In vivo proteomic imaging analysis of caveolae reveals pumping system to penetrate solid tumors.

Technologies are needed to map and image biological barriers in vivo that limit solid tumor delivery and, ultimately, the effectiveness of imaging and therapeutic agents. Here we integrate proteomic and imaging analyses of caveolae at the blood-tumor interface to discover an active transendothelial portal to infiltrate tumors. A post-translationally modified form of annexin A1 (AnnA1) is selectively concentrated in human and rodent tumor caveolae.

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues.

Ubiquitous yet distinct expression of podocalyxin on vascular surfaces in normal and tumor tissues in the rat.

Background/aims: The vasculature has become an important target in the development of therapies for increasing numbers of human diseases, yet there are few reliable markers available to identify the endothelium in rodent models. We have characterized the expression, subcellular localization and accessibility of the rat pan-endothelial marker podocalyxin (podxl) using a newly developed monoclonal antibody (mAb), G278.

Methods: podxl expression and accessibility to binding by G278 were determined in the rat by a variety of experimental approaches.

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae.

Screening phage display libraries for organ-specific vascular immunotargeting in vivo.

The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream.

Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro.

Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy.

The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation.

Targeting the proteome/epitome, implementation of subtractive immunization.

Monoclonal antibody technology has generated invaluable tools for both the analytical and clinical sciences. However, standard immunization approaches frequently fail to provide monoclonal antibodies with the desired specificity. Subtractive immunization provides a powerful alternative to standard immunization and allows for the production of truly unique antibodies.

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen.

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines.