uPAR Targeted SPECT imaging probes for detection of breast cancer

Objectives Urokinase plasminogen activator (uPA) and its cell surface receptor (uPAR) are over-expressed in breast cancer as well as in several other human cancers and play an important role in tumor growth, metastasis and cancer prognosis. The goal of this project was to develop ^99mTc labeled uPAR-targeted radiotracers as potential tumor-targeted imaging probes for non-invasive in vivo imaging of breast cancer.

Methods Two small peptides, a C-terminal His₆-tagged amino terminal fragment (ATF, ~17kDa), known to be uPAR specific, and an N-terminal NTA-nine-mer peptide (AE105, 1.4 kDa), an uPAR antagonist, were radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺ produced via an IsoLink kit. ^99mTc-ATF was purified by centrifugal ultrafiltration and its ^99mTc labeling was confirmed using a 15% SDS-PAGE gel with exposure to radiographic film overnight at 4°C; ^99mTc-AE105 was isolated by HPLC. Stability of both radiotracers was determined in PBS buffer pH 7.4 and with histidine challenge; in vitro uptake of ^99mTc-ATF in human breast cancer cell lines, MDA-MB-231 that represent a high level of uPAR expression and T47D that has a very low or negative level of uPAR, was also measured.

Results Both radiotracers were stable at PBS buffer pH 7.4 for 24 h and no significant re-oxidation, ligand exchange or decomposition were observed even at a 1000-fold molar excess of histidine for 6 h at room temperature. ^99mTc-ATF activity was detected at the 17 kDa location, which corresponds to the size of the ATF-peptide. Binding of ^99mTc-ATF peptide to the uPAR positive MDA-MB-231 cancer cells was 2.6 fold higher than that binding to uPAR negative T47D cells showing that ^99mTc-ATF retains specificity. In-vitro cell binding of ^99mTc-AE105 needs to be assessed.

Conclusions ^99mTc radiolabeling of small uPAR binding peptides provides a potentially versatile route to the development of tumor-targeting probes.

Research Support This work was supported by the NIH grant 1P50 CA128301