Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

An axoplasmic myosin with a calmodulin-like light chain.

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.