The many spaces of uPAR: delivery of theranostic agents and nanobins to multiple tumor compartments through a single target

Abstract

The urokinase plasminogen activator (uPA) system is a proteolytic system comprised of uPA, a cell surface receptor for uPA (uPAR), and an inhibitor of uPA (PAI-1) and is implicated in many aspects of tumor growth and metastasis. The uPA system has been identified in nearly all solid tumors examined to date as well as several hematological malignancies. In adults, transient expression of the uPA system is observed during wound healing and inflammatory processes while only limited expression is identified in healthy, quiescent tissue. Members of the uPA system are expressed not only on cancer cells but also on tumor-associated stromal cells. These factors make the uPA system an ideal therapeutic target for cancer therapies. To date most therapeutics targeted at the uPA system have been inhibitors of either the uPA-uPAR interaction or uPA proteolysis but have not shown robust anti-tumor activity. There is now mounting evidence that uPAR participates in a complex signaling network central to its role in cancer progression, which provides a basis for the hypothesis that uPAR may be a marker for cancer stem cells. Several new uPAR-directed therapies have recently been developed based on this new information. A monoclonal antibody has been developed that disrupts the interactions of uPAR with signaling partners and is poised to enter the clinic. In addition, nanoscale drug delivery vehicles targeted to the uPA system using monoclonal antibodies, without disrupting the normal functioning of the system, are also in development. This review will highlight some of these new discoveries and the new uPA system-based therapeutic approaches that have arisen from them.

Keywords: nanobins; theranostics; urokinase plasminogen activator.

Figure 1

Targeting uPAR for the treatment of MBC. Proteolysis-targeted approaches, shown in red, have not led to robust antitumor effects in pre-clinical studies. We have focused our therapeutic strategies on targeted delivery of nanoparticles called nanobins using a monoclonal antibody that targets uPA (ATN-291) and induces rapid internalization of the uPA-uPAR complex.

Figure 2

Co-localization of uPAR with previously described cancer stem cell markers CD133, CD34 and CD44. A multiplex IHC assay was developed for uPAR, CD133, CD34, and CD44. uPAR immunostaining of Stage IV NSCLC sections was performed using the ATN-658 antibody from Attenuon using DAB as a chromogen, which produces brown staining. CD133 staining was first optimized using two antibodies. The rabbit polyclonal antibody from AbCam demonstrated staining in formalin-fixed, paraffin-embedded tissue. Vulcan Red was used as a chromogen, and produced red staining in antigenic cells. Staining appeared nuclear and cytoplasmic, unlike the expected membrane localization. In order to produce specific staining of endothelia and rare cells, the antibody had to be used a titer that was high enough to produce week to moderate non-specific nuclear staining, especially around the edge of tissue. CD34 staining was performed using antibody clone QBEnd/10, and was localized using BCIP/NBT, a purple chromogen. Staining was membranous in nature and was localized to endothelia and, in a few specimens, cancer cells. CD44 staining was performed using antibody clone G44-26, and was localized using Vector SG, a gray chromogen. Staining was membranous and was localized to inflammatory cells and cancer cells. Red-Green-Blue color images were collected and spectrally unmixed to generate colored images for easier quantitation as described . In the spectrally unmixed images, uPAR appears blue; CD133: green; CD34: red and CD44: yellow. Panels 1, 2, and 3 are three separate Stage IV NSCLC cases that demonstrate the co-localization of uPAR with a small percentage of CD34+ and CD44+ tumor cells.

Figure 3

Disease free survival of 44 curatively resected patients according to uPAR detection on disseminated tumor cells in bone marrow taken intraopertaively. Upper curve, patients without uPAR evidence on tumor cells. MRT 60.88 ± 0.96 months, for 24 cases and 2 events. Lower curve, patients with uPAR expression on tumor cells. MRT 28.06 ± 2.44 months, for 20 cases and 14 events. Used by permission Nature Publishing Group, license number 2940350215579.

Figure 4

Internalization of ATN-291-NB(Calcein) by cells. Uptake experiments of ATN-291-NB(Calcein) in uPA positive PC3 prostate cancer cells and as determined by flow cytometry. Red dots represent untreated cells, while blue dots represent NB(Calcein), or ATN-291-NB(Calcein). The fluorescence intensity of the calcein fluorescence is plotted on the x-axis and the fluorescence intensity of the Alexa₆₄₇ is plotted on the y-axis. With increasing uptake the distribution of the blue dots will shift right on the x-axis and up the y-axis. MDA-MB-435 uPA-negative breast cancer cells did not show uptake (data not shown).