Abstract 2444: Non-invasive FMT quantification of folate receptor expression in mouse tumor xenografts with a new near-infrared fluorescent folate agent

Folate receptors (FR) are overexpressed on the surface of many different human tumor cells, including ovarian, breast, cervical, renal, colorectal and nasopharyngeal cancer cells, with little expression in normal tissues. As such, folic acid has been successfully exploited as a cancer specific targeting moiety for the efficient delivery of chemotherapeutic agents, drug carriers, photosensitizers and diagnostic reporters. Critical to the success of such agents is the determination of the level of FR expression for a given tumor, since weak FR-expressing cancers will not respond well to folate-targeted therapies. There is therefore a need for specific and quantitative imaging agents and methods for the determination of FR expression in vivo. Optical imaging in the near-infrared (NIR) range allows efficient penetration of photons through living tissue and minimizes interference from tissue autofluorescence. Combined with quantitative fluorescence molecular tomography (FMT), NIR fluorescent agents have emerged as invaluable tools for quantitative, deep tissue imaging across a range of important areas of disease research including oncology. We have developed a new, near-infrared fluorescent folate targeted agent, VM3244, and used it to noninvasively quantify FR expression in vivo by FMT of mouse tumor xenografts. Two cell types, KB and HeLa with different degrees of FR expression were employed in the present study. In vitro, FR expression levels of each cell type were visualized with anti-FR antibody by microscopy, showing high FR expression in KB cells and lower expression in HeLa cells. The binding of VM3244 to FR was quantified by flow cytometry, confirming high and low FR expression in KB and HeLa cells, with good agreement between VM3244 and the antibody. Specificity of the agent was demonstrated by blocking of the signal by addition of an excess of unlabeled folic acid. In vivo quantification was performed by injection of 2 nmol of VM3244 into nude mice mice bearing KB or HeLa tumor xenografts and imaging by FMT at 4 to 24 h post injection. KB tumors showed a high level of FR expression with approximately 200 pmol (10% injected dose) quantified in the tumors, with little fluorescence in other tissues except the kidneys at 4 h post injection biodistribution study. Consistent with the in vitro profile, HeLa tumors had less tumor fluorescence, with about 75 pmol (3.8% injected dose) quantified in the tumors. Both tumor cell lines showed significant (∼80%) knockdown of signal by co-injection of the mice with unlabeled folic acid and ex vivo colocalization with FR antibody on tumor slices, confirming the in vivo specificity of the agent. Thus, we have demonstrated the ability of VM3244 to quantify FR expression both in vitro and in vivo and, combined with FMT, to noninvasively distinguish between high and lower FR expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2444. doi:1538-7445.AM2012-2444