Recently, in vivo fluorescence imaging using indocyanine green (ICG) has actively been applied to hepatobiliary and pancreatic surgery in clinical settings. 1) Fluorescence cholangiography: fluorescence images of the extrahepatic bile ducts can be obtained by intrabiliary injection of ICG solution (0.025 mg/mL) or preoperative intravenous injection of ICG (2.5 mg). The latter technique begins to be used worldwide for confirmation of the bile duct anatomy during minimally-invasive cholecystectomy. 2) Identification of hepatic tumors: Following preoperative intravenous injection of ICG (0.5 mg/kg), it can accumulate in hepatocellular carcinoma tissues and in non-cancerous hepatic parenchyma surrounding liver metastasis, enabling intraoperative identification of subcapsular hepatic tumors by fluorescence imaging. 3) Hepatic segmentation: ICG solution (0.25 mg/5 mL) is injected into a tumor-bearing portal branch under ultrasound guidance (positive staining). ICG can also be administered intravenously following closure of a corresponding portal pedicle (negative staining). These techniques enables delineation of hepatic segmental boundaries throughout surgical procedures. 4) Assessment of blood perfusion: Fluorescence imaging following intraoperative bolus injection of ICG (2.5mg) visualizes arterial/portal blood flows and perfusion in the surrounding organs during surgeries requiring resection/reconstruction of the major vessels. for intraoperative visualization of biological structures and perfusion assessment. In addition, we have developed a novel fluorophore (glutaryl-phenylalanine hydroxymethyl rhodamine green) activated by pancreatic chymotrypsin for real-time identification of pancreatic juice leakage.
Visualization of small tumors inside biological tissue is important in cancer treatment because that promotes accurate surgical resection and enables therapeutic effect monitoring. For sensitive detection of tumor, we have been developing photoacoustic (PA) imaging technique to visualize tumor-specific contrast agents, and have already succeeded to image a subcutaneous tumor of a mouse using the contrast agents. To image tumors inside biological tissues, extension of imaging depth and improvement of sensitivity were required. In this study, to extend imaging depth, we developed a PA tomography (PAT) system that can image entire cross section of mice. To improve sensitivity, we discussed the use of the P(VDF-TrFE) linear array acoustic sensor that can detect PA signals with wide ranges of frequencies. Because PA signals produced from low absorbance optical absorbers shifts to low frequency, we hypothesized that the detection of low frequency PA signals improves sensitivity to low absorbance optical absorbers. We developed a PAT system with both a PZT linear array acoustic sensor and the P(VDF-TrFE) sensor, and performed experiment using tissue-mimicking phantoms to evaluate lower detection limits of absorbance. As a result, PAT images calculated from low frequency components of PA signals detected by the P(VDF-TrFE) sensor could visualize optical absorbers with lower absorbance.
Photoacoustic (PA) imaging is advantageous in contrast agent imaging because of high spatial resolution at depth more than several millimeter inside biological tissues. To detect small tumors specifically, we are developing small organic molecule-based activatable PA probe with mechanism similar to that of the enzyme-activatable fluorescence probe that have successfully used for rapid fluorescence imaging of small tumors. The probe can be imaged also by fluorescence imaging and the fluorescence image can be merged onto the PA images. To extend the imaging depth by increasing PA signal intensity, PA probe that produce PA signals efficiently is required. To select small organic molecules suitable for PA probe, we synthesized small-organic molecule-based contrast agents with various absorption spectra and fluorescence quantum yields and then we exhaustively evaluated their PA signal generation characteristics including PA signal generation efficiencies. To analyze PA signal generation efficiencies precisely, the absolute values of PA signal pressures produced from aqueous solutions of the contrast agents were measured by P(VDF-TrFE) piezoelectric film acoustic sensor. As a result, small organic molecule with low fluorescence quantum yield produced PA signals efficiently. Thus, as opposed to fluorescence probes, PA probes should have low fluorescence quantum yields. By considering the result and other characteristics including excitation wavelengths, we could single out the small organic molecule suitable for PA probe. We synthesized the new activatable PA probe with low fluorescence quantum yield and excitation wavelength longer than 600 nm and its specificity was examined in in vitro experiment.
Multispectral photoacoustic (MS-PA) imaging has been researched to image molecular probes in the presence of strong background signals produced from intrinsic optical absorbers. Spectral fitting method (SFM) discriminates probe signals from background signals by fitting the PA spectra that are calculated from MS-PA images to reference spectra of the probe and background, respectively. Because hemoglobin is a dominant optical absorber in visible to near-infrared wavelength range, absorption spectra of hemoglobin have been widely used as reference background spectra. However, the spectra of background signals produced from heterogeneous biological tissue differ from the reference background spectra due to presence of other intrinsic optical absorbers and effect of optical scattering. Due to the difference, the background signals partly remain in the probe images. To image the probe injected in subcutaneous tumors of mice clearly, we added the melanosome absorption spectrum to the reference background spectra because skin contains nonnegligible concentration of melanosome and the spectrum is very similar to the scattering spectrum of biological tissue. The probe injected in the subcutaneous tumor of mice was an enzyme-activatable probe which show their original colors only in the presence of γ-glutamyltranspeptidase, an enzyme associated with cancer. The probes have been successfully used for rapid fluorescence imaging of cancer. As a result of MS-PA imaging, by considering the melanosome absorption spectrum, the background signals were successfully suppressed and then clearer probe image was obtained. Our MS-PA imaging method afforded successful imaging of tumors in mice injected with activatable PA probes.
The P(VDF-TrFE) sensor which had uniform sensitivity in a frequency range of 2.9 – 19.6 MHz was developed for multispectral photoacoustic imaging (MS-PAI). A small organic molecule-based PA probe synthesized by our group had the absorption maximum at 530 nm and was used as a contrast agent. The PA probe was designed to have low quantum yield. Therefore, the PA probe efficiently converted absorbed optical energies to PA signals. The probe was injected in subcutaneous tumor of mice. Then, the subcutaneous tumor was imaged in vivo by using P(VDF-TrFE) sensor. MS-PAI successfully discriminated the probe signals from background signals produced from endogenous optical absorbers such as hemoglobin. The probe detectability of the P(VDF-TrFE) sensor was evaluated and then compared with that of lead zirconium titanate (PZT) sensors. The P(VDF-TrFE) sensor imaged the tumor more clearly than the PZT sensor with central frequency of 20 MHz, especially when the probe was accumulated in the tumor with low concentration. That was because the low-concentrated probe generated PA signals with low frequency. MS-PAI using P(VDF-TrFE) sensor which can detect PA signals with wide range of frequency is able to image various distribution of the probe and is superior to that using PZT sensor which detects PA signals with narrow frequency range.
We investigated the effects of surface charge of gold nanoparticles (Au NPs) on photoacoustic (PA) signal from cultured cancer cell. We used citrated-coated Au NPs and poly-L-lysine-coated Au NPs as the model of negatively and positively charged Au NPs, respectively. Transmission electron microscopy (TEM) were performed for cellular uptake and intracellular localization. We demonstrated PA signal measurement using ring-shaped piezopolymer (P(VDFTrFE)) film sensor coaxially arranged with an optical fiber. The PA signal intensity of the cationic Au NPs was higher than that of the anionic Au NPs over an incubation period up to 3 hours. We found that the PA signal intensities were highly dependent on the surface charge of the Au NPs because the uptake of Au NPs by cultured cancer cells was dependent on the surface charge. We also found that the aggregation of the Au NPs highly influenced the PA signal intensity. These findings are invaluable to the design and synthesis of Au NPs as PA imaging contrast agents with maximized diagnostic efficacy.
Caged compound is one of the most powerful tools for spatiotemporal control of biomolecules in cells, which can be
activated by irradiation of light. However, ultra violet light, which is required for activation of caged compounds, can
damage cells and has poor permeability into tissues. In addition, invisibility of caged compounds makes it difficult to tell
distribution of released small molecules. At the conference, we will describe the development of novel caging group and
new caged compounds which are fluorescently visible and efficiently activatable with green light. We have found that
boron dipyrromethene (BODIPY), known as a widely used fluorophore, is a potential caging group for phenol, carboxyl
acid and amine, which can be photolized with irradiation of green light at around 500 nm wavelength. Based on the
novel photo-reaction of 4-phenoxy BODIPY derivatives, we have developed caged histamine and applied it to HeLa
cells. Photo-irradiation to cells in the presence of caged histamine induced transient increase of calcium ion in cytosol,
which was specifically inhibited with pyrilamine, a H1 blocker. Also, we showed that BODIPY-caged compound can be
utilized in vivo with tissue-permeable 500 nm green light.
Tumor invasion to the peritoneum is a poor prognostic factor in cancer patients. Accurate diagnosis of disseminated
peritoneal tumors is essential to accurate cancer staging. To date, peritoneal washing cytology during laparotomy has
been used for diagnosis of peritoneal dissemination of gastrointestinal cancer, but its sensitivity has not been satisfactory.
Thus, a more direct approach is indispensable to detect peritoneal dissemination in vivo. Fluorescein diacrylate (FDAcr)
is an esterase-sensitive fluorescent probe derived from fluorescein. In cancer cells, fluorescent fluorescein generated by
exogenous application of FDAcr selectively deposits owing to its stronger hydrolytic enzyme activity and its lower
leakage rate. We examined whether FDAcr can specifically detect disseminated peritoneal tumors in athymic nude
mouse models. Intraperitoneally administered FDAcr revealed disseminated peritoneal microscopic tumors not readily
recognized on white-light imaging. These results suggest that FDAcr is a useful probe for detecting disseminated
peritoneal tumors.
KEYWORDS: Luminescence, Tumors, In vivo imaging, Molecules, Molecular imaging, Cancer, Lung, Imaging systems, Signal detection, Fluorescence resonance energy transfer
Molecular imaging probes rely on high target-to-background ratios (TBR) to achieve maximum sensitivity
and specificity. We utilized "quenchers" to turn off the background signal from the unbound probe and
investigated the ability of specific fluorophore-quencher pairs to activate at target tissues. Both fluorophore
and quencher were conjugated to a single cancer targeting molecule, either avidin or antibody. Fluorescence
signal from these targeting molecules was "turned off" by the quencher in the unbound state, but was "turned
on" only when the molecules bound to the cell surface target and was internalized.
We tested the following fluorophore-quencher combinations based on fluorescence resonance energy transfer
(FRET) pairs; OregonG-BHQ1, RhodG-BHQ1/ATTO540Q, TAMRA-QSY7/QSY21, TexRed-QSY21,
Alexa647-QSY21, Cy5.5-QSY21/BHQ3 and Alexa680-QSY21/BHQ3. Among these, only RhodGATTO540Q
and TAMRA-QSY7/21 pair showed activation upon cell binding/internalization. Among these
combinations, TAMRA-QSY7 pair showed the highest activation (40-fold and 13-fold for avidin and
antibody conjugate, respectively) as measured with an in vitro dissociation assay. The activation was
dependent on the method used to conjugate fluorophores and quenchers to the targeting molecule. In vitro
microscopic studies with TAMRA-QSY7 pair conjugated to avidin or antibody showed high fluorescent
signal inside the target cancer cells, indicating activation after internalization. In vivo imaging studies in
tumor bearing mice demonstrated that tumors could be clearly detected with low background.
Although the precise quenching mechanism remains to be determined, this activation system can achieve high
TBR in vivo molecular imaging.
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.
Nowadays, several tumor imaging modalities such as MRI, PET and fluorescence imaging techniques have been
extensively investigated. One of the central problems associated with these conventional tumor-targeted imaging
methods, however, is the fact that the signal contrast between tumor and surrounding tissues relies on the efficient
targeting to the tumor and the rapid sequestration or excretion of unbound agent. Among these modalities, only
fluorescence imaging technique has a significant feature, in that great signal activation could be achieved which
potentially leads to the selective imaging of cancer with higher tumor-to-background ratio. In this symposium, I will
present some examples of fluorescence cancer imaging based on highly activatable strategies with using precisely
designed novel fluorescence probes.
Recently, we developed highly sensitive fluorescence probes for β-galactosidase which is applicable for living cell
system. By utilizing these probes, we could establish a novel and highly activatable strategy for sensitive and selective
optical imaging of imbedded tumor in the peritoneum. We took a two step procedure in that a lectin is used to localize
β-galactosidase to cancer cells as an activating enzyme, and subsequent administration of a highly-sensitive fluorescence
probe for the enzyme have afforded remarkable fluorescence activation selectively in tumor mass. Since the
tumor-targeted enzyme can catalyze numerous substrate turnovers, a great number of fluorescent molecules could be
produced and hence the rapid and sensitive detection of tumor in vivo with high tumor-to-background ratio could be
achieved. Moreover, the consequent close-up investigation using fluorescence microscopy revealed that cancer microfoci
as small as 200 μm could be successfully visualized.
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.
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.
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