Mechanisms of Mycobacterium bovis, strain bacillus Calmette-Guerin, fibronectin attachment protein binding and internalization by bladder tumor cells

Jennifer M Sowa, Purdue University

Abstract

Instillation of Mycobacterium bovis strain bacillus Calmette-Guerin (BCG) is the most efficacious and widely used treatment for superficial bladder cancer. However, BCG treatments can induce toxicity in patients, leading to reduced anti-cancer effects and expensive patient care. In addition, long-term follow-up studies show a high rate of recurrence, approximately 70%. Previous studies from our laboratory have identified a conserved component of BCG, which mediates the binding of BCG to the bladder tumor cells, called Fibronectin Attachment Protein (FAP). In vitro studies identified a unique binding site necessary for FAP attachment to fibronectin and that FAP is sufficient to induce anti-tumor activity in the bladder. These data suggest that FAP is a potential alternative to BCG and can be used to target bladder tumor cells for therapy. The goal of this project is to develop effective therapeutic approaches for the treatment of bladder cancer using FAP as a targeting agent. We hypothesized that FAP would function as an ideal targeting ligand for bladder tumor cells based on its ability to selectively associate with fibronectin that is uniquely exposed in bladder cancer. This was tested using immuno-fluorescent microscopy and flow cytometry to detect FAP binding and internalization in bladder tumor cell lines and in vivo tumors. We also evaluated the endocytic trafficking and end-point localization of FAP. Furthermore, we determined the ability of FAP-modified liposomes to bind to tumor cells and deliver therapeutic agents for the treatment of bladder cancer. The data showed that FAP is co-localized with FN in vitro in the human T24 bladder tumor cell line and the mouse 3T3 embryonic fibroblastic cell line. Co-localization studies with rhodamine-dextran and early endosomes suggest that FAP is internalized, but to a small extent, possibly by clathrin-mediated endocytosis. Studies using flow cytometry to determine the internalized fraction of FAP also confirm that FAP is internalized at a slow rate. Studies using FAP-conjugated liposomes showed variable results, with FAP enhancing liposomal delivery in approximately 50% of experiments.

Degree

M.S.

Advisors

Ratliff, Purdue University.

Subject Area

Cellular biology|Veterinary services|Oncology

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