It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from non-target tissues is critical. Here, we achieve highly specific in vivo cancer visualization by employing a newly-designed targeted "activatable" fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pHactivatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex and in vivo imaging of HER2-positive lung cancer cells in mice were performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energyconsuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.Genetic cell labeling techniques show the possibility of detecting or tracing a single cell in vivo [1][2][3] , however, currently available injectable molecular imaging probes are limited in their ability to detect small volumes of viable cancer because of low target-to-background ratios. Generally, small molecular probes lacks specificity and low target accumulation, in contrast, larger molecules shows prolonged high retention and background 4 . However, such largemolecular complexes are cleared slowly, so a considerable amount of unbound probe remains. These pharmacokinetic characteristics result in high background signal (Scheme 1a).In order to overcome this problem, we developed an activatable fluorescence probe consisting of: 1) a cancer targeting macromolecule and 2) a small-molecular fluorescent moiety activated only within cancer cells to minimize the background signal and maximize tumor-to-normal tissue (T/N) ratio (Scheme 1b).We targeted the human epidermal growth factor type 2 (HER2) receptor with the monoclonal antibody, trastuzumab which, after binding to HER2, is internalized via the endosomsallysosomal degradation pathway 5 .The lysosome is distinct from other cellular organelles because of its low pH (pH 5-6) relative to the cytoplasm (pH ∼7.4). By designing a probe that activates in an acidic environment, the agent yields a highly tumor specific signal with greatly reduced background signal (Scheme 1c). Results Development of tunable, acidic pH-activatable fluorescent moietyTo achieve signal activation within the acidic environment of the lysosome, we required smallmolecular fluorescent molecules with the following characteristics: 1) They should be almost non-fluorescent in the extracellular environment, i.e. at pH 7.4. 2) They should become highly fluorescent under acidic conditions, i.e. pH < 6. 3) They need to be excited by long-wavelength light (≥ 500 nm) and emit a fluorescent signal which overcomes autofluorescence. 4) ...
Quantum dots can be used to perform multicolor images with high fluorescent intensity and are of a nanosize suitable for lymphatic imaging via direct interstitial injection. Here simultaneous multicolor in vivo wavelength-resolved spectral fluorescence lymphangiography is shown using five quantum dots with similar physical sizes but different emission spectra. This allows noninvasive and simultaneous visualization of five separate lymphatic flows draining and may have implications for predicting the route of cancer metastasis into the lymph nodes.
High uptake of 18F-FDG would be predictive of poor prognosis in patients with primary breast cancer, and aggressive features of cancer cells in patients with early breast cancer. 18F-FDG PET/CT could be a useful tool to pre-therapeutically predict biological characteristics and baseline risk of breast cancer.
Current contrast agents generally have one function and can only be imaged in monochrome, therefore, the majority of imaging methods can only impart uniparametric information. A single nano-particle has the potential to be loaded with multiple payloads. Such multi-modality probes have the ability to be imaged by more than one imaging technique, which could compensate for the weakness or even combine the advantages of each individual modality. Furthermore, optical imaging using different optical probes enables us to achieve multi-color in vivo imaging, wherein multiple parameters can be read from a single image. To allow differentiation of multiple optical signals in vivo, each probe should have a close but different near infrared emission. To this end, we synthesized nano-probes with multi-modal and multi-color potential, which employed a polyamidoamine dendrimer platform linked to both radionuclides and optical probes, permitting dual-modality scintigraphic and 5-color near infrared optical lymphatic imaging using a multiple excitation spectrally-resolved fluorescence imaging technique. Keywordsdendrimer; scintigraphy; near infrared; fluorescence imaging; multiple modalities; multiple colors; lymphatic imaging No imaging modality is perfect. Each has its own distinct advantages and limitations. The simultaneous use of two or more modalities can help to overcome the limitation of each individual method and increase or improve the information obtained during an examination session. The combined use of Computed Tomography (CT) and Positron Emission Tomography (PET) is a successful example of multi-modal imaging: CT provides high resolution anatomical detail and PET provides functional information 1 . Currently they are very few examples of multi-modal imaging probes that can be detected by more than one technique: dual agents for recognition by both radionuclide and optical imaging 2,3 , or Magnetic Resonance (MR) and optical imaging 4-8 . Furthermore, the conventional imaging methods are generally monochrome and only able to detect one contrast agent at a time, limiting us to single parametric data. Single photon scintigraphy has been shown to have potential for simultaneously detecting two different imaging agents, i.e. technetium-99m and thallium-201, by energy resolution 9 . However, in this case, both the spatial and the energy resolutions were poor and did not allow for the reconstruction of a precise image from each agent. Multi-color optical imaging is simple to achieve with the technique of spectrally resolved imaging. Herein, two or more optical agents can be differentiated on the basis of their different emission spectra. Multi-color imaging is already commonplace in microscopic imaging and is beginning to be utilized for in vivo imaging 10-12 . However, in vivo imaging is essentially limited to long wavelength dyes that emit in the near-infrared (NIR) range (650-850 nm), in order to maximize depth penetration and limit the autofluorescence, background signal 13 .With this in mind we have synthe...
Abstractβ-Galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TGβGal (J. Am. Chem. Soc., 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-βGal, provides a dramatic fluorescence enhancement upon reaction with β-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-βGal, after confirming that tumors in the model could be labeled with avidin-β-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-βGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 μm. This strategy has potential clinical application, for example in video-assisted laparoscopic tumor resection.
A target cell-specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in ''dequenching.'' In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures. [Cancer Res 2007;67(6):2791-9]
Purpose: Epidermal growth factor receptors (EGFR) play an important role in tumorigenesis and, therefore, have become targets for new molecular therapies. Here, we use a “cocktail” of optically labeled monoclonal antibodies directed against EGFR-1 (HER1) and EGFR-2 (HER2) to distinguish tumors by their cell surface expression profiles. Experimental Design: In vivo imaging experiments were done in tumor-bearing mice following s.c. injection of A431 (overexpressing HER1), NIH3T3/HER2+ (overexpressing HER2), and Balb3T3/DsRed (non-expression control) cell lines. After tumor establishment, a cocktail of optically labeled antibodies: Cy5.5-labeled cetuximab (anti-HER1) and Cy7-labeled trastuzumab (anti-HER2) was i.v. injected. In vivo and ex vivo fluorescence imaging was done. For comparison with radionuclide imaging, experiments were undertaken using 111Indium-labeled antibodies. Additionally, a “blinded” diagnostic study was done for mice bearing one tumor type. Results: In vivo spectral fluorescent molecular imaging of 14 mice with three tumor types clearly differentiated tumors using the cocktail of optically labeled antibodies both in vivo and ex vivo. Twenty-four hours after injection, A431 and NIH3T3/HER2+ tumors were detected distinctly by their peak on Cy5.5 and Cy7 spectral images, respectively; radionuclide imaging was unable to clearly distinguish tumors at this time point. In blinded single tumor experiments, investigators were able to correctly diagnose a total of 40 tumors. Conclusion: An in vivo imaging technique using an antibody cocktail simultaneously differentiated two tumors expressing distinct EGFRs and enabled an accurate characterization of each subtype.
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