Volume 18, Issue 3, Pages 635-642 (March 2010) Inhibition of Tumor Growth and Metastasis by a Combination of Escherichia coli– mediated Cytolytic Therapy and Radiotherapy  Sheng-Nan Jiang, Thuy X Phan, Taek-Keun Nam, Vu H Nguyen, Hyung-Seok Kim, Hee-Seung Bom, Hyon E Choy, Yeongjin Hong, Jung-Joon Min  Molecular Therapy  Volume 18, Issue 3, Pages 635-642 (March 2010) DOI: 10.1038/mt.2009.295 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Engineering of E.coli k-12 to express and secrete cytolysin A. (a) Map of the bacterial expression plasmid containing the gene encoding aspartate β-semialdehyde dehydrogenase (Asd) and the cytolysin A gene (pAClyA). (b) The E. coli asd::kan strain (HJ1020) was transformed with pAClyA (E. coli pAClyA) or the empty vector containing only the Asd gene (pAsd). The expression and secretion of ClyA (34 kd) was analyzed by western blot using an anti-ClyA antibody. Bacterial pellets (left) and culture media (right) were analyzed. (c) E. coli–expressing ClyA and Lux can lyse blood cells (left) and produce a clear bioluminescent signal in the corresponding area of hemolysis (right). Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Genetically engineered E. coli can target and deliver ClyA to subcutaneously grafted CT26 tumors. (a) Noninvasive in vivo imaging of bacterial bioluminescence in the targeted tumors of representative tumor-bearing mice (n = 2). (b,c) Detection of the 34-kd ClyA protein in CT26 tumor tissues of mice injected with E. coli–expressing ClyA (E. coli pAClyA). The ClyA protein was detected (b) using western blot analysis, and (c) by immunohistochemical and immunofluorescence staining. ClyA, cytolysin A. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Suppression of tumor growth by engineered E. coli–expressing ClyA. BALB/c mice (n = 15) were inoculated subcutaneously with 106 CT26 cells. When the tumor reached ~150 mm3 in volume or ~10 mm in a diameter, they were injected with PBS or bacteria. (a) Photographs of subcutaneous tumors in mice injected with PBS (control), or 5 × 107 CFU of E. coli carrying empty vector (E. coli pAsd) or the ClyA vector (E. coli pAClyA). Photographs of representative tumors were taken at 12, 17, 21, and 27 days after injection. (b) Effects of the injections of PBS, E. coli pAsd, or E. coli pAClyA on CT26 tumor growth rates (n = 5 each group, *P < 0.05). CFU, colony-forming units; ClyA, cytolysin A; PBS, phosphate-buffered saline. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Histologic examination of syngeneic CT26 before and after treatment. H&E stain of syngeneic CT26 tumor 4 days after intravenous injection of PBS (control), or 5 × 107 CFU of E. coli carrying empty vector (E. coli pAsd) or the ClyA vector (E. coli pAClyA) (bar = 1 mm). CFU, colony-forming units; H&E, hematoxylin–eosin; PBS, phosphate-buffered saline. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Effects of combining engineered E. coli therapy with radiation in CT26-bearing mice. BALB/c mice were inoculated subcutaneously with 106 CT26 cells. (a) Photographs of subcutaneous tumors in mice treated with PBS (no treatment) or with a combination of bacteria (BT; 5 × 107 CFU of E. coli pAClyA) and radiation (RT; 21 Gy). Photographs of representative tumors were taken at 12–42 days after the beginning of treatments. (b) Effects on the growth rates of CT26 tumors after treatment with E. coli–expressing ClyA (BT; 5 × 107 CFU) combined with various doses of radiotherapy (RT; 0, 8, 15, 21 Gy). PBS only (filled square), BT only (open square), RT (8 Gy) + BT (filled circle), RT (15 Gy) + BT (open circle) and RT (21 Gy) + BT (filled triangle). (c) Effects on the growth rates of CT26 tumors after treatment with radiation (RT; 21 Gy) and various doses of E. coli–expressing ClyA (0–5 × 107 CFU). PBS only (filled square), RT only (open square), BT (5 × 106 CFU) + RT (filled circle), BT (1 × 107 CFU) + RT (open circle), and BT (5 × 107 CFU) + RT (filled triangle) (n = 5 each group). (d) Kaplan–Meier survival curves between RT (21 Gy) and RT (21 Gy) + BT (5 × 107 CFU) are shown (n = 5 each group). *P < 0.05, **P < 0.01. BT, bacterial therapy; CFU, colony-forming units; PBS, phosphate-buffered saline; RT, radiotherapy. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Effects of combining engineered E. coli therapy with radiation in B16F10 and 4T1-bearing mice. Nude mice or BALB/c mice were inoculated subcutaneously with 106 B16F10 cells or 106 4T1 cells, respectively. Effects on (a) B16F10 and (b) 4T1 tumor growth after treatment with E. coli–expressing ClyA (5 × 107 CFU) and/or radiation (21 Gy). Controls were treated with PBS (open circle). BT, treatment with E. coli–expressing ClyA only (circle); RT, radiotherapy only (triangle); RT + BT, combination therapy with radiation and E. coli–expressing ClyA (square). (n = 5 each group, *P < 0.05, **P < 0.01). BT, bacterial therapy; CFU, colony-forming units; ClyA, cytolysin A; PBS, phosphate-buffered saline; RT, radiotherapy. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Effects of E. coli–expressing ClyA on lung metastases. BALB/c mice were injected with CT26 cells that were stably producing firefly luciferase. Mice were intravenously injected with engineered (E. coli pAClyA) or nonengineered bacteria (E. coli pAsd), or PBS at day 5 after the cancer cells were injected. The cooled CCD camera was used to analyze metastasis establishment in lungs of the mice after intraperitoneal injection of 3 mg of D-luciferin per animal. (a) Bioluminescence images of lung metastasis development in mice at 4, 7, and 11 days after each treatment. (b) Lungs were excised from the mice 6 days after the treatments. Top: photographs of representative lungs. Bottom: bioluminescence images of the same lungs. (c) Lung weights and (d) metastasis scores were measured in the mice 6 days after each treatment (n = 3 each group). (e) Kaplan–Meier survival curves are shown (n = 8 each group). *P < 0.05, **P < 0.01. CCD, charge-coupled device; ClyA, cytolysin A; PBS, phosphate-buffered saline. Molecular Therapy 2010 18, 635-642DOI: (10.1038/mt.2009.295) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions