One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with
minimal background. Here, we achieve specific in vivo tumor visualization with a newly-designed "activatable" targeted
fluorescence probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel
acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a
cancer-targeting monoclonal antibody, Trastuzumab, or galactosyl serum albumin (GSA). As proof of concept, ex and in
vivo imaging of two different tumor mouse models was performed: HER2-overexpressed lung metastasis tumor with
Trastuzumab-pH probe conjugates and lectin-overexpressed i.p. disseminated tumor with GSA-pH probe conjugates.
These pH-activatable targeted probes were highly specific for tumors with minimal background signal. Because the
acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully
visualized. Furthermore, this strategy was also applied to fluorescence endoscopy in tumor mouse models, resulting in
specific visualization of tumors as small as submillimeter in size that could hardly detected by naked eyes because of
their poor contrast against normal tissues. The design concept can be widely adapted to cancer-specific
cell-surface-targeting molecules that result in cellular internalization.
The ability to obtain multi-color fluorescent imaging in vivo simultaneously using multi-targeted imaging probes could
be of potential benefit from both a research and a clinical perspective. However, the simultaneous acquisition of more
than 2 separate organic fluorophores usually requires more than one excitation source, since a single excitation source
may not optimally excite all the fluorophores. In this study, we employed a multi-excitation approach in order to acquire
optimized images with multiple near infrared (NIR) organic fluorophores at the same time. Using 3 sets of excitation
filters (595±20nm, 640±25nm, 688±17nm) to acquire 3 distinct spectra and spectral unmixing software (CRi, Woburn,
MA), it was possible to resolve the emission spectra of each of the NIR fluorophores using commercial software
(Nuance, CRi, Woburn, MA) To demonstrate the utility of this approach 2 mouse models were investigated; In one
model, mice bearing four implanted malignancies were injected with a cocktail of 3 fluorescently labeled monoclonal
antibodies, each with its own distinct NIR fluorophore. In the second model five different lymph node drainage basins
were imaged with 5-color dendrimer-based lymphatic imaging agents tagged with 5 different NIR fluorophores. We
successfully detected each of the targeted tumors in the first model and all of the lymph nodes by their distinct color in
the second model; neither of which would have been possible using the single excitation method. In conclusion, multi-excitation
NIR spectral fluorescence imaging is feasible in a reasonable time frame and opens the possibility for in vivo
immunohistochemical imaging (IHCi).
The wavelength resolved spectral fluorescence imaging technique using a fluorescein-conjugated avidin has been reported to visualize submillimeter implants of ovarian cancer because of its highly targeted and quickly cleared pharmacokinetics. However, clinical application of avidin was hampered by its strong immunogenicity. As a clinically feasible alternative to avidin, which targets the same D-galactose receptor but is made from a nonimmunogenic source, with even better binding capability by multiplying binding sites but still maintaining a favorable characteristic of high isoelectric point, a serum albumin conjugated with 23 galactosamine and 2 rhodamine green molecules (GmSA-RhodG) was designed and synthesized. GmSA-RhodG showed more than 10-fold rapid and higher uptake by SHIN3 ovarian cancer cells than both avidin- and no galactosamine-conjugated albumin (bovine serum)–RhodG. Sensitivity and specificity of GmSA-RhodG to detect red fluorescence labeled peritoneal cancer foci in mouse cancer model were 100%/99% (n=566), respectively for ~1-mm lesions and even smaller lesions were detected in vivo. These results indicate that GmSA-RhodG is not only a clinically feasible alternative but more efficient targeting reagent for D-galactose receptors than avidin-RhodG.
Optical imaging is unique among in vivo imaging methods because it is possible to simultaneously resolve two or more probes emitting at different wavelengths of light. We employed two near-infrared (NIR) fluorescent optical probes, each labeled with a different protein, to simultaneously evaluate the pharmacokinetics of each probe. Dynamic optical imaging was performed in live mice after the coinjection of bovine serum albumin (BSA) and galactosamine-conjugated bovine serum albumin (GmSA) labeled with either Cy5.5 or Cy7 NIR dyes. The pharmacokinetics of BSA and GmSA were independently and simultaneously visualized. Next, two-color dynamic imaging of biotinylated BSA (b-BSA) and BSA labeled with Cy5.5 or Cy7 was performed before and after an avidin “chase.” Following avidin injection, fluorescently labeled b-BSA rapidly accumulated in the liver, while minimal liver uptake of BSA was noted. Thus, multicolor dynamic contrast-enhanced optical imaging can be performed to noninvasively track the pharmacokinetics of different proteins. This imaging technique can be applied to a wide variety of optically labeled proteins in order to simultaneously track their biodistribution.
KEYWORDS: Luminescence, Tumors, Cancer, Lung, Receptors, Fluorescent proteins, In vivo imaging, Target detection, Animal model studies, Control systems
Target-specific contrast agents are being developed for the molecular imaging of cancer. Optically detectable
target-specific agents are promising for clinical applications because of their high sensitivity and specificity.
Pre clinical testing is needed, however, to validate the actual sensitivity and specificity of these agents in
animal models, and involves both conventional histology and immunohistochemistry, which requires large
numbers of animals and samples with costly handling. However, a superior validation tool takes advantage of
genetic engineering technology whereby cell lines are transfected with genes that induce the target cell to
produce fluorescent proteins with characteristic emission spectra thus, identifying them as cancer cells.
Multicolor fluorescence imaging of these genetically engineered probes can provide rapid validation of newly
developed exogenous probes that fluoresce at different wavelengths. For example, the plasmid containing the
gene encoding red fluorescent protein (RFP) was transfected into cell lines previously developed to either
express or not-express specific cell surface receptors. Various antibody-based or receptor ligand-based optical
contrast agents with either green or near infrared fluorophores were developed to concurrently target and
validate cancer cells and their positive and negative controls, such as &bgr;-D-galactose receptor, HER1 and
HER2 in a single animal/organ. Spectrally resolved fluorescence multicolor imaging was used to detect
separate fluorescent emission spectra from the exogenous agents and RFP. Therefore, using this in vivo
imaging technique, we were able to demonstrate the sensitivity and specificity of the target-specific optical
contrast agents, thus reducing the number of animals needed to conduct these experiments.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.