Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth

Abstract

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.

Figure 1

Sensitivity of prostatic cell lines to 5-FU. Prostate cell lines were exposed for 6–7 days to increasing concentrations of 5-FU. Cell viability was quantified by MTS assay. For each prostate cell line, mean values ± SD are expressed as a percentage of cell viability measured in the absence of the drug. 5-FU, 5-fluorouracil.

Figure 2

Cytotoxic effect mediated by CDy-AT-MSCs in the presence of 5-FC on prostatic cell lines. In direct coculture experiments therapeutic cells were seeded in the same wells containing prostate cells in ratios from 1:500 to 1:1. As a negative control for the transgene, not transduced AT-MSCs were also seeded with the prostate cells, in ratios from 1:20 to 1:5. Cell viability was quantified by MTS assay after 6–7 days of coculture. For each prostate cell line, mean values ± SD are expressed as a percentage of the cell viability measured in the absence of transduced or untransduced AT-MSCs. AT-MSCs, adipose tissue–derived mesenchymal stem cells.

Figure 3

Tropism of therapeutic cells to prostate cell lines. Tropism of CDy-AT-MSCs to prostate cancerous and noncancerous cells was tested in vitro. Different numbers of prostate cells were seeded onto 24-well plates [in appropriate growth medium or in RPMI (Roswell Park Memorial Institute) containing 10% fetal calf serum (FCS)]. After 1 day, 8 µm pore-size cell culture inserts were added to the wells and 20,000 CDy-AT-MSCs per insert were seeded. Two days later, cells were processed as described in Materials and Methods. (a-c) Representatives fields of views (FOVs) from different inserts showing migrated CDy-AT-MSCs after staining with DAPI. In detail, fotos represent therapeutic cells migrated to (i) growth medium for EP156-T, (ii) EP156-T cells seeded in the appropriate growth medium, (iii) growth medium for LNCaP-Bicalutamide (LNCaP-Bic), (iv) LNCaP-Bic seeded in the appropriate growth medium, (v) RPMI containing 10% FCS, (vi) PC3, (vii) LNCaP-Bic, (viii) LNCaP, (ix) EP156-T, (x) Du145, (xi) BPH.1, all seeded in RPMI containing 10% FCS. (d) Quantification of the CDy-AT-MSCs migrated per each FOV, normalized to 10⁶ of prostatic cells. 5 FOVs per insert were considered. Columns represent the mean values of CDy-AT-MSCs counted (i.e., migrated to each prostatic cell line) in 3–5 different wells ± SD. AT-MSCs, adipose tissue–derived mesenchymal stem cells.

Figure 4

Local 5-FC conversion in tumors leads to tumor regression in vivo. (a) PC3 tumor cells (4 × 10⁶) alone or mixtures of PC3 cells with AT-MSCs or with various ratio of therapeutic CDy-AT-MSCs were injected s.c. into flank of each nude mouse (n = 4 in each group). All cells were mixed with the same volume of Matrigel before inoculation. All animals were treated with daily dose of 500 mg/kg of 5-FC intraperitoneally (i.p.) starting on second day after tumor appearance in control group and up to the 27th day. Animals were inspected for tumors every 2 days. At day 40 the experiments were ended. (b) PC3 cells (3 × 10⁶) or mixtures of 3 × 10⁶ of PC3 tumor cells either with 40% of AT-MSCs or with 50% and 10% of therapeutic cells were injected into flank of each nude mouse (n = 4 in each group). All animals were treated with daily dose of 500 mg/kg of 5-FC i.p. as indicated by arrows. Animals were inspected by palpation for tumors every 2 days. At day 40 the experiments were ended and animals were inspected for tumors by biopsy. AT-MSCs, adipose tissue–derived mesenchymal stem cells; 5-FU, 5-fluorouracil.

Figure 5

Systemically administered CDy-AT-MSCs exert antitumor effect in the presence of 5-FC (a) Tumors were induced with 3.0 ×10⁶ PC3 cells (without Matrigel) by subcutaneous inoculation into flank of each nude mouse in all four animal groups. Control group (n = 6) received tumor cells without any treatment. Animals in the second control group (n = 3) received equal number of tumor cells and were successively inoculated intravenously (i.v.) with 2.0 × 10⁶ of CDy-AT-MSCs and not treated with 5-FC. The third group of animals (n = 8) received 3.0 ×10⁶ PC3 cells, were inoculated i.v. with a single dose of 2.0 × 10⁶ of CDy-AT-MSCs, and were treated daily with 500 mg/kg of 5-FC intraperitoneally starting on fourth day after therapeutic cells inoculation. On day 24, animals were killed and tumors examined by histopathology. The fourth group of animals (n = 6) received two doses of therapeutic cells, as indicated in the figure by the orange arrows. On day 36 animals were killed. Tumor volumes were measured at the indicated time points and expressed as mean ± SD. *P < 0.05. (b) Hematoxylin-eosin staining of PC3 tumors from control animals (upper panel), from animals treated with single dose of CDy-AT-MSCs (middle panel), and from animals treated with two doses of CDy-AT-MSCs (lower panel). (c) Nude mice were inoculated with 3.0 × 10⁶ PC3 cells mixed with the same volume of Matrigel. Control group animals (n = 6) received tumor cells only. Animals in the second group (n = 9) were inoculated i.v. with one dose of 2.0 × 10⁶ of CDy-AT-MSCs. These two groups of animals were killed on day 14, tumor volume was estimated and histopathologic examinations performed by hematoxylin-eosin staining. Mice of the third group (n = 9) received a second injection of 1 × 10⁶ of CDy-AT-MSCs on day 18. Tumor volumes were measured on day 32 and expressed as mean ± SD. *P < 0.05. AT-MSCs, adipose tissue–derived mesenchymal stem cells; 5-FU, 5-fluorouracil.