Distribution of the Formulated Poly IC in the Cells

In order to verify selective entrance of the complexes into the target U87MGwtEGFR cells and its release from endosomes fluorescent labeling of poly IC was performed (Methods). U87MGwtEGFR and U87MG cells were transfected with the labeled poly IC/PEI-PEG-EGF complex either in presence or absence of PEI-Mel (Figure 2). Figure 2A showsefficient transfection of the target U87MGwtEGFR cells and virtually no signal in U87MG cells. To examine the intracellular distribution, U87MGwtEGFR cells incubated with fluorescently labeled poly IC/PEI-PEG25-EGF or poly IC/PEIPEG25- EGFþPEI-Mel complexes, were washed, and viewed live to rule out fixation artifacts, using confocal microscopy (Figure 2B). After 4 h of incubation, poly IC/PEI-PEG25-EGF complexes appeared in a punctate intracellular pattern, suggesting entrapment within vesicles (Figure 2B). In contrast, poly IC/PEI-PEG25-EGFþPEI-Mel complexes showed fluorescence dispersed throughout the cytoplasm (Figure 2B). The cytoplasmic fluorescence of the Melittin-containing complexes suggests that Melittin indeed facilitated release of the complex into the cytoplasm, by lysis of intracellular vesicles.

Involvement of PKR in Cell Death

The fast killing effect of the complexes was significantly inhibited by 2-aminopurine (2-AP), a potent inhibitor of PKR [15], suggesting that this effect is largely mediated by PKR (Figure 3A). At later time points, 48 and 72 h after transfection, the protective effect of 2-AP was significantly less pronounced, suggesting that additional cell killing mechanisms were activated.

In Vitro Bystander Effect

We next investigated whether the poly IC-transfected cells exerted a bystander effect on untransfected cells (Methods). Growth medium from poly IC-transfected U87MGwtEGFR cells inhibited the growth of U87MG and U87MGDEGFR cells (Figure 3B), which are themselves insensitive to the (poly IC)PEI-PEG-EGFþPEI-Mel complex (Figure 1A). The bystander effect was less pronounced when U87MGwtEGFR cells were transfected with targeted poly IC at higher concentrations (Figure S2), probably because the transfected cells died before they could exert the full effect. The bystander effect was further confirmed by co-culture of U87MGwtEGFR and U87MGDEGFR cells in the same wells (Figure 3C). A large fraction of the U87MGDEGFR cells were killed, although they are themselves almost insensitive to the poly IC complex.

Double-stranded RNA is known to induce the expression of interferons and other anti-proliferative cytokines. To investigate whether the bystander effect was caused by interferon, we incubated medium from transfected U87MGwtEGFR cells with neutralizing IFNa antibody. This pre-incubation reduced the bystander effect on all cell lines by 20%–30% (Figure 3B), indicating that IFN-a was indeed responsible for part of the bystander effect. The partial reduction in the bystander effect suggests that additional cytokines, other than IFN-a, were also expressed. Using ELISA (IBL, Hamburg, Germany), we confirmed the presence of IFNa in growth medium from poly IC-transfected cells. Up to 4 pg/ml IFNa were generated in the medium of 500,000 U87MGwtEGFR cells transfected with 2.5 lg/ml of poly IC, but not in the medium of U87MG or U87MGDEGFR cells treated with the same complex. No IFN-a was detected in medium from cells transfected with poly IC at higher concentrations, suggesting that these cells were killed before they could secrete the cytokine. IFN-a was also generated in vivo (600 pg/g of tumor tissue), specifically in U87MGwtEGFR tumor xenografts (Figure 4). Using a Human Cytokine Microarray (RayBiotech, Norcross, Georgia, United States), we sought to identify additional cytokines in the medium of poly ICtransfected cells. Both GROa [16] and IP-10 [17] were detected. The production of IP-10 and Gro-alpha at high concentrations in vitro and selectively in tumors in vivo, was confirmed by cytokine specific ELISAs (Figure 4). These cytokines are chemokines responsible for the recruitment of T-cells to area of expression [16,17].