Bystander Effect In Vivo

In the clinical setting, some GBM cells over-express wt EGFR, whereas neighboring cells express lower levels of EGFR or over-express truncated EGFR (DEGFR) [2]. As described above, in culture the EGFR-targeted poly IC-transfected cells induced the killing of neighboring, untransfected cells (Figure 3B and 3C). We therefore examined bystander killing in vivo (Methods). We implanted a mixture of 5000 U87MGwtEGFR and 5000 U87MGDEGFR cells in mice brains (Figure 5C). The mixed tumors that developed 10 d later were treated with the targeted poly IC at various doses (Figure 5C). Control animals survived for no more than 34 d, while mice that received the lowest dose of poly IC complex (0.2 lg/h) survived more than 2 times longer. Animals that received higher doses of formulated poly IC (0.4 lg/h and 0.8 lg/h) are alive and well on the day of submission of this paper (day 297þ). This very encouraging result is probably due to the strong bystander effect induced by the slow delivery of the formulated poly IC. Elimination of the mixed tumor was not caused by immune reaction since we did not detect increased infiltration of immune cells into the poly IC treated tumors (Figure 6A).

Formulated Poly IC Induces Apoptosis in U87MGwtEGFR Cells In Vivo

We next examined the mode of U87MGwtEGFR cell death induced by the targeted poly IC in vivo. U87MGwtEGFR cells were injected into mice and the established tumors were treated with formulated poly IC. EGFR expression was monitored with FITC-conjugated EGFR antibody (green fluorescence). Apoptosis was monitored with the TMR Red Cell Death Kit (Roche). Apoptosis occurred exclusively in cells over-expressing the EGFR (Figure 6B). No significant red signal was observed in the surrounding tissue, demonstrating the high selectivity and low toxicity of the treatment regimen.

Effect of Formulated Poly IC on 15-d-Old Intracranial Xenografts

Although 10-d tumors used in the survival experiments are clinically relevant to newly diagnosed GBM in humans (R. Catane and R. Pfeffer, Division of Neuro-oncology, Tel Hashomer/Sheba Hospital, personal communication), we examined whether formulated poly IC is effective against much larger tumors (Figure 5A). For this purpose we synthesized Mel-PEI25-PEG-EGF (MPPE, Methods). Although not observed, there is a possibility of PEI-Mel dissociation from (poly IC)PEI-PEG-EGFþPEI-Mel complexes. This may result in decreased release of poly IC to cytoplasm. When both Melittin and EGF are covalently bound to the same PEI molecule, the vector should be more efficient and even less toxic, because Melittin is now covalently linked to the complex, and therefore there is no possibility of PEI-Mel leakage. It was also observed that PEI25 conjugates are significantly smaller in size than PEI2 or PEI2þPEI25 conjugates (M.O., unpublished data), leading to higher in vivo transfection efficiency.

In order to obtain larger tumors, the intracranially implanted 10,000 U87MGwtEGFR cells were left to grow for 15 d. The resulted tumors were 13–15 times bigger than the 10-d-old tumors (Figures 5A and 7A). (poly IC)Mel-PEI-PEGEGF and (poly IC)MPPE complexes were delivered directly into the tumors at a constant rate for 3, 4, and 5 d, using Alzet osmotic micropumps. Animals that were treated with MPPE alone (no poly IC), survived for no longer than 33 d, as did untreated animals (Figure 7A). In contrast, the (poly IC)MPPE-treated animals in all groups are still alive on the day of submission of this manuscript (day 244þ), and show no symptoms of GBM.