Cetuximab or Panitumumab (against EGFR) or Bevacizumab (against VEGF). Imaging of tumors after a first labeled test dose could potentially predict response to targeted chemotherapy. On the other hand, antibodies may confer allergic reactions after systemic application, and their diffusion across epithelial borders and delivery to target structures is slow due to their high molecular weight. Peptides are low molecular weight molecules that consist of a few amino acids in length and face fewer of these limitations(4
). The challenge in developing these peptides is to select unique sequences that have high specificity and affinity towards the target structures. Antibody fragments could serve as an alternative. Nanoparticles such as quantum dots and metallic nanoparticles can be coated with significantly stronger fluorophores. In animal and cell culture studies, they allow targeting of even minute amounts of target structures, and can be loaded with ligands to multiple biomarkers. Pharmacotoxic considerations have so far precluded widespread clinical testing.
Comparison of different molecular probe classes
The contrast agents described above all rely on direct binding to their target site. Depending on their affinity and the biodistribution of the target they may show a significant background signal of unbound or non-specifically bound agent. In contrast, “smart” probes are activated by specific biomarkers that are selectively upregulated in the tissue of interest. Imaging probes have been designed to be activatable by proteases overexpressed in tumors(5
). In their native state, the fluorescent activity is quenched by energy resonance transfer among fluorophores or by a molecular quencher. After cleavage by tumor-associated proteases, these probes show a significant increase of fluorescence intensity in the tumor. pH-activatable probes have also been developed that can be linked to target specific structures on the tumor cell surface that are to be internalized after probe binding(6
). After subsequent integration into the acidic lysosomes, the pH-sensing fluorophore is activated. Both quenching in the quiescent state and tumor-specific activation optimize the signal-to-background ratio for fluorescent imaging.
In order to obtain a specific signal, the molecular probe has to gain access to the region of interest. Eventually the choice for either route of application will be determined by the distribution and accessibility of the target structure. Systemic application may be preferred if even distribution throughout the body is sought. Potential side effects of intravenous application may be higher than with topical application, and the timing of imaging after contrast application has to be well standardized to ensure optimal binding to the target while at the same time minimizing background signal. With topical application, e.g. via a spraying catheter during colonoscopy(4
), a region of interest has to be identified a priori, and specific binding of the molecular probe to the target has to occur within a time frame that is compatible with the endoscopy procedure, usually within a few minutes. Targets have to be on the luminal surface of the tissue, or the fluorescent probe has to permeate rapidly towards the target structure to ensure binding.
None of the currently tested molecular probes has yet undergone extensive pharmacodynamic and safety assessments. However, in radiology and nuclear medicine molecular imaging with radioactively labeled probes has become part of the routine workup for certain indications. Similarly, protocols for the application of fluorescently labeled probes in GI endoscopy are now being tested.