Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less α-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

A methylated membrane protein of 97 kDa was suggested on the basis of mutant analysis to transduce signals from the phototaxis receptor sensory rhodopsin I to the flagellar motor in Halobacterium halobium. Here we report isolation of the proposed transducer protein, cloning of its gene based on partial protein sequences, the complete gene sequence, and analysis of the encoded primary structure. The 1611-basepair gene termination codon overlaps the initiator ATG of the sop1 gene, which encodes the sensory rhodopsin I apoprotein. The predicted size of57 kDa for the methylated protein indicates an aberrant electrophoretic migration on SDS/polyacrylamide gels, as occurs with other acidic halophilic proteins. Putative promotor elements are located in an A+T-rich region upstream of the gene. Comparison of the translated nucleotide sequence with N-terminal sequence of the purified protein shows the protein is synthesized without a processed leader peptide and the N-terminal methionine is removed in the mature protein. The deduced protein sequence predicts two transmembrane helices near the N terminal that would anchor the protein to the membrane. Beyond this hydrophobic region of 46 residues, the remainder of the protein (536-amino acid residues total) is hydrophilic. The C-terminal 270 residues contain a region homologous to the signaling domains of eubacterial transducers (e.g., Escherichia coli Tsr protein), flanked by two regions homologous to the methylation domains of the transducer family. The protein differs from E. coil Tsr in that it does not have an extramembranous-receptor binding domain but instead has a more extended cytoplasmic region. Coexpression ofthe methylaccepting protein gene (designated Mr!) and sop! restores sensory rhodopsin I phototaxis to a mutant (Pho8l) that contains a deletion in the hlal/sopI region. These results extend the eubacterial transducer family to the archaebacteria and substantiate the proposal that the methylated membrane protein functions as a signal-transducing relay between sensory rhodopsin I and cytoplasmic sensory-pathway components.Sensory rhodopsin I (SR-I) is a retinal-containing intrinsic membrane protein that functions as a phototaxis receptor in the archaebacterium Halobacterium halobium (also known as Halobacterium salinarium) (1). SR-I controls swimming behavior of the cells by modulating the frequency of reorientation ("reversals") of their swimming direction (for reviews, see refs. 2 and 3). Orange light generates attractant signals that suppress reversals, whereas near-UV light generates repellent signals that induce reversals. Several early events in the signaling process have been elucidated. Photon absorption by SR-I (Amax, 587 nm) causes isomerization around the C13==C14 double bond of the retinal chromophore (4), which is essential for receptor activation (5). The photoisomerization energy is transferred to the protein in a process requiring steric interaction between the retinal C13 methyl group and protein residues (6). These events produce in <...

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20 years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.