Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals such as mice has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans incorporating biliverdin as the chromophore can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation/emission maxima of 684/708 nm respectively, extinction coefficient > 90,000 M −1 cm −1 , and quantum yield 0.07. IFPs express well in mammalian cells and mice, and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water and lipids as well as light scattering (1,2). Thus, genetically encoded infrared fluorescent proteins would be particularly valuable for whole-body imaging in cancer and stem cell biology (3,4), gene therapy etc. However, excitation and emission maxima of FPs have not yet exceeded 598 and 655 nm respectively (5-7). Somewhat longer wavelengths (644 nm excitation, 672 nm emission) have been observed in a phytochrome-based FP that incorporates phycocyanobilin (PCB) as the chromophore (8). However, neither incorporation of exogenous phycocyanobilin nor transfer of its biosynthetic pathway into animal cells has yet been demonstrated. Bacterial phytochromes are more promising because they incorporate biliverdin IXα (BV) instead of PCB (9), and BV is the initial intermediate in heme catabolism by heme oxygenase in all aerobic
High-resolution imaging of molecules intrinsically involved in malignancy and metastasis would be of great value for clinical detection and staging of tumors. We now report in vivo visualization of matrix metalloproteinase activities by MRI and fluorescence of dendrimeric nanoparticles coated with activatable cell penetrating peptides (ACPPs), labeled with Cy5, gadolinium, or both. Uptake of such nanoparticles in tumors is 4-to 15-fold higher than for unconjugated ACPPs. With fluorescent molecules, we are able to detect residual tumor and metastases as small as 200 μm, which can be resected under fluorescence guidance and analyzed histopathologically with fluorescence microscopy. We show that uptake via this mechanism is comparable to that of other near infrared protease sensors, with the added advantage that the approach is translatable to MRI. Once activated, the Gd-labeled nanoparticles deposit high levels (30-50 μM) of Gd in tumor parenchyma with even higher amounts deposited in regions of infiltrative tumor, resulting in useful T 1 contrast lasting several days after injection. These results should improve MRI-guided clinical staging, presurgical planning, and intraoperative fluorescence-guided surgery. The approach may be generalizable to deliver radiationsensitizing and chemotherapeutic agents.Molecular navigation | dendrimeric nanoparticles | molecular amplification | targeted imaging agent | transgenic tumor model C linical cancer staging currently depends mainly on anatomical imaging with x-ray computed tomography (CT) and MRI. Some tumors can be imaged by PET of glucose uptake, but modest spatial resolution, high cost, exposure to radiation, and imperfect correlation of glucose uptake with malignancy limit the usefulness of PET and its more recent combination with CT. MRI is a particularly attractive imaging modality due to its moderate cost, relatively widespread availability, high spatial resolution tomography, excellent anatomical detail, and lack of radioactivity. Most clinical MRI is either T 1 -or T 2 -weighted, for which the standard contrast agents are, respectively, gadolinium (Gd) chelates and superparamagnetic iron oxide particles. The difficulty in using MRI for molecular imaging of specific biomolecules rather than for anatomy is sensitivity, because the detection limit is on the order of 10 −5 M Gd chelate or Fe, respectively (1, 2). Therefore several orders of magnitude of molecular amplification are necessary to detect tumor markers at low nanomolar abundance. T 2 -weighted MRI has the additional disadvantages that contrast is usually negative and the iron oxide particles are largely confined to the intravascular and reticuloendothelial compartments. Recently, there has been interest in designing T 1 magnetic resonance (MR) contrast agents that give information beyond that of a standard blood pool agent and detect tumor neovascularization (3, 4), folate receptor (5), and various antigens (6-8). Recent attempts at in vivo MRI of matrix metalloproteinase (MMP) activity have been based on...
The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative wholebody tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.intraoperative fluorescence imaging | molecular navigation | long-term survival | molecular imaging | surgical margin
Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signaling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to down-regulate dormancy, quiescence, and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting LIFR:STAT3 signaling confers a dormancy phenotype in breast cancer cells disseminated to bone.
Summary Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, due to absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the “optical window” above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.
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