Preventing growth of brain tumors by creating a zone of resistance - PubMed (original) (raw)

Preventing growth of brain tumors by creating a zone of resistance

Casey A Maguire et al. Mol Ther. 2008 Oct.

Abstract

Glioblastoma multiforme (GBM) is a devastating form of brain cancer for which there is no effective treatment. Here, we report a novel approach to brain tumor therapy through genetic modification of normal brain cells to block tumor growth and effect tumor regression. Previous studies have focused on the use of vector-based gene therapy for GBM by direct intratumoral injection with expression of therapeutic proteins by tumor cells themselves. However, as antitumor proteins are generally lethal to tumor cells, the therapeutic reservoir is rapidly depleted, allowing escape of residual tumor cells. Moreover, it has been difficult to achieve consistent transduction of these highly heterogeneous tumors. In our studies, we found that transduction of normal cells in the brain with an adeno-associated virus (AAV) vector encoding interferon-beta (IFN-beta) was sufficient to completely prevent tumor growth in orthotopic xenograft models of GBM, even in the contralateral hemisphere. In addition, complete eradication of established tumors was achieved through expression of IFN-beta by neurons using a neuronal-restricted promoter. To our knowledge this is the first direct demonstration of the efficacy of targeting gene delivery exclusively to normal brain cells for brain tumor therapy.

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Figures

Figure 1. Pretreatment of brain with AAV-CBA-IFN-β prevents aggressive glioma growth in nude mice

(a) Mice were infused with 1011 genome copies (gc) of either AAV-empty vector (AAV-ev) or AAV-hIFN-β by stereotaxic injection of the striatum (six mice per group). Two weeks after injection, 105 U87 glioma cells stably expressing luciferase and mCherry as well as expressing a constitutively active epidermal growth factor receptor (EGFR) variant III (U87fluc-mCherry-EGFRvIII) were implanted into the same site as vector and mice were imaged for tumor-associated bioluminescent signal at the indicated time points post-tumor injection. ND=, not detectable. (b) Representative bioluminescent images of mice injected with AAV-ev (left image) or AAV-CBA-hIFN-β (right image), both at day 21 post-tumor implantation. Corresponding hematoxylin and eosin–stained coronal brain sections from either AAV-ev-treated animals (left image, day 21 post-tumor) or AAV-CBA-hIFN-β-treated animals (right image, day 53 post-tumor).CBA, chicken β-actin; hIFN-β, human interferon-β.

Figure 2. Pretreatment of brain with AAV-CBA-hIFN-b provides 100% survival against glioma challenge

Kaplan–Meier survival analysis of mice pretreated as in (Figure 1a) and later implanted with (a) 5 × 105 U87fluc-mCherry cells, or (b) 105 U87fluc-mCherry-EGFRvIII cells. Solid line indicates AAV-CBA-IFN-β-treated mice, while dashed line indicates AAV-empty vector–treated mice (six mice per group). AAV, adeno-associated virus; CBA, chicken β-actin; EGFRvIII, epidermal growth factor receptor variant III; hIFN-β, human interferon-β.

Figure 3. Antitumor effect of different doses of AAV-CBA-hIFN-β

AAV-CBA-hIFN-β was infused into the striatum of three groups of mice (n = 4) at the following doses: 1011 genome copies (gc), 1010 gc, 5 × 109 gc. Two weeks later, 5 × 105 U87fluc-mCherry cells were injected at the same coordinates used for vector injection and tumor growth was monitored by bioluminescent imaging. #Only one of four mice in the 5 × 109 gc AAV-CBA-hIFN-β group gave detectable signal at days 12 and 25. AAV-ev, adeno-associated virus–empty vector; CBA, chicken β-actin; EGFRvIII, epidermal growth factor receptor variant III; hIFN-β, human interferon-β; ND, not detected.*P < 0.05.

Figure 4. Intracranial injection of AAV-CBA-hIFN-β causes regression of distal tumors in the brain

Mice were infused with 5 × 1010 genome copies of either AAV-empty vector (AAV-ev) or AAV-CBA-hIFN-β by stereotaxic injection of the striatum (five mice per group). Two weeks post-injection, 5 × 105 U87fluc-mCherry glioma cells were implanted into the striatum of the contralateral hemisphere and mice were imaged for tumor-associated bioluminescent signal at the indicated time points post-tumor injection. AAV, adeno-associated virus; CBA, chicken β-actin; hIFN-β, human interferon-β. *P < 0.01.

Figure 5. AAV-NSE-hIFN-β vector function and in vivo levels

Interferon-β (IFN-β) levels in striatum after injection with either AAV-CBA-hIFN-β or AAV-NSE-hIFN-β. Nude mice were infused into the striatum with 1011 genome copies of the indicated vectors, and 2 weeks later human IFN-β (hIFN-β) was detected by enzyme-linked immunosorbent assay and immunoblot analysis (inset). A band of the expected 25-kd MW was detected in lysate from brains transduced with both AAV-CBA-hIFN-β vector (lane 1) and AAV-NSE-hIFN-β vector (lane 2). AAV-ev, adeno-associated virus–empty vector; NSE, neuron-specific enolase.

Figure 6. AAV-NSE-GFP vector expresses exclusively in brain parenchyma

(a, b; large tumor) 105 U87fluc-mCherry-EGFRvIII cells were implanted into the striatum of nude mice and 1 week later, 1010 genome copies AAV-NSE-GFP was infused intratumorally. Seven days later, brains were analyzed as above. Transduced cells are shown in green and tumor cells in red due to stable mCherry expression. Note the transduction of brain parenchyma on the perimeter of the tumor and also the few GFP+ cells within the tumor which did not co-localize with mCherry. Scale bar = 200 μm. (c, d; small tumor) Mice were injected with 104 U87fluc-mCherry cells and were intratumorally injected with AAV-NSE-GFP 7 days later. Ten days later, mice were killed and brains analyzed for transduction. Note the excellent transduction of brain which completely surrounds the tumor. Scale bars = 200 µm. AAV, adeno-associated virus; EGFRvIII, epidermal growth factor receptor variant III; GFP, green fluorescent protein; NSE, neuron-specific enolase.

Figure 7. Expression of human interferon-β (hIFN-β) from nontumor tissue causes regression of established tumors

(a) 5 × 105 U87fluc-mCherry cells were implanted into nude mice (four mice per group) and 2 weeks later, mice were infused with 4 × 1011 genome copies of AAV-empty vector (AAV-ev) or AAV-NSE-hIFN-β. Mice were imaged as in Figure 1a. (b) Representative bioluminescent images of mice injected with AAV-ev (left image) or AAV-NSE-hIFN-β (right image), both at day 14 post-vector. Hematoxylin and eosin–stained coronal brain sections from either AAV-ev-treated animals (left image, day 38 post-tumor) or AAV-NSE-hIFN-B-treated animals (right image, day 49 post-tumor). Arrow points to glial scar.

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Preventing growth of brain tumors by creating a zone of resistance - PubMed (original) (raw)