Tumor-targeting Salmonella typhimurium A1-R inhibits human prostate cancer experimental bone metastasis in mouse models.

Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting.


INTRODUCTION
Prostate cancer is diagnosed in more than 500,000 men worldwide [1]. Bone metastases from prostate cancer can cause chronic pain, hypercalcemia, pathologic fractures, and nerve compression [2]. Zoledronic acid (ZOL), a bisphosphonate, has been used to prevent the development of metastatic bone lesions. Although bisphosphonates, irradiation, and surgical resection of tumors are used as treatments of bone metastases, the outcomes of the treatments are still unsatisfactory.
In the present study, the efficacy of S. typhimurium A1-R and the combination of S. typhimurium A1-R and ZOL was assessed in nude mice models of solitary and multiple bone metastases of prostate cancer.

Efficacy of S. typhimurium A1-R on human prostate cancer cells in vitro
To determine the efficacy of A1-R on prostate cancer cells, PC-3-RFP cells were incubated in 35 mm dishes for 24 h, and the cells were treated with S. typhimurium A1-R for 1 h. The cells were observed with a Fluoview FV1000 confocal microscope (Olympus Corp., Tokyo, Japan). Fluorescence imaging demonstrated that S. typhimurium A1-R expressing GFP selectively invaded and replicated intracellularly and killed PC-3-RFP cells (Figure 1). Clonogenic assays demonstrated that S. typhimurium A1-R inhibited proliferation of PC-3-RFP cells in a dosedependent manner ( Figure 2).

Efficacy of S. typhimurium A1-R therapy on a mouse model of multiple bone metastasis
Nude mice were injected in the left ventricle with PC-3-GFP cells (5 × 10 5 ). One week after intracardiac injection, half of the mice were treated once a week for 3 weeks with an i.v. injection of S. typhimurium A1-R

Efficacy of S. typhimurium A1-R on a mouse model of solitary bone metastasis of prostate cancer
PC-3-RFP (5 × 10 5 ) cells were injected into the intramedullary cavity of the tibia in nude mice ( Figure 4a). One week after the injection, the mice were divided into 4 groups: a control group, a ZOL group, an A1-R group, and a ZOL+ S. typhimurium A1-R group. ZOL-group mice were treated with subcutaneous injection of ZOL (120 mg/ kg) (Sigma-Aldrich. St. Louis, MO) 5 times a week for 4 weeks (Figure 4b). S. typhimurium A1-R group mice were treated with weekly i.v. injections of S. typhimurium A1-R (5 × 10 7 CFU) for a total of 3 weeks. ZOL+ S. typhimurium A1-R-group mice were treated with both s.c. injection of ZOL and i.v. injection of S. typhimurium A1-R at the same dosages listed above. Fluorescence imaging was performed with an iBOX Scientia Imaging System (UVP, LLC, Upland, CA) every week. The fluorescence images demonstrated that the control group had rapid growth of metastatic bone cancer, whereas the ZOL group, S. typhimurium A1-R group, and the ZOL+S. typhimurium A1-R group mice had reduced metastatic growth ( Figure  4c). The fluorescent tumor area of the control-group mice was 54.9 ± 6.1 mm 2 , ZOL group at 4 weeks mice was 29.1 ± 9.2 mm 2 , S. typhimurium A1-R-group mice was 23.8 ± 6.1 mm 2 , and ZOL+ S. typhimurium A1-R group mice was 18.4 ± 5.3 mm 2 (Figure 4d). The S. typhimurium A1-R group and the ZOL+ S. typhimurium A1-R group had significant inhibition of tumor growth compared to the control group. Four weeks after the injection of cancer cells, the mice were sacrificed and tumor weight was measured. Tumor weights of the control group was 2.6 ± 0.6 g; ZOL group was 0.5 ± 0.2 g; S. typhimurium A1-R group was 1.1 ± 0.6; and ZOL+ S. typhimurium A1-R group mice at 4 weeks was 0.3 ± 0.1 g (Figure 4e).
The present study demonstrates that S. typhimuium A1-R could significantly inhibit or prevent prostate cancer metastasis in the bone. These results indicate a promising approach to a currently highly treatment-resistant disease.

Animal care
Athymic nude mice (nu/nu) (AntiCancer Inc, San Diego, CA) were used in this study. Mice were maintained in a barrier facility of high efficiency particulate air-filtered racks. The animals were fed an autoclaved laboratory rodent diet. Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals under National Institutes of Health assurance number A3873-01.

Preparation of S. typhimurium A1-R
GFP-expressing S. typhimurium A1-R bacteria (AntiCancer Inc., San Diego, CA, USA) were grown overnight on LB medium (Fisher Sci., Hanover Park, IL, One week after intratibial injection, fluorescence imaging was performed to confirm the growing RFP-expressing tumor using the iBOX Scientia Small Animal Imaging System (UVP LLC, Upland, CA, USA). The S. typhimuium A1-R group was administered S. typhimurium A1-R (5 × 10 7 CFU, i.v.) once a week for 3 weeks. The control group was administered the same volume of PBS. Fluorescence imaging was performed on treated and untreated mice. RFP fluorescent area was recorded every week for 5 weeks using the iBOX. c. Time-course imaging of the RFP-expressing bone tumors in the mouse model of solitary bone metastasis after treatment with ZOL and S. typhimuium A1-R. d. Fluorescence area of bone tumors in the control group and A1-R group mice. *p < 0.05, ** p < 0.01 compared with the control group. e. Tumor weight at the end of the experiment in mice injected intratibially with PC-3-RFP cells. Data are expressed as the mean ± SE. Differences between groups were analyzed with ANOVA. * p < 0.05 compared with the untreated control group. www.impactjournals.com/oncotarget USA) and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then diluted in PBS [3,5].

S. typhimurium A1-R killing of prostate cancer cells in vitro
PC-3-RFP cells were plated in 35 mm dishes (5 × 10 2 cells/dish). S. typhimurium A1-R-GFP was added to the cancer cells (1 × 10 7 or 1 × 10 8 CFU/dish). After 1 h incubation at 37°C, the cells were rinsed and cultured in medium containing gentamycin sulfate (100 µg/ml) to kill external but not internal bacteria. Invasion and destruction of PC-3-RFP cells by S. typhimurium A1-R-GFP was visualized with a Fluoview FV1000 confocal microscope (Olympus Corp., Tokyo, Japan). Eight days after treatment with S. typhimurium A1-R, PC-3-RFP colonies were fixed in methanol and stained with 1% crystal violet as previously described [40]. ImageJ (National Institute of Mental Health, Bethesda, Maryland, USA) was used to quantify the area of the colonies of the cells.

Efficacy of S. typhimurium A1-R therapy on a mouse model of multiple bone metastases
PC-3-GFP cells (5 × 10 5 ) were injected intracardially in nude mice. One week after injection, mice (treatment group) were administered S. typhimurium A1-R (5 × 10 7 CFU, i.v.) once a week for 3 weeks. The remaining mice (control group) were administered the same volume of PBS. To evaluate metastasis-free survival, GFP-expressing lesions were initially observed using the Illumatool imaging system(Lighttools Research, Encinitas, CA) every 2 days. Metastasis-free survival was defined as the time from intracardiac injection of cancer cells to the time of detection of bone metastases with the Illumatool.

Efficacy of S. typhimurium therapy on a mouse model of solitary bone metastases
A midline skin incision (5 mm) was made just below the knee joint to expose the tibial tuberosity ( Figure 4a). Matrigel (5 µL) (BD Bioscience, San Jose, CA) and PC-3-RFP cells (5 × 10 5 ) were co-injected into the intramedullary cavity of the tibia with a 1.0 mL 28 G latex-free insulin syringe (BD and Company, Franklin Lakes, NJ). The skin was closed with a 6-0 suture. One week after injection, fluorescence imaging was performed to confirm the RFP-expressing tumor was growing, using the iBOX Scientia (UVP LLC, Upland, CA). The study mice were randomly divided into the following groups: control group; ZOL group; S. typhimuium A1-R group; and ZOL+ S. typhimuium A1-R-group. In the ZOL group, mice were treated with subcutaneous injection of 120 mg/kg ZOL for 5 times a week for 4 weeks (Figure 4b). S. typhimuium A1-R-group mice were administered S. typhimurium A1-R (5 × 10 7 CFU, i.v.) once a week for 3 weeks. ZOL+ S. typhimuium A1-R-group mice were administered both treatments. Fluorescence imaging was performed on treated and untreated mice, and GFPexpressing areas were recorded every week for 5 weeks using the iBOX. At the end of the follow-up, the mice were sacrificed and the metastatic tumors were excised. Tumor weight was compared to evaluate the efficacy of ZOL and S. typhimuium A1-R.

Statistical analysis
Data showing comparisons among 3 or more groups were assessed using analysis of variance (ANOVA). The Kaplan-Meier method was used for bone metastasis-free survival and overall survival. Log-rank tests were used for statistical significance of the difference between the two groups. Differences were considered significant when p < 0.05. Data are expressed as mean ± SE. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).