Abstract 699: Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer
Authors: Brennen W. Nathaniel , Levy Oren , Ranganath Sudhir , Schweizer Michael , Rosen Marc , Billet Sandrine , Bhowmick Neil , Denmeade Samuel , Karp Jeffrey , Isaacs John 
Published: May 2015

Abstract
Mesenchymal stem cells (MSC) traffic to sites of inflammation, such as those commonly found at sites of prostate and other cancers. Using multi-parameter flow cytometry, our group has shown that MSCs represent 0.01-1.1% of the total cells present at sites of primary prostate cancer (PCa). Allogeneic MSCs have been safely administered to &gt;1000 patients in over 350 clinical trials worldwide for a variety of diseases. Coupled with their ability to evade the immune system, this suggests that allogeneic MSCs can be used as cell-based delivery vectors for anti-cancer agents.
Towards this goal, a method has been developed to load MSCs with PLGA microparticles (MP) encapsulating PSA-activated proaerolysin. Proaerolysin is a highly potent (low pM) bacterial pore-forming toxin that selectively kills PSA-expressing cells in a proliferation-independent manner, which is critical due to the low proliferative index of PCa. Proaerolysin has been modified using site-directed mutagenesis to replace the wildtype activation domain with a PSA cleavage sequence. Though prodrug delivery will be enriched at cancer sites using MSC ‘Trojan horses’, entrapment in non-malignant tissue, such as the lung, following systemic administration is expected and must be addressed; here, using a prodrug strategy. Importantly, enzymatically active PSA is only present in the prostate and at sites of PCa, including metastases. Circulating PSA is inactive due to covalent binding to serum protease inhibitors. Therefore, toxicity to non-target tissues will be minimized through both selective delivery and prodrug activation.
The prodrug is released from the MPs in a controlled manner over at least a 1 week period in vitro. Hemolysis assays in the presence of MP-conditioned supernatant demonstrated that MP fabrication does not neutralize drug toxicity. Furthermore, incubation of LNCaP (PSA+) and PC3 (PSA-) cells with MP-conditioned supernatant demonstrates selective toxicity to PSA-expressing cells at low nM concentrations. MP internalization by MSCs has been confirmed using both flow cytometry and confocal microscopy. Chitosan-modification of the MPs permitted increased loading of MSCs (100 ug/mL of MPs). For long-term development, preclinical studies such as these have raised the question of tumor homing efficiency in humans. Therefore, an ongoing FDA-approved first-in-man pre-prostatectomy clinical trial to quantify the number of systemically-delivered allogeneic MSCs that traffic to sites of primary PCa has been initiated. BEAMing (digital PCR) technology will be used to accurately quantify donor MSCs based on differential SNP profiles (detection threshold: ≥0.01%). Data from this trial will be used to determine the amount of prodrug-loaded MPs necessary to deliver per MSC to achieve a therapeutic effect. This data will subsequently be used in ongoing animal studies to model clinical relevance in efficacy studies prior to further translation.
Citation Format: W. Nathaniel Brennen, Oren Levy, Sudhir Ranganath, Michael Schweizer, Marc Rosen, Sandrine Billet, Neil Bhowmick, Samuel Denmeade, Jeffrey Karp, John Isaacs. Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2014-699