We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

We have devised and tested a new strategy for selectively delivering molecules to tumor cells. Cellular association of polyargininebased, cell-penetrating peptides (CPPs) is effectively blocked when they are fused to an inhibitory domain made up of negatively charged residues. We call these fusions activatable CPPs (ACPPs) because cleavage of the linker between the polycationic and polyanionic domains, typically by a protease, releases the CPP portion and its attached cargo to bind to and enter cells. Association with cultured cells typically increases 10-fold or more upon linker cleavage. In mice xenografted with human tumor cells secreting matrix metalloproteinases 2 and 9, ACPPs bearing a far-red-fluorescent cargo show in vivo contrast ratios of 2-3 and a 3.1-fold increase in standard uptake value for tumors relative to contralateral normal tissue or control peptides with scrambled linkers. Ex vivo slices of freshly resected human squamous cell carcinomas give similar or better contrast ratios. Because CPPs are known to import a wide variety of nonoptical contrast and therapeutic agents, ACPPs offer a general strategy toward imaging and treating disease processes associated with linker-cleaving activities such as extracellular proteases.cancer ͉ molecular imaging ͉ polycation ͉ transduction

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...

A glowing new era in cancer surgery may be dawning. Using fluorescently labelled markers, surgical molecular navigation means that tumours and nerves can be displayed in real time intra-operatively in contrasting pseudocolours, which allows more complete tumour resection while preserving important structures. These advances can potentially cause a paradigm shift in cancer surgery, improving patient outcome and decreasing overall health-care costs.