Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice.

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R.


INTRODUCTION
For more than 200 years, there have been reports that cancer patients went into remission after recovering from bacterial infections [1]. From the late 19th to early 20th century, William Coley, an American physician, treated cancer patients with both live and heat-killed bacteria such as Streptococcus pyogenes and Serratia marcescens as firstline therapy for sarcoma and lymphoma. The combination of Coley's heat-killed bacteria was referred to as "Coley's toxins, which remained in clinical use for sarcoma patients until 1963 [1]. Since 1976, BCG (Bacillus Calmette-Guerin) www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 33), pp: 54616-54628

Research Paper
Oncotarget 54617 www.impactjournals.com/oncotarget bacteria have been used to treat superficial bladder cancer [2], which is still in clinical use [3].
There has been a great resurgence in preclinical research on bacterial therapy of cancer in the last 20 years [4]. Salmonella species are the most extensively studied bacteria in the field of tumor targeting. They are Gramnegative facultative-anaerobic bacteria that can grow and replicate inside host cells. Some Salmonella strains can preferentially colonize solid tumors [5][6][7][8].
Salmonella typhimurium VNP20009 (VNP20009) is attenuated with a lipid A-mutation (msbB), purine auxotrophy (purI) and amino acid autotrophy [9]. In a Phase I clinical trial on patients with metastatic melanoma and renal-cell carcinoma in the United States, VNP20009 was not toxic but poorly colonized the patients' tumors, perhaps because it was over attenuated [10]. At the highest tolerated dose, some tumor colonization was observed [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 [16].
In the present report, we compare efficacy, tumor targeting and toxicity of S. typhimurium A1-R and VNP20009, since VNP20009 has been tested to be safe in a Phase I clinical trial [10].

Correlation between optical sensitivity and colony counting to quantitate bacteria
In order to determine viable bacterial counts, we analyzed the relationship between the OD 600 value and bacteria colony forming units (CFU) on LB agar. A linear relationship between OD 600 and CFU was observed for S. typhimurium A1-R: Y = 20.141x + 2.0578, R 2 = 0.9473) ( Figure 1).

Effect of a single-dose S. typhimurium A1-R on body weight of non-tumor-bearing BALB/c mice
Six-to 8-week-old BALB/c mice without tumors, with an average body weight 20 to 22 gram, were used in the study. A single-dose of S. typhimurium A1-R at 1 × 10 5 -5 × 10 5 CFU/mouse was administred i.v. Acute weight loss of 10% to 20% from day 0 was observed in all cohorts. However, body weight began recover by day 6. However, 1 × 10 6 CFU/mouse did cause severe weight loss ( Figure  2A, 2B). No signs of delayed toxicity were observed. S. typhimurium A1-R doses at 1 × 10 5 and 5 × 10 5 CFU did not cause mouse death, but 1 × 10 6 CFU did ( Figure 2C, 2D).
Effect of a single dose of S. typhimurium A1-R on body weight of tumor-bearing BALB/C mice Single doses of 1 × 10 5 , 5 × 10 5 and 1 × 10 6 were given to BALB/c mice bearing subcutaneous CT26 murine colon tumors. The body weight loss in the tumorbearing mice was less than in the normal mice. The tumorbearing mice which were given the 1 × 10 6 CFU dose of S. typhimurium A1-R rapidly recovered unlike the nontumor bearing mice ( Figure 3A, 3B). However, although no deaths were observed at the two lower doses, the 1 × 10 6 CFU dose caused death ( Figure 3C, 3D).

Effect of multiple doses of S. typhimurium A1-R on the body weight of tumor-bearing mice
Based on the single-dose toxicologic results, we also assessed the toxicity of multiple doses of S. typhimurium A1-R compared with PBS administered to tumor-bearing mice ( Figure 4). Weekly doses of S. typhimurium A1-R at 2.5, or 5 × 10 5 CFU/mouse, were administered for 3 weeks. The 2.5 × 10 5 CFU weekly doses did not cause noticeable weight loss. The 5 × 10 5 CFU/mouse caused a body weight loss by day-4 which was recovered by day-11. Weekly doses of VNP20009 at 5 × 10 5 or 1 × 10 6 CFU/ mouse were administered for 3 weeks without noticeable weight loss.

Clearance of S. typhimurium A1-R from the circulation
To quantitatively assess the clearance of bacteria from the circulation, GFP-labeled S. typhimurium A1-R (S. typhimurium A1-R-GFP) was intravenously injected into non-tumor-bearing and tumor-bearing BALB/c mice. S. typhimurium A1-R-GFP in the blood was collected, cultured, and counted at various times after injection using three mice per time point. Greater than 95% of the bacteria were cleared from the circulation within 1 day in non-tumor-bearing mice, and the number of CFU fell to barely detectable levels over the next 7 days ( Figure 5). Bacteria in tumor-bearing mice were cleared more slowly ( Figure 5).

Biodistribution of bacteria
To determine the distribution of bacteria, normal and tumor-bearing BALB/c mice were injected with S. typhimurium A1-R GFP. Organs and tumors were Oncotarget 54618 www.impactjournals.com/oncotarget    Oncotarget 54620 www.impactjournals.com/oncotarget collected, homogenized and S. typhimurium A1-R was cultured on LB agar. The number of CFU was counted from organs starting 1 hour after injection. Bacteria colonies from organs and tumors were subsequently observed by fluorescence microscopy. In non-tumorbearing mice, significant S. typhimurium A1-R-GFP colonies from organs were present at 1 h after injection, but were undetectable 1week later. The largest amount of S. typhimurium A1-R-GFP was present in the liver and spleen, but was cleared by 7 days. The other organs were cleared by 3 days (Figure 6A).
In the tumor-bearing mice, S. typhimurium A1-R-GFP in tumors survived much longer than in the normal organs ( Figure 6B).
S. typhimurium A1-R-GFP and VNP20009-RFP were isolated from the viable and necrotic tumor regions and cultured in LB agar. S. typhimurium A1-R survived in tumor necrotic regions more than VNP20009 at day-7 when the bacteria were administered i.v. (p<0.01) ( Figure  7). FP Fluorescent colonies of S. typhimurium A1-R-GFP isolated from the tumor, spleen and liver at various time points were imaged.
Fluorescent colonies are seen derived from the tumor at all time points from 1 hour to day 7. Fluorescent colonies can be seen isolated from the liver and spleen only at 1 hours and day 1 and not at day 3 and day 7 (Figure 8).

Effect of S. typhimurium A1-R on organ weight and histology
Each mouse received a single intravenous injection of S. typhimurium A1-R (5 × 10 5 CFU). In non-tumorbearing mice, only the spleen significantly increased in weight after treatment with S. typhimurium A1-R (5 × 10 5 CFU) beginning at day 3 ( Figure 9A).
In female BALB/c mice bearing subcutaneous CT26 mouse colon tumors, S. typhimurium A1-R (5 × 10 5 CFU) treatment also caused the spleen to gain significant weight beginning at day 7, compared to control mice ( Figure 9B).
Histopathologic analysis of liver and spleen at day 3 post-treatment of S. typhimurium A1-R or VNP20009 showed pathological changes in both organs ( Figure 9C).
Previously-developed concepts and strategies of highly-selective tumor targeting can take advantage of molecular targeting of tumors, including tissue-selective therapy which focuses on unique differences between normal and tumor tissues [33][34][35][36][37][38].

Cell lines and other reagents
Murine CT26 colon cancer cells were obtained from ATCC (The American Type Culture Collection) and grown in RPMI 1640 Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan City, UT) at 37°C with 5% CO 2 .

Mice
Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4-6 weeks old, were used in this study. Animals were housed in a barrier facility on a high efficacy particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet. All mouse surgical procedures and imaging were performed with the animals anesthetized by subcutaneous injection of a ketamine mixture (0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate). The response of animals during surgery was monitored to ensure adequate depth of anesthesia. The animals were observed on a daily basis and humanely sacrificed by CO 2 inhalation if they met the following humane endpoint criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant body weight loss, difficulty breathing, rotational motion and body temperature drop. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under Assurance Number A3873-1.

Comparison of OD 600 and colony counting to quantify bacteria
GFP-expressing S. typhimurium A1-R were grown overnight in LB medium and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then serial diluted 10-fold for six times in PBS. OD 600 measurements were performed on a spectrophotometer. Dilutions of bacteria were plated on LB agar and incubated at 37°C overnight. Colonies were counted under fluorescence microscopy to determine colonr forming units (CFU/ml) at each time point.

Tumor inoculation
Murine CT26 colon cancer cells (2×10 6 ) were injected subcutaneously into the right flank of BALB/c mice. Tumor volume was calculated as length × width 2 × 0.5. In general, 10-14 days were required for tumors to reach the target size of 100 mm 3 for treatment initation.

Dose-tolerance experiments
The dose-tolerance relationship was examined in BALB/c mice for a single bolus or multiple doses once weekly for 3 consecutive weeks of S. typhimurium A1-R or VNP20009. Animal weights were compared with day 0 (first day of treatment administration) to determine percentage weight change. Mice were sacrificed at either 25% weight loss from day 0 or at the end of the observation period, which ever came first.
After administration of S. typhimurium A1-R or VNP20009 or control treatments, mice were closely monitored and weighed daily for 8 days, then at day 10 and 12, and twice weekly until the end of the observation period. At this stage, the animals were euthanized by CO 2 inhalation.

Biodistribution of S. typhimurium A1-R-GFP
At various time points after injection of S. typhimurium A1-R, mice were sacrificed by CO 2 narcosis at 1 h, 1 day, 3 days, 7 days, 14 days and 28 days postinjection, and their blood (0.1 ml), brain, heart, lungs, liver, spleen, kidneys, muscle, bone, small and large intestines, and tumor were harvested. Tissues were removed, weighed, and homogenized in phosphate buffered saline (PBS), at 2 ml per 200 mg of tissue using an homogenizer (Fisher Scientific, USA) at a speed of ~24,000 rpm for 30-60 s. The resulting suspension was serial diluted by 10× dilutions in PBS and carefully spread onto LB agar. Blood was obtained from intracardiac puncture and mixed with 9 volumes of PBS, after which a 100 μl suspension was spread on LB agar plates. LB agar plates were incubated for 18 h at 37°C. Following incubation, plates were removed from the incubator and colonies were counted. At least three mice were used for each time point. S. typhimurium A1-R-GFP colonies were visualized with an Olympus OV100 Small Imaging System with a CCD camera [39].

Histological analysis
Fresh tumor and organ samples were fixed in 10% formalin and embedded in paraffin before sectioning and staining. Tissue sections (5 μm) were deparaffinized in xylene and rehydrated in an ethanol series. Hematoxylin and eosin (H &E) staining was performed according to standard protocols. Histological examination was performed with a BHS System Microscope (Olympus Corporation, Tokyo, Oncotarget 54626 www.impactjournals.com/oncotarget Japan). Images were acquired with INFINITY ANALYZE software (Lumenera Corporation, Ottawa, Canada) [28].

Anti-tumor efficacy
BALB/c mice bearing CT26 murine colon carcinoma xenografts were administered S. typhimurium A1-R or VNP20009 (i.v) with weekly doses indicated in Figure 10. Mice were monitored and weighed as described in the toxicity studies. Tumors were measured twice weekly using calipers along the x and y plane of the tumor. Tumor volume was determined using the formula volume = a x (b 2 )/2, where a is the largest diameter and b is the smallest diameter. Antitumor assessment of treatments was determined by reduction in tumor growth rate compared with controls.

Statistical analysis
All statistical analyses were performed using SYSTAT 12.0 (SYSTAT, Inc., Chicago, IL, USA). The experimental data are expressed as the mean ± SD. The two-tailed Student's t-test was used for statistical analysis, with α equal to 0.05. P<0.05 was considered statistically significant.

CONCLUSION
S. typhimurium A1-R has a similar toxicity pattern as VNP20009 which was previously determined to be safe in a Phase I clinical trial. However, S. typhimurium A1-R had significantly greater tumor targeting and anti-tumor efficacy than VNP20009. The results of the present study, therefore, indicate the promising clinical potential of S. typhimurium A1-R.
Bacterial therapy offers many advantages over conventional chemotherapy, including, but not limited to tumor-targeting, ability to deliver therapeutic cargoes, does not depend entirely on the tumor vascular system to reach the tumor since bacteria are motile, can stimulate the immune system against the tumor, can serve as adjuvant treatment after surgery to eliminate residual disease and can enhance chemotherapy by decoying quiescent drugresistant cancer cells in tumors to attempt to cycle, thereby becoming sensitive to chemotherapy [4].
With regard to bacterial labeling, small molecular fluorescent probes or radionuclides may offer advantages over GFP to label S. typhimurium A1-R regarding translation to the clinic [40,41]. Whether S. typhimurium A1-R can pass the blood-brain barrier is still an open question. Although, S. typhimurium A1-R was found at early time-points in the brain in the present study, it is possible that bacteria were actually from brain blood vessels in which the bacteria have not passed to the brain tissue itself. On the other hand, S. typhimurium A1-R prevented brain metastasis in a breast cancer mouse model [14]. It is not clear if the bacteria had to cross the bloodbrain barrier for this anti-brain-metastasis efficacy. Future experiments will address this point.

DEDICATION
This paper is dedicated to the memory of A. R. Moossa, M.D., and Sun Lee, M.D.