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The Quigley Laboratory


Identification of Molecules and Pathways that Function in Human Tumor Metastasis and Angiogenesis for Possible Therapeutic Application

Our laboratory is conducting studies on the mechanism of human tumor metastasis using an in vivo model system that employs human tumor cells disseminating to specific organs in the developing chick embryo. This very rapid, fascile and quantitative in vivo model system is used as our initial molecular discovery-based model. When specific contributing molecules or pathways are identified in the chick system, distinct mouse metastasis models are employed to affirm these discoveries and identifications. The ongoing mouse models include both orthotopic tissue implantations and i.v. inoculations of tumor cells directly into the circulation.

A technique known as subtractive immunization is employed to generate in an unbiased manner unique antibodies directed against antigens on the surface of metastatic human tumor cells which are then tested for their ability to modulate metastatic spread in the various live animal models. Tumor cell surface antigens that are functionally involved in metastasis are being identified by these methods. The quantitation of metastasis, using real time qPCR with human specific primers, has allowed for the in vivo selection and isolation of unique tumor cell variant pairs from human fibrosarcomas and prostate, epidermoid and pancreatic carcinomas that greatly differ in their metastatic capabilities. Comparative analyses of the gene and protein expression levels of the human tumor variants are providing distinct information about metastasis-specific molecules.

Also under way in the laboratory is an investigation of a specific early step in metastasis, namely, the process of intravasation, which is the entry of primary tumor cells into the vasculature and is likely a rate-limiting step in tumor dissemination. Three pairs of isogenic human tumor variants have now been isolated and shown to have a 25-100 fold differential in their respective ability to enter the vasculature and disseminate in vivo. The cellular and molecular analysis of these variants using array technology, proteomic approaches, and intravital microscopy has allowed us to identify specific contributory molecules that are functionally important in tumor cell/vascular interactions and impact the intravasation step in the metastatic cascade.

Our laboratory is also examining the differential expression of specific proteolytic enzymes and how these enzymes may mediate angiogenesis and tumor cell migration and invasion. The experimental approach is to employ chemical-based assays to detect those enzymes which are distinctly activated in angiogenic tissue, or around invading tumor cells. We then generate specific mutant constructs, inhibitors or neutralizing antibodies to the enzymes, all of which are then tested in various in vivo model systems that manifest the angiogenic or invasive phenotype. This approach allows for the identification of specific proteolytic enzymes that are functionally involved in distinct physiological and pathological processes. Two enzyme families presently under study include the matrix metallo proteases (e.g. MMP-9, MMP-1) and the serine proteases, (e.g. uPA and plasmin). New cell surface substrates for these proteases have recently been identified and their cleavage has been shown to activate important signaling cascades.

We also have ongoing studies in the lab examining the role of inflammatory neutrophils that influx into developing primary tumors. The influx of these “first responder” cells appears to be driven by the inappropriate overexpression by tumor cells of specific cytokines that attract various types of inflammatory cells. We are presently focusing on neutrophil influx since these cells deliver to the developing tumor tissue a distinct proteolytic enzyme (TIMP-free MMP-9) that is a potent angiogenic agent. The biochemical mechanisms behind the angiogenesis-inducing capabilities of this enzyme are currently being investigated.