br the right flank tumor in order
the right flank tumor in order to assess whether synergy could also be seen in uninjected tumors. In subsequent experiments using HCT116 unilateral xenografts created as above, I-131 dose was reduced to 2.0 mCi. As above, 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.
Tumors harvested at euthanasia were formalin fixed for 2 weeks, embedded in paraffin, and cut into 5-mm-thick sections. H&E stain-ing was done for routine histopathological examination. Slides were deparaffinized and applied for heat-mediated antigen retrieval per manufacturer’s protocol (IHC World, Ellicott City, MD, USA). Tumor sections were then permeabilized with methanol, and Tris-NaCl-blocking buffer (PerkinElmer, Waltham, MA, USA) was used to decrease background for 20 min. Rabbit anti-vaccinia Nocodazole antibody 1:200 in Tris-NaCl-blocking buffer (category no. ab35219; Abcam, Cambridge, MA, USA) was added overnight in a humidified chamber at 4 C. The next day, tumor sections were secondarily stained with Alexa Fluor 488-conjugated goat anti-rabbit (category no. ab150080; Abcam, Cambridge, MA, USA) for 1 h at room temper-ature. Finally, DAPI was added for nuclear staining, and images were obtained using EVOS FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
Statistical analysis was performed using GraphPad Prism (Version 7.01, La Jolla, CA, USA). Student’s t test or one-way ANOVA was
used to evaluate for statistical significance. p < 0.05 was considered significant.
Supplemental Information can be found online at https://doi.org/10.
S.G.W. conceptualized and directly supervised projects, drafted the manuscript, and refined figures. S.-I.K., S.C., M.P.O., J.L., and V.S. as-sisted with conceptualization of experiments, performed experiments, and revised and edited the manuscript. N.G.C. designed and con-structed the viruses. J.L. helped with generation and production of the viruses. S.-I.K. kept raw data and created many raw figures. Y.W., N.G.C., and Y.F. assisted with conceptualization of the project, assisted with direction of experiments, and revised and edited the manuscript.
CONFLICTS OF INTEREST
The authors declare no competing interests.
The authors are very grateful to Junie Chea and the City of Hope Small Animal Imaging Core for their assistance with radioisotope injections and imaging. S.G.W.’s work is supported by American Cancer Society Mentored Research Scholar Grant MRSG-16-047-01-MPC. The authors are grateful to Dr. Indra M. Newman for her scientific editorial expertise.
Molecular Therapy: Oncolytics
Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer. Gene Ther. 23, 357–368.
33. Workenhe, S.T., and Mossman, K.L. (2014). Oncolytic virotherapy and immunogenic cancer cell death: sharpening the sword for improved cancer treatment strategies. Mol. Ther. 22, 251–256.
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