Volume 21, Issue 11, Pages 1985-1995 (November 2013) Engineering of Bacteria for the Visualization of Targeted Delivery of a Cytolytic Anticancer Agent  Sheng-Nan Jiang, Seung-Hwan Park, Hee Jung Lee, Jin Hai Zheng, Hyung-Seok Kim, Hee-Seung Bom, Yeongjin Hong, Michael Szardenings, Myung Geun Shin, Sun-Chang Kim, Vasilis Ntziachristos, Hyon E Choy, Jung-Joon Min  Molecular Therapy  Volume 21, Issue 11, Pages 1985-1995 (November 2013) DOI: 10.1038/mt.2013.183 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Plasmids used in this study. (a) A schematic feature of Tet system including regulatory region. The tet operators O1 and O2 overlap the divergent tetA and tetR promoters. (b) Brief depiction of the plasmids constructed in this study. See Materials and Methods for detailed description. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Doxy-inducible bilateral expression of rluc and fluc in ppGpp-defective S. typhimurium. Salmonellae transformed with pJL84 (tetAP::rluc, tetRP::fluc) were cultured in the presence of varying concentrations of Doxy for 4 hours. Luminescence of RLuc and FLuc was measured by luciferase assay immediately after adding the appropriate substrate, coelenterazine and D-luciferin, respectively, to the transformed bacteria (SLRLuc/FLuc). (a) Activity of RLuc and FLuc in the presence of increasing concentrations of Doxy (0, 10, 50, 100, 200, 500, and 1000 ng/ml). (b) Correlation between RLuc and FLuc activity in the presence of increasing concentrations of Doxy, P = 0.0001. (c) In vivo expression of RLuc and Fluc in tumor-colonized Salmonellae before and after (12 hours) Doxy induction. BALB/c mice inoculated subcutaneously with 1 × 106 CT26 cells were injected with engineered Salmonellae via the tail vein. Mice (n = 3 per group) were administered Doxy at 1 dpi (upper panel) or 3 dpi (lower panel). Bioluminescence imaging was performed following i.v. injection of coelenterazine (0.7 mg/kg, RLuc activity) and intraperitoneal (i.p.) injection of D-luciferin (150 mg/kg, FLuc activity) in each animal. Arrowhead indicates activity from the liver. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Doxy-induced expression and secretion of ClyA in engineered S. typhimurium. S. typhimurium (ΔppGpp) was transformed with pJL87 (tetAP::clyA, tetRP::rluc8). (a) The indicated concentrations (~200 ng/ml) of Doxy were added to fresh bacterial cultures (1 hour) at an A600 of 0.5–0.7. After 3 hours of incubation, approximately 4 × 107 CFU were collected and analyzed by immunoblotusing an anti-ClyA antibody (lower panel). Ponceau staining indicated equal loading of protein (upper panel). (b) The indicated concentrations (~200 ng/ml) of Doxy were added to bacterial culturesatan OD600 of 0.5–0.7. After 4 hours of incubation, coelenterazine (1 μg) was added to the cultures, and bioluminescence was measured immediately using a cooled CCD camera. (c) The expression and secretion of ClyA (34 kDa) was verified by immunoblotusing an anti-ClyA antibody. Bacterial pellets and filtered culture medium (Filtrate) were collected 4 hours after induction with or without Doxy (200 ng/ml). Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 In vivo Doxy-induced gene expression in CT26 by engineered S. typhimurium. BALB/c mice (n = 5) were injected subcutaneously with CT26 (1 × 106). When the tumors reached a volume of approximately 150 mm3, tumor-bearing mice were injected with engineered S. typhimurium carrying pJL87 followed by oral administration of Doxy at 3 dpi. Bioluminescence imaging was performed following i.v. injection of coelenterazine before and after (12 hours) Doxy administration. Immunoblot analysis (n = 3) and histologic study (n = 2) was done after excision of CT26 tumor grafts 5 days after treatment with Salmonellae and 2 days after the start of Doxy administration. (a) Expression of RLuc8 in CT26 tumors of mice treated with engineered Salmonella was verified by in vivo bioluminescence imaging. (b) Immunoblot analysis of the expression of ClyA in CT26 tumor tissue from mice injected with engineered Salmonellae before (–) and after (+) Doxy induction. (c) H&E staining revealed three regions in the engrafted tumor: necrotic (N), border (B), and viable (V) zones. Scale bar = 100 μm. (d) Immunofluorescence staining of indicated H&E stained areas. Sections were stained with anti-salmonella antibody (green), anti-ClyA antibody (red) and DAPI/Antifade (blue). A merged image is shown (Merged). Scale bar= 20 μm. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Effect of engineered Salmonellae on tumor growth and survival. BALB/c or BALB/c athymic nu−/nu− mice were injected subcutaneously with CT26 (1 × 106) or Hep3B2.1–7 cells (1 × 107), respectively. When the tumors reached a volume of approximately 150 mm3, CT26 or Hep3B2.1-7 tumor-bearing mice were treated with PBS (n = 6), S. typhimurium carrying empty vector (SL) (n = 8), or engineered S. typhimurium carrying pJL87 (SLClyA/RLuc8) (n = 8). In a separate group of tumor-bearing mice (n = 14 for CT26, n = 12 for Hep3B2.1-7), 17 mg/kg of Doxy was administered orally everyday, starting 4 days after treatment with engineered S. typhimurium (SLClyA/RLuc8 + Doxy). (a) Images of representative subcutaneous tumors. (b) Change in tumor volume. Arrows indicate time of therapeutic injection. Left panel; effect on CT26 tumor growth. *P = 0.003 (PBS versus SL, SLClyA/RLuc8, or SLClyA/RLuc8 + Doxy at day 20), **P = 0.004 (SLClyA/RLuc8 versus SLClyA/RLuc8 + Doxy at day 35), Right panel; effect on Hep3B2.1-7 tumor growth. *P = 0.001 (PBS versus SL, SLClyA/RLuc8, or SLClyA/RLuc8 + Doxy at day 35), **P = 0.002 (SL versus SLClyA/RLuc8 + Doxy at day 80), ***P = 0.014 (SLClyA/RLuc8 versus SLClyA/RLuc8 + Doxy at day 80) (c) Kaplan-Meier survival curves of mice bearing CT26 (*P < 0.0001) or Hep3B2.1–7 tumors (*P = 0.0076, **P = 0.0005, ***P = 0.0003) are shown. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Localization and therapeutic effect of engineered Salmonellae in a mouse lung metastases model. On day 7 after i.v. injection of CT26FLuc (1 × 104), BALB/c mice were injected i.v. with engineered S. typhimurium carrying pJL87 (SLClyA/RLuc8) or with PBS. Oral administration of Doxy was started 2 days after bacterial injection (SLClyA/RLuc8 + Doxy). (a) D-luciferin bioluminescence imaging of lung metastases after the indicated treatments. (b) Coelenterazine bioluminescence imaging of bacterial gene expression on day 5 after Doxy administration. (c) Lungs were excised 7 days after treatment. Top panel: photographs of the lungs; middle: bioluminescence (FLuc) images of the same lungs; bottom: India-ink staining of lungs from separate animals. (d,e) Effects on CT26 lung metastasis. (d) Lung weights were measured 7 days after the indicated treatments (n = 5 per group). (e) Metastasis scores were measured 7 days after the indicated treatments (n = 5 per group). The fraction of lung surface covered by fused metastases was scored as follows: 0 = 0%, 1 = less than 20%, 2 = 20–50%, 3 = more than 50%. (f) Kaplan-Meier survival curves. *P = 0.042,**P = 0.0001. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Systemic toxicity of engineered Salmonellae expressing ClyA. BALB/c mice (n = 5 in each group) were injected s.c. with CT-26 cells. After tumors reached a volume of 150 mm3, the tumor-bearing mice were injected with PBS, S. typhimurium carrying empty vector (SL) or S. typhimurium carrying pJL87 followed by oral administration of Doxy (SLClyA/RLuc8 + Doxy) at 0 dpi or 3 dpi. The level of serum aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, plasma C-reactive protein, and procalcitonin were measured at 5 dpi. Boxes represent the quartiles and whiskers mark the 10th and 90th percentiles. *P < 0.01. Normal values: ALT 17-77 IU/L, AST 54-298 IU/L, blood urea nitrogen 8–33 mg/dl, creatinine 0.2–0.9 mg/dl, CRP < 0.5 mg/dl, procalcitonin < 0.5 ng/ml. Yellow-shaded areas = normal range of measured parameters. Molecular Therapy 2013 21, 1985-1995DOI: (10.1038/mt.2013.183) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions