Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer

Abstract

Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo.

Keywords: Cancer therapy; Gene therapy; Hyperthermia; Magnetic core–shell nanoparticles; Stem cell therapy.

FIGURE 1

Mild Magnetic Hyperthermia-Activated Stem Cell-Based Gene Therapy. A) MCNPs composed of a ZnFe₂O₄ magnetic nanoparticle (MNP) core and a mesoporous silica (mSi) shell (i) are functionalized with polyethyleneimine (PEI) to allow for complexing with a heat-inducible therapeutic plasmid (iii). The MCNPs enhance delivery of the heat-inducible plasmid into the adipose-derived mesenchymal stem cells (AD-MSCs) via magnetically-facilitated uptake (iv–v). These engineered ADMSCs can then be injected in vivo (vi), where they innately home to the tumors/metastases. Finally, mild magnetic hyperthermia, via exposure of the MCNPs to an alternating magnetic field (AMF), can be used to specifically activate the heat-inducible secretion of therapeutic TRAIL from the AD-MSCs (vii). B) The heat-inducible plasmid is composed of a HSP70B’ promoter and a secreted form of TRAIL (sTRAIL) that is fused to an EGFP reporter.

FIGURE 2

Characterization of the MCNPs. A) HR-TEM image of the MCNPs. B) Higher magnification HR-TEM image of the MCNPs shows that the pores are about 3 nm in size. C) Size was determined using TEM and dynamic light scattering (DLS). Moreover, the Zeta potential was confirmed. The values in the chart are given as mean ± standard deviation. D) The MCNPs (25 µg/mL) can be heated to temperatures as high as 47°C after exposure to an alternating magnetic field (5 kA/m, 225 kHz) for one hour.

FIGURE 3

Characterization of the Heat-Inducible Plasmid. A) Schematic depicting the sTRAIL-EGFP plasmid (i), which expresses a sTRAIL-EGFP fusion that is constitutively activate, and the HSP-sTRAIL plasmid (ii), which expresses the same sTRAIL-EGFP fusion under the control of a heat-inducible HSP70B’ promoter. B) RT-PCR demonstrating the successful synthesis of the sTRAIL-EGFP plasmid, which was transfected into A2780 ovarian cancer cells. C) Proof-of-concept demonstrating that the HSP-sTRAIL plasmid can be specifically activated by heat (1 hour at 41°C in a water bath) as seen via fluorescence imaging due to fusion of TRAIL with EGFP. Scale bar = 50 µm. D) Confirmation of heat-specific TRAIL activation was obtained using qPCR (*p < 0.05) and was normalized to transfected cells that were incubated at 37°C. GAPDH was used as the housekeeping gene.

FIGURE 4

Proliferation of AD-MSCs Engineered with MCNP-PEI/Plasmid Complexes. A) The proliferation of unengineered (control) and engineered (MCNP-PEI/plasmid) AD-MSCs was evaluated using Ki-67 (red). The nuclei were stained with Hoechst (blue). Scale bar = 50 µm. B) Approximately 20% of the AD-MSCS expressed Ki-67 and there was no statistically significant difference between the two groups (p > 0.05).

FIGURE 5

Differentiation and Migration of AD-MSCs Engineered with MCNP-PEI/plasmid Complexes. A) To evaluate osteogenic differentiation, engineered or unengineered AD-MSCs were differentiated for three weeks. Osteogenesis was then quantified via Alizarin Red staining. B) Quantification of staining suggested that there was no statistically significant difference between the two groups (p > 0.05). C) qPCR of key osteogenic genes demonstrated that all four genes were highly expressed over non-differentiated control and that no significant difference was found between the engineered and unengineered AD-MSCs (p > 0.05). GAPDH was used as the housekeeping gene. D) Timeline of the studies used to evaluate the tumor homing ability of the engineered and unengineered AD-MSCs. E) Luciferase was used to identify the A2780 cells. Luminescence imaging shows the establishment of disseminated A2780 tumors. The luminescence intensity goes from blue to red, wherein blue is the weakest and red is the strongest. F) One week after the injection of AD-MSCs, tumors were collected. Fluorescence imaging shows the DiD-labeled engineered and unengineered AD-MSCs. The fluorescence intensity goes from dark red to yellow, wherein dark red is the weakest and yellow is the strongest. G) Luminescence imaging of the conglomerated tumors demonstrates that the AD-MSCs are able to colocalize with the tumors.

FIGURE 6

Engineered AD-MSCs Can Effectively Induce Apoptosis When Exposed to Heat. A) Timeline of the in vitro study. B) Mild magnetic hyperthermia with an average temperature of 41.5°C was maintained for one hour by periodically exposing the engineered AD-MSCs to an AMF (5 minutes on, 5 minutes off). C) Mild magnetic hyperthermia alone did not significantly affect AD-MSC viability. Moreover, the process of engineering the AD-MSCs with MCNP-PEI/plasmid complexes did not significantly affect cell viability. D) To test therapeutic efficacy, A2780 ovarian cancer cells were treated with conditioned media from the engineered AD-MSCs that were exposed to mild magnetic hyperthermia. A2780 cells showed a remarkable decrease in cell viability when compared to those treated with conditioned media from engineered AD-MSCs that had not been exposed to mild magnetic. E) To confirm the mechanism of action, qPCR for caspases, which are downstream of TRAIL, was performed. F) To evaluate in vivo efficacy, we injected half a million AD-MSCS engineered with MCNP-PEI/plasmid complex, wherein the plasmid was sTRAIL-EGFP. Unengineered AD-MSCs and a single dose of recombinant TRAIL (5 mg/kg) were injected as controls. Tumor volume was followed over two weeks, we found that the size of the tumors decreased significantly (max value at day 0 was 6 × 10⁴ whereas the max value on day 14 was 8 × 10³) when treated with the engineered AD-MSCs. G) Quantification of luminescence intensity shows that the engineered AD-MSCs are significantly better than treatment with a single dose of recombinant TRAIL.