br HMGB Cells were infected with CF hNIS MOI Superna
HMGB1. Cells were infected with CF33-hNIS (MOI 5). Superna-tants were collected at different time points and concentrated using a column with 10-kDa size cutoff. The concentrated supernatants were loaded (15 mL/well) for western blotting. HMGB1 was detected using a rabbit anti-HMGB1 antibody (category no. ab18256; Abcam) at 1:100 dilution followed by an HRP-labeled goat anti-rabbit second-ary antibody (category no. ab205718; Abcam) at 1:5,000 dilution.
In Vivo Studies
Animal studies were performed under the City of Hope Institutional Animal Care and Use Committee (IACUC)-approved protocol.
Establishment of Colorectal Cancer Flank Xenografts
Six-week-old Hsd:Athymic Nude-Foxn1nu female mice (Envigo, Indi-anapolis, IN, USA) were purchased and acclimatized for 1 week. Bilat-eral flank tumors were generated by injecting 2–5 106 HCT116 or HT29 Concanamycin A in a total of 100 mL PBS containing 50% matrigel for each tumor. When average tumor size approached 150 mm3, mice were divided into experimental groups.
In Vivo Efficacy of CF33-hNIS
Mice were divided into the following groups (n = at least 4 mice per group): PBS control HT29, PBS control HCT116, CF33-hNIS HT29, and CF33-hNIS HCT116. When tumors reached average volume of 150 mm3, mice were treated with 105 PFU of CF33-NIS or PBS, and experiments were also repeated at 104 PFU. In all groups, tumor volume was measured twice weekly with tumor volume V (mm3) = (1/2) A2 B, where A is the shortest and B is the longest measure-ment. Percent tumor change was calculated based upon initial tumor size at time of intervention. At the time of euthanasia, tumors and solid organs were harvested. Tissues were split into halves formalin-fixed for immunohistochemical analysis. Two mice from each group were sacrificed at day 10 following viral injection. The remaining mice were euthanized on day 50.
In Vivo I-124 Uptake Measured by PET/CT
Mice were divided into imaging and control groups (n = 4 mice). To analyze tumor imageability after intratumoral delivery, mice received an intratumoral injection of 104 PFU per tumor of either CF33-hNIS, CF33-Fluc, or PBS when tumors reached 100 mm3. At 7, 15, and 22 days post-viral infection, mice in each group received 200 mCi of I-124 injected per tail vein. The radioisotope was obtained from the City of Hope Small Animal Imaging Core Radiopharmacy. PET imaging was then obtained 2 h following injection using the small-animal PET scanner (microPET R4; Siemens Corporation), which provides fully three-dimensional PET imaging with spatial resolution
of better than 2.0 mm and quantitative accuracy for measurement of tissue activity concentration on the order of 10%. The 8-cm axial field of view is adequate for simultaneous whole-body imaging of mice. Advanced image reconstruction software provides resolution ap-proaching 1.0 mm. Quantitative accuracy is supported by scatter, dead time, and measured attenuation corrections. The system in-cludes a fully developed image analysis package that supports volu-metric regions of interest and fusion of PET with co-registered anatomic CT. In order to protect mouse thyroids from radioiodine ablation, all mice received T4 supplementation with 5 mg levothyrox-ine/L of water beginning 1 week prior to radioiodine administration.
In Vivo Combination Therapy of CF33-hNIS and I-131
Bilateral flank xenografts were implanted in athymic nude female mice using 3 106 HT29 cells in 100 mL of PBS containing 50% matrigel. Ten days later, when tumors reached average volume of 150 mm3, they were randomized to four treatment groups: PBS control, i.v. I-131 alone, intratumoral CF33-hNIS alone, or combination of i.v. I-131 with intratumor (i.t.) CF33-hNIS. I-131 injection was i.v., whereas viral injection was intratumoral. In the combination group, I-131 injection occurred 7 days after viral infection. Doses of 1 104 PFU of i.t. CF33-NIS were used at day zero in both groups using virus. In the radioisotope alone group, I-131 was administered on the same date as virus. Based upon previous publications exploring radioisotope