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. 2017 Nov 9;8:1395. doi: 10.1038/s41467-017-01385-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Ad-TD-nsIL-12 is an effective and safe treatment for orthotopic PaCa in Syrian hamsters. Six days after Hap-T1 cells were seeded into the tail of the pancreas, hamsters were injected i.p. with 500 µl PBS, 1 × 10⁹ PFU Ad-TD-LUC, Ad-TD-IL-12 or Ad-TDnsIL-12 (a) or 2.5 × 10⁹ PFU Ad-TD-LUC, 2.5 × 10⁹ PFU Ad-TD-nsIL-12 or 5 × 10⁸ PFU Ad-TD-IL-12 (b) (n = 7/group) on days 0, 2, 4, 6, 8, and 10. Kaplan–Meier survival curves were generated. Significance was assessed using the log-rank (Mantel–Cox) test. *p < 0.05, **p < 0.01. c−h Animals were treated as b (n = 3/time point/group) and killed on day 3, 7, and 14 after last viral treatments. c Representative images of immunohistochemical staining for Hexon at day 7. Hexon-positive cells were counted in five high-power fields (HPF) from each tumor section (×200). ND: not detected. d Infectious virion recovery from tumor tissue was determined by TICD50 using JH293 cells. e Tumors, lung, and livers were analyzed by qPCR for the copy numbers of the viral E1A gene after treatment with Ad-TD-nsIL-12 at 2.5 × 10⁹ PFU/injection on day 0, 2, and 4. The sensitivity of the assay is illustrated by the dotted line. Mean and SEM are shown for each group and compared using an independent t-test. *p < 0.05, **p < 0.01. f Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05. g Representative images of immunohistochemical staining for CD3 at day 7. Quantitative scores of lymphocyte infiltration within tumors are shown (right panel) from five HPF from each tumor section (×200). The scoring was conducted within the tumor and stroma and necrotic areas were avoided. The extent of positive cells was categorized into the following four grades: 1, <15 cells/HPF; 2, 16–30 cells/HPF; 3, 31–45 cells/HPF; 4, >45 cells/HPF. ND: not detected. h IL-12 expression in sera was detected by ELISA. i Representative histopathology of HE staining of livers after three i.p injections of virus into hamsters bearing orthotopic PaCa tumors (n = 3/group) on day 0, 2, and 4, using the same dose as in b. Livers were collected one day following the last injection and analyzed using HE staining (×200)