Engineered human mesenchymal stem cells for neuroblastoma therapeutics

Abstract

Drug-resistant neuroblastoma remains a major challenge in paediatric oncology and novel and less toxic therapeutic approaches are urgently needed to improve survival and reduce the side effects of traditional therapeutic interventions. Mesenchymal stem cells (MSCs) are an attractive candidate for cell and gene therapy since they are recruited by and able to infiltrate tumours. This feature has been exploited by creating genetically modified MSCs that are able to combat cancer by delivering therapeutic molecules. Whether neuroblastomas attract systemically delivered MSCs is still controversial. We investigated whether MSCs engineered to express tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) could: i) cause death of classic and primary neuroblastoma cell lines in vitro; ii) migrate to tumour sites in vivo; and iii) reduce neuroblastoma growth in xenotransplantation experiments. We observed that classic and primary neuroblastoma cell lines expressing death receptors could be killed by TRAIL-loaded MSCs in vitro. When injected in the peritoneum of neuroblastoma-bearing mice, TRAIL-MSCs migrated to tumour sites, but were unable to change the course of cancer development. These results indicated that MSCs have the potential to be used to deliver drugs in neuroblastoma patients, but more effective biopharmaceuticals should be used instead of TRAIL.

Figure 1.

Classic and patient-derived neuroblastoma cell lines exhibit variable expression of TRAIL death receptors. Western blot analysis revealing the expression of DR4 and DR5 in a panel of classic (A) and patient-derived (B) neuroblastoma cell lines in the presence or absence of BTZ. α-tubulin was used as a loading control. Quantification of DR4 and DR5 expression relative to control (α-tubulin) is displayed at the bottom of each set of western blots. TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; BTZ, Bortezomib; CTR, control.

Figure 2.

Combination of TRAIL-MSCs with BTZ causes synergistic cell death of classic and patient-derived neuroblastoma cell lines. (A) MSCs infected with a TRAIL lentivirus, or parental cells, were subjected to flow cytometric analysis with a TRAIL antibody. Cell death assays; (B) classic or (C) patient-derived neuroblastoma cell lines were cultured in the presence or absence of recombinant TRAIL (r-TRAIL), BTZ, mesenchymal stem cells engineered to express secreted TRAIL (TRAIL-MSCs) or their combinations. Error bars indicate the standard errors of the means of three (Kelly and SKNAS) or two (primary cell lines) experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. MSCs, mesenchymal stem cells; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; BTZ, Bortezomib.

Figure 3.

TRAIL-MSCs home into neuroblastoma tumours growing in vivo. TRAIL-MSCs were stained with the vital dye DiR and injected intraperitoneally into mice with tumours growing in the right flank. IVIS Lumina Imaging System (Caliper Life Sciences) confirmed that human TRAIL- MSCs infiltrated subcutaneous neuroblastoma tumour masses. The colour scale indicates the intensity of DiR signal, with the yellow colour indicating the strongest intensity. MSCs, mesenchymal stem cells; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; BTZ, Bortezomib.

Figure 4.

Bortezomib, but not TRAIL-MSCs, treatment slow down the growth of neuroblastoma xenografts. (A) Tumour growth curves. SKNAS cells were injected in the right flanks of mice and treatments were started after tumours reached ~50 mm³. The results are expressed as the tumour volume means ± SD (n=10, *P=0.05). (B) Survival curves. Log-rank test indicates that there is no significant effect on mice survival after TRAIL-MSCs or bortezomib treatments (P=0.051). MSCs, mesenchymal stem cells; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; BTZ, Bortezomib.