EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice

Abstract

Background: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells.

Methods and findings: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors.

Conclusion: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

Figure 1. (poly IC)PEI-PEG-EGF(+PEI-Mel) Complexes Selectively Kill U87MGwtEGFR Cells

(A) Cells were seeded in duplicate onto a 96-well plate at a density of 5,000 cells in 0.2 ml of medium per well and grown overnight. Cells were then transfected as described [6,9] with poly IC at the indicated concentrations using either PEI-PEG-EGF or PEI-PEG-EGF+PEI-Mel (w/w ratio PEI-PEG-EGF:PEI-Mel = 1:10) complexes. Viability was measured by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions, at 1 h after transfection. (B and C) Fast induction of apoptosis by (poly IC)PEI-PEG-EGF+PEI-Mel complexes. Apoptotic death was detected 1 h after transfection by Annexin (B) and TUNEL (C) assays as described in Methods.

Figure 2. Distribution of the Complexes

Cells were seeded into 6-cm plates at a density of 300,000 cells in 2 ml of medium per plate and grown overnight. Cells were then transfected with the fluorescently labeled poly IC (5 μg/ml) using the indicated conjugates. After 4 h, cells were analyzed by fluorescent microscopy (Methods) for selectivity of the transfection (A) and intracellular distribution of the complex in the U87MGwtEGFR cells (B).

Figure 3. Cell Killing Mechanisms

(A) Protection of the cells by 2-AP. Cells were grown as in Figure 1A. Cells were then transfected with poly IC at the indicated concentrations using PEI-PEG-EGF+PEI-Mel complexes. Where indicated, 2-AP (5 mM) was added 18 h before transfection and the medium was replaced every 24 h with medium containing fresh 2-AP. Viability was measured by the Methylene Blue assay [8]. (B) In vitro bystander effect. U87MGwtEGFR cells were grown and transfected as described in Methods. U87MG and U87MGΔEGFR “indicator” cells were grown in duplicates in 96-well plates. Medium of the “indicator cells” was then partially replaced by the medium collected from the transfected (+poly IC) or untransfected (-poly IC) U87MGwtEGFR cells (Methods). Where indicated the medium was preincubated with neutralizing polyclonal anti IFNα antibody. In NT samples, medium was not replaced. (C) A total of 4,000 U87MGwtEGFR and U87MGΔEGFR cells were seeded in duplicate onto a 96-well plate at the indicated ratios and grown overnight. Cells were then transfected with poly IC at the indicated concentrations using PEI-PEG-EGF+PEI-Mel conjugates. Cell survival was measured by the Methylene Blue assay 96 h after transfection.

Figure 4. Targeted Poly IC Induces Expression of Cytokines

For this assay, 10,000 U87MGwtEGFR cells were implanted into the brain of nude mice. After 15 d, poly IC was injected with Alzet micropumps for 24 h at 0.8 μg/hr. After 48 h, animals were sacrificed and the brains were extracted. Tumors (average weight 17.6 mg) and surrounding brain tissue (average weight 253 mg) were resuspended in Tris-HCl buffer (100 mM Tris, [pH 8.1] with 1% Triton X-100) at 1:10 w/v. Samples were homogenized on ice by sonication, triturated through 19 gauge needles and spun at 20,000 × g. Protease inhibitors were added to supernatant, which was then subjected to cytokine-specific ELISA.

Figure 5. Targeted Poly IC Eliminates Intracranial GBM Models

(A and B) (poly IC)PEI-PEG-EGF+PEI-Mel complexes eliminate U87MGwtEGFR xenografts. Intracranial U87MGwtEGFR tumors were established and treated with formulated poly IC as described in Methods. (A) Sizes of the tumors before and after the treatment. (B) Survival of the animals (Methods). (C) In vivo bystander effect. A mixture of 5,000 U87MGwtEGFR and 5,000 U87MGΔEGFR cells was implanted into the brain of nude mice as described in Methods. The mixed tumors were treated with formulated poly IC (poly IC-/conjugate) at the indicated doses, and control animals received equivalent doses of PEI-PEG-EGF+PEI-Mel complexes without poly IC (Conjugate only). Survival of the animals was analyzed as above. Mice receiving 0.2μg/h of poly IC die up to day 75, whereas those receiving 0.4 μg poly IC/h and 0.8 μg poly IC/h are still alive.

Figure 6. In Vivo Selectivity of the Approach

(A) Pathological analysis of the brains. Intracranial tumors were established and treated as described in Methods. After 24 h, animals were sacrificed and ultrathin slices of the brains were prepared. Slices were stained with H & E and analyzed by light microscopy (20× magnification) for development of pathological signs and immune cell infiltration (yellow arrow). Bottom panel shows the example of slices from the brains treated with (poly IC)PEI-PEG-EGF+PEI-Mel in the experiment described in Figure 5A (7 d post-treatment). (B) Poly IC induces apoptosis in intracranial xenografts. Intracranial tumors were established and treated as described in Methods. Apoptotic death was detected using Cell Death Detection kit-TMR Red (Methods). White dashed lines represent borders of the tumors.

Figure 7. Targeted Poly IC Eliminates Three Types of EGFR Over-Expressing Tumors

(A) (poly IC)Mel-PEI-PEG-EGF (MPPE) complexes prolong survival of mice bearing large intracranial U87MGwtEGFR xenografts. Cells were implanted into the brains of 16 mice. 15 d later, two animals were sacrificed to measure the tumors (Methods). Other animals received the indicated treatments. The daily doses of complexes were similar to the doses in Figure 3A and 3B. Survival of the animals was analyzed as above. (B) Formulated poly IC selectively kills A431 and MDA-MB-468 cells. Cells were seeded in duplicate onto a 96-well plate at a density of 5,000 cells in 0.2 ml of medium per well and grown overnight. Cells were then transfected as described [6,9] with poly IC at the indicated concentrations using either MPPE or PEI-PEG-EGF+PEI-Mel. Cell survival was measured by the Methylene blue assay at 48 h after transfection. (C) (poly IC)MPPE complexes eliminate A431 and MDA-MB-468 xenografts in mice. A431 and MDA-MB-468 tumors were established and treated with formulated poly IC as described in Methods. Tumors were measured daily. Control animals were euthanized at day 33 after treatment initiation. Poly IC treated mice were kept alive to detect possible late recurrence of the tumors.