Aerosolized BC-819 Inhibits Primary but Not Secondary Lung Cancer Growth

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819

Effect of BC-819 on cell growth in different lung cancer cell lines.

<p>NCI-H460, NCI-H358 and A549 cells were transfected, using Lipofectamine 2000, with BC-819 and co-transfected with a plasmid, encoding for the reporter enzyme luciferase. As early as 24 h after transfection, luciferase activity (indirectly indicating cell growth) was reduced by at least >90% when 350 ng BC-819 was used (A). 48 hours later luciferase activity was decreased by more than 98%, except for the cell line NCI-H358. However, a luciferase decrease of more than 98% was observed in all cell lines when the amount of BC-819 was increased to 700 ng (B). The influence of BC-819 on bioluminescent A549-C8-luc was examined as well and revealed decreased luciferase and therefore reduced cell growth (more than 50%) as early as after 24 hours (C). Maximum inhibition of cell growth was reached by 48 hours after transfection, becoming apparent through more than 75% reduction in luciferase activity.</p

Survival of untreated and treated tumor-bearing mice.

<p>Mice bearing intrabronchially induced lung tumors showed a significant survival benefit (p = 0.01) when treated with BC-819 compared to animals which received no treatment (A). No survival benefit was observed in mice suffering from intravenously induced lung tumors, independent of treatment with BC-819 (B).</p

In-situ-hybridization for H19-activity in lung tumors.

<p>An intrabronchially induced lung tumor was stained for H.E. (A) and screened for H19-activity using ISH (B). In the given sample a strong expression of H19 was found, indicated by a brownish colorization of the examined tissue.</p

BLI of untreated and treated tumor-bearing mice at different time points.

<p>Growth of lung tumors in untreated mice was characterized by initial tumor appearance in the neck region and subsequently continuous dissemination into the lungs (A). This growth pattern differed in mice which were treated with BC-819 where tumors either decreased in size or in completely vanished during treatment.</p

Histology of orthotopically induced lung tumors.

<p>Tumors were detectable as early as 10 days after intrabronchial implantation in the peribronchiolar region of the lungs (A). End stages of intrabronchially induced lung tumors were characterized by multiple, peribronchiolar and parenchymal localized tumors (B) and by tumor tissue within the luminal lung regions as well (C). Intravenous injection of A549-C8-luc, however, resulted in tumors predominately growing in the parenchyma of the lungs but not peribronchiolar or within the bronchi (D). Figure D shows parenchymal tumor tissue close to a bronchus, but not peribronchiolar. Alveolar tissue localized between the tumor and the bronchus however is condensed by the adjacent tumor nodule.</p