Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice.

Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.


INTRODUCTION
Bone metastasis is found in more than 80% of patients in advanced stages of breast cancer [1] and is highly treatment resistant and results in extreme pain and high mortality. More effective mouse models and treatment are necessary.
S. typhimurium, which is a facultative anaerobe, was previously attenuated with purine and other auxotrophic mutations, for cancer therapy [2]. In a Phase I clinical trial on patients with metastatic melanoma and renal cell carcinoma, the S. typhimurium strain tested (VNP20009) was attenuated by lipid A-modified (msbB) and purine auxotrophic (purI) mutations. VNP20009 was safely administered to patients but colonized the patients' tumors to a limited extent, perhaps because it was over-attenuated [3].
Another strain of S. typhimurium, A1-R, has been developed by our laboratory which has increased antitumor efficacy. S. typhimurium A1-R is auxotrophic for Leu-Arg which prevents it from mounting a continuous infection in normal tissues, but does not inhibit tumor targeting and virulence. S. typhimurium A1-R has no other attenuating mutations as does VNP20009 and, therefore, may have higher tumor virulence. The ability to grow in viable tumor tissue may account, in part, for the unique antitumor efficacy of S. typhimurium strains [4].
S. typhimurium A1-R was able to eradicate primary and metastatic tumors in monotherapy in nude mouse models of prostate, breast, and pancreatic cancer, as well as sarcoma and glioma [4][5][6][7][8][9][10]. Tumors with a high degree of vascularity were more sensitive to S. typhimurium A1-R, and vascular destruction appears to play a role in S. typhimurium A1-R antitumor efficacy [11,12]. Tumor vessel destruction and tumor-growth inhibition was enhanced by primer dosing of S. typhimurium A1-R in immunocompetent transgenic mice expressing the nestindriven green fluorescent protein (ND-GFP), which is selectively expressed in nascent blood vessels [13].
S. typhimurium A1-R targeted the Lewis lung carcinoma (LLC) growing subcutaneously in nude mice [14] whereby the bacterially-infected cancer cells greatly expanded and burst and lost viability [15].
In the present study, we demonstrate that S. typhimurium A1-R can prevent human breast cancer bone metastasis using a metastatic variant in nude mouse models.

Efficacy of S. typhimurium A1-R on MDA-MB-435 cells in vitro
To determine the efficacy on breast cancer cells, MDA-MB-435 cells, were treated with S. typhimurium A1-R for 1 h. Fluorescence imaging demonstrated that S. typhimurium A1-R expressing GFP selectively invaded and replicated intracellularly in the MDA-MB-435 cells expressing RFP (Fig. 1a). Clonogenic assays demonstrated that S. typhimurium A1-R inhibited proliferation of MDA-MB-435-GFP cells in a dose-dependent manner (Fig. 1).  marrow and other organs.One, 2, 3, 7, 14, and 28 days after injection of S. typhimurium A1-R, the non-tumor-bearing mice were sacrificed and blood, spleen, and bone marrow were harvested and irrigated with PBS. PBS containing S. typhimurium A1-R from each tissue was cultured on LB agar. a) Collection of bone marrow. After the resection of the femur, two holes were made in both ends. After irrigation, a portion of the PBS was cultured on LB agar for 24 hours. b) Colony formation of GFP-expressing S. typhimurium A1-R obtained from blood, spleen, and bone marrow which were harvested 14 days after S. typhimurium A1-R treatment. S. typhimurium A1-R fluorescent colonies were imaged with the OV100 Small Animal Imaging System. www.impactjournals.com/oncotarget

S. typhimurium A1-R survives in bone marrow
After injection of S. typhimurium A1-R (5×10 7 CFU, i.v.) to non-tumor-bearing nude mice, blood, spleen, and bone marrow were cultured on Luria-Bertani (LB) agar (Fig. 2a). The presence of S. typhimurium A1-R was confirmed by GFP-expressing colony formation 24 hours after culture (Fig. 2b). These results showed that S. typhimurium A1-R survived for 2 weeks in bone marrow, compared to only 3 days in spleen. Blood had no colony formation on LB agar.

Efficacy of S. typmimurium A1-R on breast cancer cell growth in the tibia
MDA-MB-435-GFP-BM4 cells (5×10 5 ) were injected into the intramedullary cavity of the tibia in nude mice (n=12) (Fig. 5a). Two weeks after intratibial injection of cancer cells, six mice were treated three times with weekly i.v. S. typhimurium A1-R (Fig. 5b).   Matrigel (BD Bioscience, San Jose, CA) were injected in the intramedullary cavity of the tibia. b) Study protocol. Two weeks after intratibial injection, fluorescence imaging was performed to confirm the growing GFP-expressing tumor using the iBOX Scientia Small Animal Imaging System (UVP LLC, Upland, CA, USA). Mice (n=5) (treatment group) were administered S. typhimurium A1-R (5×10 7 CFU, i.v.) once a week for 3 weeks. The remaining mice (untreated control group, n=5) were administered the same volume of PBS. Fluorescence imaging was performed on treated and untreated mice. GFP fluorescent areas were recorded every 2 weeks for 12 weeks using the iBOX. c) Time-course imaging of the GFP-expressing bone tumors in S. typhimurium A1-R therapy treated mice and untreated mice. d) Fluorescence area of bone tumors in untreated control and S. typhimurium treated mice. Data are expressed as the mean ± SE. Differences between groups were analyzed with the Student's t-test. **p < 0.01 compared with the untreated control group.
Fluorescence imaging demonstrated that the control mice had rapid tumor growth of the tibial tumor. In contrast, the S. typhimurium A1-R-treated mice had slow growth (Fig. 5c). The fluorescent tumors area of the untreated control mice and S. typhimurium A1-R-treated mice 10 weeks after bacterial therapy was 196.6 ± 22.1 mm 2 and 61.8 ± 15.0 mm 2 , respectively (P < 0.001) (Fig. 5d). The bone marrow was observed with an FV1000 confocal microscope to confirm the presence of GFP-expressing cancer cells.
The present study demonstrates that S. typhimuium A1-R has and could significantly inhibit or prevent bone metastasis. These results indicate a promising approach to a currently highly treatment-resistant disease.

Preparation of S. typhimurium A1-R
GFP-expressing Salmonella typhimurium A1-R bacteria (AntiCancer Inc., San Diego, CA, USA) were grown overnight on LB medium (Fisher Sci., Hanover Park, IL, USA) and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then diluted in PBS [4,5].

S. typhimurium A1-R killing of breast cancer cells in vitro
MDA-MB-435-GFP or MDA-MB-435-RFP cells were planted in 35 mm dishes (2×10 3 ). S. typhimurium A1-R-GFP was grown in LB medium and added to the cancer cells (1×10 8 or 1×10 9 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 MDA-MB-435-RFP cells by S. typhimurium A1-R-GFP was visualized with the FV1000 confocal microscope. Eight days after treatment with S. typhimurium A1-R, MDA-MB-435-GFP colonies were fixed in methanol and stained with 1% crystal violet as previously described [26]. ImageJ (National Institute of Mental Health, Bethesda, Maryland, USA) was used to quantify the colonies of the cells.

Distribution of S. typhimurium A1-R in blood, spleen, and bone marrow tissue
Tumor-free nude mice were injected with S. typhimurium A1-R (5×10 7 CFU, i.v.). On days 1, 2, 3, 7, 14, and 28 after S. typhimurium A1-R GFP injection, femurs were taken from 3 mice and irrigated with PBS. Spleens were minced and mixed with PBS, as was blood. The PBS, including S. typhimurium A1-R from each tissue, was plated on LB agar containing 50 µg/mL ampicillin to identify S. typhimurium A1-R in each tissue. Fluorescent S. typhimurium A1-R colonies were observed with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan).

In vivo cycle selection of highly metastatic breast cancer cells
MDA-MB-435-GFP cells were harvested from subconfluent cell culture plates, washed with PBS, and resuspended in PBS. Cells (2×10 5 ) were injected into the left cardiac ventricle of female nude mice using a 27G needle. Mice were anesthetized with a ketamine mixture (10 µl ketamine, HCL, 7,6 µl xylazine, 2.4 µl acepromazine maleate and 10 µl H 2 O) before injection. A successful injection was characterized by the pumping of arterial blood into the syringe. Development of bone metastases was initially monitored with an Illumatool imaging system (Lightools Research, Encinitas, CA, USA). To isolate cancer cells from the bone metastasis, mice were sacrificed, and the affected bones were excised. Both ends of the bones were cut open. A one ml syringe with a 27G needle was filled with PBS and inserted into one end of the bone. Mouse bone marrow cells as well as cancer cells were forced out from the other end by applying pressure to the syringe. Cells were collected by centrifugation and washed once with PBS before being cultured at 37°C. After two weeks of culture, a pure population of human cancer cells was obtained as confirmed by fluorescence imaging. After 4 cycles of this procedure, we obtained a highly metastatic cells line,

Efficacy of S. typhimurium A1-R therapy on early-stage experimental bone metastasis
MDA-MB-435-GFP-BM4 cells (2×10 5 ) were injected intracardially in nude mice. One week after injection, mice (n=5) (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 every week. Metastatic bone lesions were also imaged every two weeks using the iBOX Scientia Imaging System (UVP LLC, Upland, CA, USA). Metastasis-free survival was defined as the time from cardiac injection of cancer cells to the time of detection of bone metastases with the Illumatool. At the end of the follow-up, the metastases were excised and the bone marrow was washed with PBS to confirm the presence of GFP-expressing cancer cells.

Efficacy of S. typhimurium therapy on breast cancer cell growth in the tibia
A midline skin incision (5 mm) was made just below the knee joint to expose the tibial tuberosity (Fig.  6). Matrigel (5 µL) (BD Bioscience, San Jose, CA) and MDA-MB-435-GFP-BM4 cells (5×10 5 ) were co-injected into the intramedullary cavity of the tibia with a 0.5 mL 28 G latex-free insulin syringe (0.5 mL 28 G) (TYCO Health Group LP, Mansfield, MA). The skin was closed with a 6-0 suture. Two weeks after injection, fluorescence imaging was performed to confirm the GFP-expressing tumor was growing, using the iBOX. Nude mice (n=5) were administered S. typhimurium A1-R (5×10 7 CFU, i.v.) once a week for 3 weeks. The remaining mice (untreated control group) were administered the same volume of PBS. Fluorescence imaging was performed on treated and untreated mice, and GFP-expressing areas were recorded every 2 weeks for 12 weeks using the iBOX.

Statistical analysis
Data showing comparisons between two groups were assessed using the Student's t-test. Comparisons among more than two groups were assessed using analysis of variance (ANOVA). The Kaplan-Meier method was used for bone metastasis-free survival and log-rank test was used for statistical significance of the difference between the two groups. Differences were considered significant when p < 0.05. Data are expressed as mean ± SEM. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).

DEDICATION
This paper is dedicated to the memory of A. R. Moossa, MD.