Advantages of bioluminescence imaging to follow siRNA or chemotherapeutic treatments in osteosarcoma preclinical models

Abstract

Osteosarcoma is the most common malignant primary bone tumor for which pertinent preclinical models are still needed to develop new therapeutic strategies. As osteosarcoma growth is strongly supported by bone resorption, previous studies have inhibited the cytokine receptor activator of nuclear factor-kB ligand using antibodies or recombinant proteins. However, its expression has not yet been inhibited using genetic approaches using small interfering RNA. To optimize the delivery of small interfering RNA to its cellular target and demonstrate their efficiency in vivo, two new osteosarcoma models expressing the firefly luciferase enzyme were developed. These luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice and were detectable by in vivo bioluminescence imaging. In comparison with measurement of tumor volume, bioluminescence analysis enabled earlier tumor detection and revealed extensive cell death in response to ifosfamide treatment. Finally, by targeting the luciferase expression into osteosarcoma, we established a protocol for in vivo administration of small interfering RNA combined with cationic liposome.

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

“…27 Using these plasmids, lentiviral particles were produced to stably transduce HEK 293 cells. We selected two modified HEK 293 cell lines that express similar levels of the recombinant LucF transcript associated with either WT or Mut exon 23 (data not shown).…”

“…For LacZ-and Mut-shRNAs synthesis, sequences of corresponding siRNAs were used to design 2 oligonucleotides (Eurogentec) with an overlapping sequence as described by Chang et al Oligonucleotide duplexes (sequences in Figure 1a, Sigma, Saint-Quentin Fallavier, France) corresponding to 54 base pairs of the WT or the Mut Brtl Col1a1 exon 23 sequence were cloned at the MluI site in the 3' untranslated region of the firefly luciferase gene (LucF) in the pLNT-LucF plasmid. 27 Using the ViraPower Lentiviral Expression System (Invitrogen, Saint Aubin, France), the resulting plasmids were used for lentiviral particles production. To measure lentiviral titration and transduction efficiency, EGFP encoding particles were coproduced utilizing pFG12.…”

“…Transduction efficiency was assessed by EGFP expression analysis by flow cytometry (FC500, Beckman Coulter, Villepinte, France) and luciferase activity measurement, as previously described. 27 siRNA transient transfection …”

Orthotopic murine model of a primary malignant bone tumor in the spine: functional, bioluminescence, and histological correlations

“…6 Although aggressive surgical treatment of osteosarcoma is associated with improved survival, 13,20 there remains a need for further development of preclinical models to develop new therapeutic approaches. 16 We report the development of an orthotopic nude mouse model in which the human osteosarcoma cell line, Krib-1, was implanted directly into the VB. We demonstrate that the implanted mice subsequently experience a progressive neurological decline in a reproducible and predictable time frame correlating with increasing severity of spinal canal compression by pathological evaluation.…”

siRNA transfection by dendritic core–shell nanocarriers

“…[42,44] Preclinical experiments have already demonstrated that synthetic siRNAs are potent inhibitors of a variety of diseases in vivo, [43,45] including hypercholesterolaemia, [46][47] liver cirrhosis, [48][49][50] hepatitis B virus (HBV), [51][52][53][54] human papillomavirus, [53,55] ovarian cancer, [56][57] and bone cancer. [58][59][60] Since the therapeutic benefit of RNAi inevitably relies on the delivery of synthetic siRNA into cells, and its systemic delivery to target cells is complex, the development of efficient gene delivery systems represents a challenging hurdle in the field of gene therapy. [1,[61][62] …”