Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br HMGB Cells were infected with CF hNIS MOI Superna

    2019-10-21


    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 Concanamycin A 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