Mice

All animal studies were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Emory University, Atlanta, USA. C57Bl/6, Rag^−/−, μMT, C3^−/−, CD36 ^−/−, CD18^−/−, and Itgam^−/− mice were purchased from The Jackson Laboratory. C1q^−/− mice were provided by M. Diamond (Washington University, St. Louis, MO). All mice were on C57Bl/6 genetic background, matched by age, and housed in specific pathogen–free facilities. NCr/nude athymic female nude mice were purchased from Taconic Biosciences, and NSG mice were purchased from The Jackson Laboratory. More details and genotypes are in table S1.

Analysis of antitumor activity of viruses in mouse tumor models. For subcutaneous tumor model, 8-week-old female NSG mice (The Jackson Laboratory) were injected subcutaneously with 5 × 10⁶ A549-Luc-C8 (A549-Luc) cells. For establishing the PDX tumor models, the PDX-bearing mice were purchased from The Jackson Laboratory (PDX models used in the study: TM00302, TM00784, TM00219, and TM00233). Upon reaching a volume of ~1500 mm³, the PDX tumors were excised, cleaned of connective and/or necrotic tissue, sliced in small pieces, and subcutaneously implanted in naïve NSG mice (48). To test virus transduction efficiency, small pieces of PDX tissues were placed in standard growing medium and incubated with Ad5-3M virus in 12-well plates. Virus-dependent GFP expression was monitored, and pictures were taken with either wide-field microscope or scanned on IncuCyte Zoom live-cell analysis system (Essen Bioscience) 3 to 5 days after infection. The tumor growth of PDX- or A549-Luc–derived tumors was closely monitored, and when subcutaneous tumors reached mean volumes of 37.79 ± 23.27 mm³ (PDX-TM00302), 443.2 ± 135.7 mm³ (PDX-TM00784), and 298.1 ± 146.3 mm³ (A549-Luc), mice were randomized and divided in cohorts. The Ad5-3M–treated cohorts were injected intravenously via tail vein with three doses of the Ad5-3M virus, each dose containing 2 × 10¹⁰ virus particles (vp) and separated by 48 hours. The buffer cohort mice were injected with the same regiment, but with a buffer solution. In Ad5-WT–treated cohorts, mice were injected with two doses of virus, each dose containing 2 × 10¹⁰ vp, and all mice succumbed to virus-induced hepatotoxicity before the third dose injection. The tumors were measured with electronic calipers, and tumor volume was calculated by the formula ((tumor width (mm))² × tumor length (mm))/2. The mice were euthanized when tumor volume reached 2000 mm³ or tumor-burden score > 17 (Emory University IACUC guidelines).

For the orthotopic disseminated cancer model, 8- to 12-week-old female athymic NCr nude mice (Taconic Biosciences) were injected intravenously via tail vein with 3 × 10⁶ A549-Luc cells, expressing firefly luciferase gene. In 5 weeks after tumor cell implantation, the tumor burden was measured by in vivo bioluminescent imaging (BLI). Only tumor-bearing mice with luciferase activity measured at 2 × 10⁶ to 8 × 10⁶ photons s⁻¹ cm⁻² sr⁻¹ were enrolled in the experiment. Enrolled mice were randomized and divided in cohorts. The Ad5-3M cohort mice were injected with three doses of the virus intravenously via tail vein, each dose containing 1 × 10¹¹ vp and administered over 48-hour intervals. In the Ad5/35-3M cohort, mice received the same regimen, but each dose was equal to 5 × 10¹⁰ vp per mouse. Mice from the buffer cohort were injected similarly, but with a virus-diluent buffer only. In the Ad5-WT cohort, mice were injected with 1 × 10¹¹ vp/mouse; however, all mice succumbed to virus-induced hepatotoxicity after the first virus dose, and the planned injection of a second virus dose was not performed. The tumor burden was measured by BLI weekly, and mice were monitored for signs of endpoint criteria (measured firefly luciferase activity at least 1 × 10⁸ photons s⁻¹ cm⁻² sr⁻¹ and 20% body weight loss or episodes of dyspnea).