Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into Ewing sarcoma tumors

Abstract

Background: This study evaluated the therapeutic efficacy of interleukin 12 (IL-12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL-12 gene delivery.

Methods: MSCs were isolated from murine bone marrow cells. Cells were phenotyped using flow cytometry. Cultured MSCs differentiated into osteocytes and adipocytes using the appropriate media. Freshly isolated MSCs were transfected with adenoviral vectors containing either the beta-galactosidase (Ad:beta-gal) or the IL-12 (Ad:IL-12) gene. Expression of IL-12 was confirmed using reverse transcription polymerase chain reaction. Mice with TC71 Ewing sarcoma tumors were then treated intravenously with MSCs transfected with Ad:beta-gal or Ad:IL-12. Tumors were measured and analyzed by immunohistochemical analysis for expression of IL-12 protein.

Results: Expression of both p35 and p40 IL-12 subunits was demonstrated in MSCs transfected in vitro with Ad:IL-12. IL-12 expression was seen in tumors from mice treated with MSCs transfected with Ad:IL-12. Tumor growth was also significantly inhibited compared with that in mice treated with MSCs transfected with Ad:beta-gal.

Conclusions: MSCs can be transfected with the IL-12 gene. These transfected cells localize to tumors after intravenous injection and induce local IL-12 protein production and the regression of established tumors.

Figure 1. MSCs were established from BM marrow cells

BM adherent cells were isolated from the hind femurs of nude mice. Cells were cultured in tissue-culture flasks for various time periods as described. On day 35, cells were identified as MSCs by immunophenotyping and by osteogenic and adipogenic differentiation. Magnification = 20x.

Figure 2. Immunophenotypic profile of MSCs derived from adult murine BM

Flow-cytometric histograms show the expression (unshaded) of CD29, CD44, CD49E, and Sca-1. Cells were negative for CD31, CD11b, CD34, CD45, and CD62E. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3. Transfected MSCs migrate into the peripheral TC71 tumor

(A) MSCs were transfected with Ad:GFP for 48 h. (B) Mice with subcutaneous TC71 Ewing’s sarcoma tumors received 2 i.v. injections of Ad:GFP MSCs given 4 days apart. Mice were sacrificed 10 days later, and tumors were excised, frozen, and analyzed by IHC. (C–H) CM-Dil-stained MSCs were injected in the tail veins of nude mice. Organ tissue was examined under fluorescence microscopy 10 days later. CM-Dil-positive cells were detected in lungs, livers, and spleens but not kidney, heart or brain.

Figure 4. Expression of IL-12 after transfection

MSCs were incubated with Ad: β-gal at 100 MOI or various MOIs of Ad:IL-12 for 48 h. Total RNA was extracted from 10⁶ MSCs after transfection. IL-12 expression was determined by RT-PCR.

Figure 5. Effect of Ad:IL-12 MSCs on tumor growth

Mice bearing TC71 tumors were treated i.v. with Ad:IL-12 MSCs, Ad: β-gal MSCs, or untransfected MSCs. (A) Frozen sections of tumors were analyzed by IHC. Expression of IL-12 was only observed in the Ad:IL-12 MSC-treated group. (B) Tumors were measured by calipers and the tumor area calculated as the geometric mean of perpendicular diameters. Day 25: MSC vs. Ad:IL-12 MSC, P = .024; and Ad: β-gal MSC vs. Ad:IL-12 MSC, P = .041. Day 29: MSC vs. Ad:IL-12 MSC, P = .04; and Ad: β-gal MSC vs. Ad:IL-12 MSC, P = .038.