Adjuvant treatment with tumor-targeting Salmonella typhimurium A1-R reduces recurrence and increases survival after liver metastasis resection in an orthotopic nude mouse model.

Colon cancer liver metastasis is often the lethal aspect of this disease. Well-isolated metastases are candidates for surgical resection, but recurrence is common. Better adjuvant treatment is therefore needed to reduce or prevent recurrence. In the present study, HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used to establish liver metastases in nude mice. Mice with a single liver metastasis were randomized into bright-light surgery (BLS) or the combination of BLS and adjuvant treatment with tumor-targeting S. typhimurium A1-R. Residual tumor fluorescence after BLS was clearly visualized at high magnification by fluorescence imaging. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase survival and disease-free survival after BLS of liver metastasis. The results suggest the future clinical potential of adjuvant S. typhimurium A1-R treatment after liver metastasis resection.


INTRODUCTION
Colon cancer liver metastasis is often the lethal aspect of this disease [1]. Well-isolated metastases are candidates for surgical resection, but recurrence is common [2]. Better adjuvant treatment is therefore needed to reduce or prevent recurrence.
Bacterial therapy of cancer has a long history [3][4][5]. The bacteria, now known as Streptococcus pyogenes [3][4][5], was found in cancer patients who had remission and was used for therapy in the late 19 th and early 20 th centuries [3][4][5], especially under William B. Coley.
Salmonella typhimurium (S. typhimurium) is a facultative anaerobe, which in contrast to obligate anaerobes, allows growth in the viable regions as well as necrotic regions of tumors [20]. S. typhimurium-VNP20009, with msbB and purI mutations, was found safe in a Phase I clinical trial of metastatic melanoma and renal carcinoma [21].
The present report demonstrates adjuvant treatment efficacy of S. typhimurium A1-R after bright-light surgery (BLS) of liver metastasis.

BLS cannot resect the entire liver metastasis
Residual tumor fluorescence was detected on the surgical resection bed after BLS of HT-29-RFP liver metastasis ( Figure 1). There was no significant difference in residual tumor area between the control group (0.237 ± 0.094 mm 2 ) and BLS group (0.250 ± 0.120 mm 2 ).
Liver metastasis of colon cancer is the major lethal event of this disease. In many cases, diffuse liver metastasis is inoperable. However, isolated liver metastasis provides an opportunity for resection, but BLS very often results in residual cancer cells. The present report demonstrates that S. typhimurium A1-R can eradicate sufficient residual cancer cells after BLS to significantly increase disease-free survival and overall survival. Future experiments will also use S. typhimurium A1-R as neoadjuvant chemotherapy to convert inoperable tumors to those that are resectable.
In a recent study, we found that Salmonella typhimurium A1-R was active as monotherapy on liver metastasis in the orthotopic HT-29 mouse model. The results of that study demonstrated the potential of S. typhimurium A1-R targeting of liver metastasis [44].
The recurrence rate after liver metastasis resection is high, up to 75% [1,2,45]. Adjuvant therapy for colon cancer liver metastasis is usually based on 5-fluorouracil and oxaliplatin and it is not highly effective [1,2,45]. Therefore, novel approaches are necessary to improve adjuvant therapy of colon cancer liver metastasis.
The present study indicated that S. typhimuirum A1-R can be curative as adjuvant treatment for liver metastasis. Clinical trials of S. typhimurium A1-R for adjuvant therapy of patients with liver metastasis resection would have high potential.
Previously developed concepts and strategies of highly selective tumor targeting [46][47][48][49][50][51] can take advantage of bacterial targeting of tumors, including tissue-selective therapy which focuses on unique properties of normal and tumor tissues [46,51]. S. typhimurium A1-R can possibly overcome de-differentiation of a tumor leading to resistance to targeted chemotherapy, where the targeted protein or pathway may no longer be expressed [51], since S. typhimurium A1-R does not depend on Oncotarget 41858 www.impactjournals.com/oncotarget such targets [46,48]. S. typhimurium A1-R may also be effectively combined with teratogens which could selectively affect cancer cells that are dedifferentiated [47]. Since S. typhimurium A1-R can decoy quiescent cancer cells to begin to cycle, S. typhimurium A1-R could be effectively combined with agents which selectively target proliferating cancer cells [49], where normal cells are protected by agents which induce wild type p53 [50].

Mice
Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4-6 weeks old, were used in this study. All animal studies were conducted with an AntiCancer Institutional Animal Care and Use Committee (IACUC)protocol specifically approved for this study and in accordance with the principals and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. In order to minimize any suffering of the animals, anesthesia and analgesics were used for all surgical experiments. Animals were anesthetized by subcutaneous injection of a 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 when they met the following humane endpoint criteria: prostration, significant body weight loss, difficulty breathing, rotational motion and body temperature drop. The use of animals was necessary to evaluate S. typhimurium A1-R efficacy in vivo. Animals were housed with no more than 5 per cage. Animals were housed in a barrier facility on a high efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet.

Establishment of RFP-labeled HT29
The pDsRed-2 vector (Clontech Laboratories Inc., Palo Alto, CA) expressing RFP and the neomycinresistance gene was used to stably transfect HT-29 cells to express RFP. For RFP gene transfection, 25% confluent HT-29 cells were incubated with a mixture of retroviral Oncotarget 41859 www.impactjournals.com/oncotarget supernatants of PT67-RFP packaging cells and DMEM for 24 h. Fresh medium was replenished at this time, and cells were allowed to grow in the absence of retrovirus for 12 h. This procedure was repeated until high levels of RFP expression were achieved. Cells were then harvested with trypsin-EDTA and subcultured into selective medium that contained G418 (200 µg/ml) (Geneticin, Invitrogen Corp., Carlsbad, CA). The level of G418 was increased to 2,000 µg/ml stepwise. Clones expressing high levels of RFP were isolated and were amplified and transferred using conventional culture methods. High RFP-expression clones were isolated in the absence of G418 for 10 passages to select for stable expression of RFP [52,54,55].

Preparation of S. typhimurium A1-R
GFP-expressing S. typhimurium A1-R bacteria (AntiCancer Inc.) 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 [23].

Initial establishment of liver metastases
HT-29-RFP cells were harvested by trypsinization and washed twice with serum-free medium. Cells (5×10 5 in 50 μl serum-free medium with 50% Matrigel) were injected into the superior and inferior pole of the spleen in mice. Three weeks after injection, liver metastases were established.

Surgical orthotopic implantation of liver metastasis
Established liver metastases were resected and cut into blocks (3 mm 3 ). A single tumor fragment was orthotopically implanted into the left lobe of the liver in other nude mice. Four weeks later, liver metastasis was observed in the implanted site.

Efficacy for adjuvant S. typhimurium A1-R treatment on liver metastasis after resection
Four weeks after orthotopic implantation to the liver, the liver metastasis was resected along the tumor margin under bright light. Twelve mice treated with standard bright-light surgery (BLS) were randomized into 2 groups: a control group (n=6) and an S. typhimurium A1-R adjuvant group (n=6). S. typhimurium A1-R (5 × 10 7 CFU/body, iv, weekly, 3 weeks) was administered to the mice as adjuvant treatment beginning one week after BLS. Tumor fluorescence was visualized with the OV100 variable magnification small animal fluorescence imaging system (Olympus Corp., Tokyo, Japan) before and right after BLS. Recurrence was monitored by weekly noninvasive tumor fluorescence evaluation with the LT-9900 Illumatool (Lightools Research, Encinitas, CA, USA) until the end of the experiment.

Statistical analysis
SPSS statistics (version 21.0) was used for all statistical analyses (IBM, New York City, NY, USA). Survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test. A probability value of P < 0.05 was considered statistically significant.

ACKNOWLEDGEMENT
This study was supported by National Cancer Institute grant CA132971 and CA142669 and JSPS KAKENHI grant 26830081 to YH, 26462070 to IE and 24592009 to KT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Dedication
This paper is dedicated to the memory of A. R. Moossa, M.D.