Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Efficacy of Tumor-Targeting Salmonella A1-R on a Melanoma Patient-Derived Orthotopic Xenograft (PDOX) Nude-Mouse Model

  • Mako Yamamoto,

    Affiliations AntiCancer, Inc., Ostrow Street, San Diego, California, United States of America, Department of Surgery, University of California San Diego, West Arbor Drive, San Diego, California, United States of America

  • Ming Zhao,

    Affiliation AntiCancer, Inc., Ostrow Street, San Diego, California, United States of America

  • Yukihiko Hiroshima,

    Affiliations AntiCancer, Inc., Ostrow Street, San Diego, California, United States of America, Department of Surgery, University of California San Diego, West Arbor Drive, San Diego, California, United States of America

  • Yong Zhang,

    Affiliation AntiCancer, Inc., Ostrow Street, San Diego, California, United States of America

  • Elizabeth Shurell,

    Affiliation Division of Surgical Oncology, University of California Los Angeles, Los Angeles, California, United States of America

  • Fritz C. Eilber,

    Affiliation Division of Surgical Oncology, University of California Los Angeles, Los Angeles, California, United States of America

  • Michael Bouvet,

    Affiliation Department of Surgery, University of California San Diego, West Arbor Drive, San Diego, California, United States of America

  • Makoto Noda,

    Affiliation Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan

  • Robert M. Hoffman

    all@anticancer.com

    Affiliations AntiCancer, Inc., Ostrow Street, San Diego, California, United States of America, Department of Surgery, University of California San Diego, West Arbor Drive, San Diego, California, United States of America

Efficacy of Tumor-Targeting Salmonella A1-R on a Melanoma Patient-Derived Orthotopic Xenograft (PDOX) Nude-Mouse Model

  • Mako Yamamoto, 
  • Ming Zhao, 
  • Yukihiko Hiroshima, 
  • Yong Zhang, 
  • Elizabeth Shurell, 
  • Fritz C. Eilber, 
  • Michael Bouvet, 
  • Makoto Noda, 
  • Robert M. Hoffman
PLOS
x

Abstract

Tumor-targeting Salmonella enterica serovar Typhimurium A1-R (Salmonella A1-R) had strong efficacy on a melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse model. GFP-expressing Salmonella A1-R highly and selectively colonized the PDOX melanoma and significantly suppressed tumor growth (p = 0.021). The combination of Salmonella A1-R and cisplatinum (CDDP), both at low-dose, also significantly suppressed the growth of the melanoma PDOX (P = 0.001). Salmonella A1-R has future clinical potential for combination chemotherapy with CDDP of melanoma, a highly-recalcitrant cancer.

Introduction

Melanoma is a recalcitrant cancer. When melanoma metastasizes to regional lymph nodes, the 5-year survival rate is 29% and when it metastasizes to organs, the survival rate is 7% [1]. Although recently-developed immunotherapy has extended survival to some extent, the 5-year survival rates have not been significantly increased [2]. Decarbazine and cisplatinum have been used to treat melanoma with limited efficacy [3, 4]. Therefore, more effective approaches to melanoma treatment are needed.

Immunotherapy involving PD-1/PD-L1 blockade has had some success with melanoma but is limited by lack of sufficient tumor infilation of activated lymphocytes to kill the cancer cells within the tumor in the majority of patients tested [5].

Tumor-targeting Salmonella A1-R developed by our laboratory is auxotrophic (leucine-arginine dependent) which prevents it from continuously infecting normal tissues. Monotherapy using Salmonella A1-R was able to regress or eliminate primary and/or metastatic tumors in models of mouse highly aggressive of prostate [6, 7], breast [810], lung [11, 12], pancreatic [1317], ovarian [18, 19], stomach [20], and cervical cancer [21], as well as sarcoma [2226] and glioma [27, 28].

Patient-derived orthotopic xenograft (PDOX) models were developed by our laboratory [29, 30]. In the PDOX models, the patient’s tumor is transplanted in the organ of nude or other immunocompetent mice corresponding to its origin and thereby metastasizes such that the tumor mimics the complexity of tumor behavior in patients. Our laboratory has developed PDOX models of all major tumor types including colon [29], pancreatic [31], breast [32], ovarian [33], lung [34], cervical [21], stomach cancer [35], as well as mesothelioma [36] and sarcoma [25, 26, 37, 38].

The aim of the current study was to determine the efficacy of Salmonella A1-R on a PDOX model of melanoma compared to and in combination with standard chemotherapy.

Materials and Methods

Animal Experiments and Ethics Statement

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 principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. 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-efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet (S1 File). In order to minimize any suffering of the animals, anesthesia and analgesics were treated for all surgical procedures. Animals were anesthetized by subcutaneous (s.c.) 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. Ibuprofen (7.5 mg/kg orally in drinking water every 24 hours for 3 days post-surgery) was used in order to provide analgesia post-operatively in the surgically-treated animals. The animals were carefully observed on a daily basis and would be humanely sacrificed by CO2 inhalation if they met the following humane endpoint criteria: prostration, skin lesions, significant bodyweight loss, difficulty breathing, epistaxis, and rotational motion. Individual cages housed animals only in the same treatment group with no more than five mice per cage.

Melanoma Specimen Collection

The patient had given written informed consent for experimental research on residual tumor tissue available after histopathologic and cytogenetic analyses. The written informed consent document was recorded in a special binder for such documents. The consent procedure was approved by the Institutional Review Board of the UC San Diego Medical Center. This study was also conducted under the approval of the UCSD IRB.

Patient-Derived Orthotopic Xenograft (PDOX) Melanoma Model

Tumor tissue was obtained from a patient at the time of surgery at the UCSD Medical Center. The harvested tumor was cut into fragments (3-mm3) and transplanted into the back skin of five nude mice with two mice transplanted with two tumors. Animals were sacrificed at the end of the experiment. Tumors were harvested and fragments were transplanted to one or two sides of the back skin for the next passage and/or for analysis.

Histology

Four tumors were harvested from four mice. Harvested tumor samples were fixed with 10% formalin solution, embedded into paraffin and sectioned. Hematoxylin and eosin (H&E) staining was performed with standard protocol. For immunohistochemistry, paraffin-embedded tumor sections were stained with a rabbit anti-human MHC class I antibody (1:100; ab52922, Abcam, Cambridge, MA) and a mouse anti-MHC class I H2 Kd + H2 Dd antibody (1:100; ab24229, Abcam). Immunohistochemistry was performed using anti-rabbit and anti-mouse secondary antibodies and avidin/biotin/horseradish peroxidase complex (Dako Denmark A/S, Glostrup, Denmark) and developed with the DAB kit (BD Biosciences, San Diego, CA) [16]. Five microscopic fields were inspected for each tumor.

Salmonella A1-R and Chemotherapy Drugs

Green fluorescent protein (GFP)-expressing Salmonella A1-R bacteria were grown overnight in LB medium and then diluted in 1:10 with LB medium. Bacteria were harvested at late-log phase, washed with phosphate buffered saline (PBS), and diluted in PBS [68]. Two weeks after transplantation, bacteria and/or chemotherapy were started. 5-fluorouracil (5-FU, Kyowa Hakko Kirin, Co., Tokyo, Japan) and cisplatinum (CDDP, Nippon Kayaku, Co., Tokyo, Japan) solutions were administered via intraperitoneal (i.p.) injection at a dose of 10 mg/kg (5-FU) and 3 or 5 mg/kg (CDDP) once a week. Salmonella A1-R was injected intravenously (i.v.) at a dose of 3 or 5 × 107 CFU/body once a week. The approximate volume of the mass was measured with a caliper twice a week and calculated with the following formula: Tumor volume = 4/3π × (d/2)2 × D/2, where d is the minor tumor axis and D is the major tumor axis. Treatment efficacy was indicated as a ratio of the tumor volume at each time point compared with the tumor volume at the beginning of the treatment. Body weight of the mice was measured on a balance twice a week.

Detection of GFP-Labeled Salmonella A1-R Bacteria in Tumor and Organs

Two days before harvesting tumors and normal organs, mice were injected intravenously with Salmonella A1-R (5 × 107 CFU/body). Two days later, PDOX tissues from melanoma tumor and normal organs (liver, spleen and blood) were harvested and weighed. The tumor and organs were minced and diluted in 1:1, 1:10 and 1:100 with 100 μl PBS, respectively. Each dilution (10 μl) was spotted on an LB agar plate containing 50 μg/mL ampicillin and the plates were incubated at 37°C for 24 hours. Relative colony numbers are calculated by actual colony number divided by mg of tissue. One tumor and normal organs from one mouse were harvested and the experiment was repeated three times.

Imaging

The iBox Imaging System (UVP LLC, Upland, CA) was used for imaging GFP-labeled S. typhimurium A1-R [3942]. The BHS System Microscope (Olympus) was used for H&E staining and immunohistochemistry.

Statistical Analysis

STATA 12.0 SE was used for further data analysis. Time points were chosen a priori: 28 days was selected as a final end point, and 10 days was chosen as a midway representative time point. However, data were collected from 9 time points throughout the 28 days, at regular intervals. A repeated-measures regression model was used to assess the tumor volume of the animals over these regular intervals. The within-subject covariance structure of the data was compound symmetric, therefore we proceeded with repeated measure ANOVA including the repeated option to compute p-values for conservative F-tests. Greenhouse-Geisser correction was performed to correct for any violations of sphericity. The treatment-by-time interaction was significant (p = 0.0000) as were the main-effects for treatment and time (p = 0.0000) for Salmonella A1-R, CDDP and Salmonella A1-R and CDDP combined. The specific comparisons of interest remained significant within this model. Graph of adjusted predictions of interaction of treatment group and time with 95%CI and graph of mean change in tumor volume over time.

Results

Patient-Derived Melanoma Growing Orthotopically in Nude Mice

Four weeks after transplantation of the patient melanoma, solid tumors were found growing in the back skin of the nude mice. The tumors were harvested from the mice and used for the next passage and histological analysis (Fig 1A).

thumbnail
Fig 1. Establishment of a melanoma patient-derived orthotopic xenograft (PDOX) model.

A) Schematic diagram of the experimental protocol. B) Representative cross-sections of transplanted tumor 28 days after transplantation obtained from an orthotopically-transplanted patient’s melanoma. Scale bar: 10 mm. C) Immunohistochemical characterization of PDOX melanoma after being grown in nude mice. H&E-stained sections (left column) and immunohistochemistry for human MHC class I (HLA; middle column) and mouse MHC class I (H2 KdtH2 Dd; right column). Strong staining for HLA was observed in the cancer cells (middle column), whereas strong staining for H2 KdtH2 Dd was observed in the stromal cells (right column). Magnified views of boxed region in the upper rows are indicated at the middle rows and magnified views of boxed region in the middle rows are indicated in the lower rows. Black arrowhead indicates necrotic region of the tumor. Scale bars: (top and middle row) 200 μm; (bottom row) 100 μm.

https://doi.org/10.1371/journal.pone.0160882.g001

To determine whether the grown tumor is completely derived from melanoma patients’ specimen, immunohistochemistry analysis was performed. The melanoma PDOX strongly expressed human MHC class I protein (Fig 1C), whereas cells around blood vessels or stromal cells only reacted with mouse MHC class I antibody (Fig 1C). These data indicate that the growing PDOX tumor was human.

Salmonella A1-R is Highly Effective on the PDOX Melanoma in Nude Mice

Salmonella A1-R was administrated intravenously to the melanoma PDOX two weeks after transplantation at a dose of 5 × 107 CFU/body, qW×4. The relative tumor volume on day-28, compared to day-0, of untreated control was 8.46 ± 1.95 and in the Salmonella A1-R-treated mice, the tumor volume ratio was 1.68 ± 0.37 (p = 0.021) (Fig 2B). There were five mice in each group and the experiment was repeated twice.

thumbnail
Fig 2. Salmonella A1-R targeting and efficacy on the melanoma PDOX model.

A) Schematic diagram of the experimental protocol. B) Efficacy of Salmonella A1-R is indicated by the volume ratio of the transplanted tumor at day 28 after injection compared with the tumor at the beginning of the treatment. Tumor size of the Salmonella A1-R-treated group was significantly decreased compared with the untreated control group. The values are mean relative tumor volume ± SEM (bars). There were five mice per group. *p < 0.05 compared to the untreated group. C) Distribution of GFP-labeled Salmonella A1-R in tumor and organs. Representative images of GFP-labeled Salmonella A1-R bacteria isolated and cultured from the tumor and the normal organs (blood, liver and spleen) of the mice treated with Salmonella A1-R. Fluorescence imaging with the iBox small animal imaging system (UVP LLC). Scale bar: 10 mm. D) Colony number of each sample is indicated per mg of harvested tissue. Tissues were collected from three different mice. GFP-labeled Salmonella A1-R was clearly detected in the tumor. A small number of GFP-labeled Salmonella A1-R was detected in the liver and no GFP-labeled Salmonella A1-R was detected in blood and spleen.

https://doi.org/10.1371/journal.pone.0160882.g002

Extensive GFP-labeled Salmonella A1-R could be isolated from the tumor and could not be isolated from the blood and spleen and only very small amounts could be isolated from the liver (Fig 2C and 2D). These results indicated that Salmonella A1-R selectively and effectively colonized and targeted the tumor.

Efficacy of Salmonella A1-R and Chemotherapy on the PDOX Melanoma

Two weeks after tumor transplantation, mice were treated with the following groups: (1) untreated control (Control); (2) 5-fluorouracil (5-FU; 10 mg/kg, i.p., qW×4); (3) cisplatinum (CDDP; 5 mg/kg, i.p., qW×4); (4) Salmonella A1-R (5 × 107 CFU/body, i.v., qW×4) and (5) Salmonella A1-R (3 × 107 CFU/body, i.v., qW×4) + CDDP (CDDP, 3 mg/kg, i.p., qW×4) (Fig 3A).

thumbnail
Fig 3. Effect of a tumor-targeting Salmonella A1-R and chemotherapy on the melanoma PDOX.

A) Schematic diagram of the experimental protocol. (1) untreated control (Control); (2) 5-fluorouracil (5-FU; 10 mg/kg, intraperitoneal injection (i.p.), qW×4); (3) cisplatinum (CDDP; 5 mg/kg, i.p., qW×4); (4) Salmonella A1-R (5 × 107 CFU/body, intravenously (i.v.), qW×4) and (5) Salmonella A1-R (3 × 107 CFU/body, i.v., qW×4) + CDDP (CDDP; 3 mg/kg, i.p., qW×4). B) Growth curves of the melanoma PDOX tumor treated with various drugs as described above. B1: Mean change in tumor volume plotted against time; Control, n = 9; 5-FU, n = 4; CDDP, n = 5; Salmonella A1-R, n = 9; Salmonella A1-R + CDDP, n = 8. B-2: Data plotted are linear prediction versus time with adjusted predictions of interaction of treatment group and time with 95% Cis. The treatment-by-time interaction was significant (p = 0.0000) as were the main-effects for treatment and time (p = 0.0000) for Salmonella A1-R, CDDP and Salmonella A1-R and CDDP combined. C) Comparison of body weight of nude mice transplanted PDOX tumors after Salmonella A1-R and/or chemotherapy. All values represent mean ± SEM; Control, n = 8; 5-FU, n = 4; CDDP, n = 4; Salmonella A1-R, n = 7; Salmonella A1-R + CDDP, n = 4. **p < 0.01, compared with the untreated control group.

https://doi.org/10.1371/journal.pone.0160882.g003

The relative tumor volume on day 28, compared with day 0, of each group was as follows: (1) untreated control: 9.63 ± 1.37; (2) 5-FU: 6.86 ± 0.52; (3) CDDP: 2.25 + 0.32 (p = 0.0001); (4) Salmonella A1-R: 2.29 ± 0.35 (p = 0.0001); (5) Salmonella A1-R + CDDP: 2.90 ± 0.47. The treatment-by-time interaction was significant (p = 0.0000) as were the main-effects for treatment and time (p = 0.0000) for Salmonella A1-R, CDDP and Salmonella A1-R and CDDP combined. These data suggest that Salmonella A1-R and/or CDDP treatment is highly effective in the melanoma PDOX and the efficacy appears from the very early period of the treatment (Fig 3). Regarding the choice of chemotherapy drugs, CDDP was used as a positive control and 5-FU as a negative control in addition to the untreated control, since 5-FU is known not to be effective against melanoma [43].

The relative body weight on day 28 compared with day 0, of each group was as follows: (1) untreated control: 1.13 ± 0.012; (2) 5-FU: 1.12 ± 0.016; (3) CDDP: 1.02 ± 0.033; (4) Salmonella A1-R: 1.07 ± 0.032 and (5) Salmonella A1-R + CDDP: 1.07 ± 0.022. Only the body weight of CDDP-treated mice was significantly decreased compared with untreated control (p = 0.0001) at the end of this experiment (Fig 3C).

Discussion

Salmonella has been previously used for effective cancer therapy of a melanoma and other cell lines [4458]. The Salmonella strain (VNP20009) was attenuated by a lipid A–mutation (msbB), purine auxotrophy (purI) and amino acid auxotrophy [44]. VNP20009 was safely administered to patients in a Phase I clinical trial on patients with metastatic melanoma. However it was poorly colonized in the tumors since it might be over-attenuated [58]. Our results are the first to demonstrate efficacy of Salmonella treatment on a patient melanoma tumor models (PDOX).

Salmonella A1-R was also previously shown to be active in syngeneic mouse tumor models: we recently determined the efficacy of Salmonella A1-R on the Lewis lung (LLC) in C57BL/6 (C57) immunocompetent mice and observed anti-metastatic efficacy [59] as well as against primary tumors [60]. These results suggest that Salmonella A1-R is also active in animals with an intact immune system and a syngeneic rather than xenografted tumor. The present and previous results suggest the potential of Salmonella A1-R alone or in combination with CDDP to treat melanoma patients in the future.

Supporting Information

S1 File. ARRIVE checklist.

https://doi.org/10.1371/journal.pone.0160882.s001

(PDF)

Acknowledgments

Dedication

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

Author Contributions

  1. Conceptualization: MY MZ YH RMH.
  2. Data curation: MY RMH.
  3. Formal analysis: MY ES FCE.
  4. Funding acquisition: RMH MB.
  5. Investigation: MY MZ YH YZ.
  6. Methodology: MY MZ YH RMH.
  7. Project administration: RMH.
  8. Resources: RMH MB MN.
  9. Supervision: RMH MN MB.
  10. Validation: MY MZ YH ES FCE.
  11. Visualization: MY MZ YH YZ.
  12. Writing - original draft: MY RMH.
  13. Writing - review & editing: MY RMH.

References

  1. 1. Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. (2012) Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 380:358e365.
  2. 2. Gandidi S, Massi D, Mandala M. (2016) PD-L1 expression in cancer patients receiving anti PD-1/PD-L1 antibodies: a systemic review and meta-analysis. Crit Rev Oncol Hematol, in press.
  3. 3. Chapman PB, Einhorn LH, Meyers ML, Saxman S, Destro AN, Panageas KS, et al. (1999) Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol 17:2745–2751. pmid:10561349
  4. 4. Rabik CA, Dolan ME. (2007) Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treatment Rev 33:9–23.
  5. 5. Tang H, Wang Y, Chlewicki LK, Zhang Y, Guo J, Liang W, et al. (2016) Facilitating T Cell infiltration in tumor microenvironment overcomes resistance to PD-L1 blockade. Cancer Cell 29:285–296. pmid:26977880
  6. 6. Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S, et al. (2005) Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 102:755–760. pmid:15644448
  7. 7. Zhao M, Geller J, Ma H, Yang M, Penman S, Hoffman RM. (2007) Monotherapy with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic mouse models of human prostate cancer. Proc Natl Acad Sci USA 104:10170–10174. pmid:17548809
  8. 8. Zhao M, Yang M, Ma H, Li X, Tan X, Li S, et al. (2006) Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice. Cancer Res 2006;66:7647–7652. pmid:16885365
  9. 9. Zhang Y, Tome Y, Suetsugu A, Zhang L, Zhang N, Hoffman RM, et al. (2012) Determination of the optimal route of administration of Salmonella typhimurium A1-R to target breast cancer in nude mice. Anticancer Res 2012;32:2501–2508. pmid:22753706
  10. 10. Zhang Y, Miwa S, Zhang N, Hoffman RM, Zhao M. (2015) Tumor-targeting Salmonella typhimurium A1-R arrests growth of breast-cancer brain metastasis. Oncotarget 6:2615–2622. pmid:25575815
  11. 11. Uchugonova A, Zhao M, Zhang Y, Weinigel M, König K, Hoffman RM. (2012) Cancer-cell killing by engineered Salmonella imaged by multiphoton tomography in live mice. Anticancer Res 32:4331–4339. pmid:23060555
  12. 12. Liu F, Zhang L, Hoffman RM, Zhao M. (2010) Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity. Cell Cycle 9:4518–4524. pmid:21135579
  13. 13. Nagakura C, Hayashi K, Zhao M, Yamauchi K, Yamamoto N, Tsuchiya H, et al. (2009) Efficacy of a genetically-modified Salmonella typhimurium in an orthotopic human pancreatic cancer in nude mice. Anticancer Res 29:1873–1878. pmid:19528442
  14. 14. Yam C, Zhao M, Hayashi K, Ma H, Kishimoto H, McElroy M, et al. (2010) Monotherapy with a tumor-targeting mutant of S. typhimurium inhibits liver metastasis in a mouse model of pancreatic cancer. J Surg Res 164:248–255. pmid:19766244
  15. 15. Hiroshima Y, Zhao M, Zhang Y, Maawy A, Hassanein MK, Uehara F, et al. (2013) Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells. Cell Cycle 12:2774–2780. pmid:23966167
  16. 16. Hiroshima Y, Zhao M, Maawy A, Zhang Y, Katz MH, Fleming JB, et al. (2014) Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX). J Cell Biochem 115:1254–1261. pmid:24435915
  17. 17. Hiroshima Y, Zhang Y, Murakami T, Maawy AA, Miwa S, Yamamoto M, et al. (2014) Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograph (PDOX) and cell line mouse models. Oncotarget 5:12346–12357. pmid:25402324
  18. 18. Matsumoto Y, Miwa S, Zhang Y, Hiroshima Y, Yano S, Uehara F, et al. (2014) Efficacy of tumor-targeting Salmonella typhimurium A1-R on nude mouse models of metastatic and disseminated human ovarian cancer. J Cell Biochem 115:1996–2003. pmid:24924355
  19. 19. Matsumoto Y, Miwa S, Zhang Y, Zhao M, Yano S, Uehara F, et al. (2015) Intraperitoneal administration of tumor-targeting Salmonella typhimurium A1-R inhibits disseminated human ovarian cancer and extends survival in nude mice. Oncotarget 6:11369–11377. pmid:25957417
  20. 20. Yano S, Zhang Y, Zhao M, Hiroshima Y, Miwa S, Uehara F, et al. (2014) Tumor-targeting Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle as visualized by FUCCI imaging and become sensitive to chemotherapy. Cell Cycle 13:3958–3963. pmid:25483077
  21. 21. Hiroshima Y, Zhang Y, Zhao M, Zhang N, Murakami T, Maawy A, et al. (2015) Tumor-targeting Salmonella typhimurium A1-R in combination with Trastuzumab eradicates HER-2-positive cervical cancer cells in patient-derived mouse models. PLoS One 10: e0120358. pmid:26047477
  22. 22. Hayashi K, Zhao M, Yamauchi K, Yamamoto N, Tsuchiya H, Tomita K, et al. (2009) Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium. J Cell Biochem 106:992–998. pmid:19199339
  23. 23. Hayashi K, Zhao M, Yamauchi K, Yamamoto N, Tsuchiya H, Tomita K, et al. (2009) Systemic targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma in nude mice with a tumor-selective strain of Salmonella typhimurium. Cell Cycle 8:870–875. pmid:19221501
  24. 24. Miwa S, Zhang Y, Baek K-E, Uehara F, Yano S, Yamamoto M, et al. (2014) Inhibition of spontaneous and experimental lung metastasis of soft-tissue sarcoma by tumor-targeting Salmonella typhimurium A1-R. Oncotarget 5:12849–12861. pmid:25528763
  25. 25. Murakami T, DeLong J, Eilber FC, Zhao M, Zhang Y, Zhang N, et al. (2016) Tumor-targeting Salmonella typhimurium A1-R in combination with doxorubicin eradicate soft tissue sarcoma in a patient-derived orthotopic xenograft PDOX model. Oncotarget 7:12783–12790. pmid:26859573
  26. 26. Kiyuna T, Murakami T, Tome Y, Kawaguchi K, Igarashi K, Zhang Y, et al. (2016) High efficacy of tumor-targeting Salmonella typhimurium A1-R on a doxorubicin- and dactolisib-resistant follicular dendritic-cell sarcoma in a patient-derived orthotopic xenograft nude mouse model. Oncotarget, in press.
  27. 27. Kimura H, Zhang L, Zhao M, Hayashi K, Tsuchiya H, Tomita K, et al. (2010) Targeted therapy of spinal cord glioma with a genetically-modified Salmonella typhimurium. Cell Proliferation 43:41–48. pmid:19922490
  28. 28. Momiyama M, Zhao M, Kimura H, Tran B, Chishima T, Bouvet M, et al. (2012) Inhibition and eradication of human glioma with tumor-targeting Salmonella typhimurium in an orthotopic nude-mouse model. Cell Cycle 11:628–632. pmid:22274398
  29. 29. Fu X, Besterman JM, Monosov A, Hoffman RM (1991) Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens. Proc Natl Acad Sci USA 88:9345–9349. pmid:1924398
  30. 30. Hoffman RM. (2015) Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts. Nature Reviews Cancer 15:451–452. pmid:26422835
  31. 31. Fu X, Guadagni F, Hoffman RM (1993) A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically from histologically intact patient specimens. Proc Natl Acad Sci USA 89:5645–5649.
  32. 32. Fu X, Le P, Hoffman RM (1993) A metastatic orthotopic-transplant nude-mouse model of human patient breast cancer. Anticancer Res 13:901–904. pmid:8352558
  33. 33. Fu X, Hoffman RM (1993) Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens. Anticancer Res 13:283–286. pmid:8517640
  34. 34. Wang X, Fu X, Hoffman RM (1992) A new patient-like metastatic model of human lung cancer constructed orthotopically with intact tissue via thoracotomy in immunodeficient mice. Int J Cancer 51:992–995. pmid:1639545
  35. 35. Furukawa T, Fu X, Kubota T, Watanabe M, Kitajima M, Hoffman RM (1993) Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue. Cancer Res 53:1204–1208. pmid:8439965
  36. 36. Astoul P, Wang X, Colt HG, Boutin C, Hoffman RM (1996) A patient-like human malignant pleural mesothelioma nude-mouse model. Oncology Reports 3:483–487. pmid:21594397
  37. 37. Hiroshima Y, Zhang Y, Zhang N, Uehara F, Maawy A, Murakami T, et al. (2015) Patient-derived orthotopic xenograft (PDOX) nude mouse model of soft-tissue sarcoma more closely mimics the patient behavior in contrast to the subcutaneous ectopic model. Anticancer Research 35:697–701. pmid:25667448
  38. 38. Hiroshima Y, Zhao M, Zhang Y, Zhang N, Maawy A, Murakami T, et al. (2015) Tumor-targeting Salmonella typhimurium A1-R arrests a chemo-resistant patient soft-tissue sarcoma in nude mice. PLoS One 10:e0134324. pmid:26237416
  39. 39. Miwa S, Yano S, Zhang Y, Matsumoto Y, Uehara F, Yamamoto M, et al. (2014) Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice. Oncotarget 5:7119–7125. pmid:25216526
  40. 40. Miwa S, Matsumoto Y, Hiroshima Y, Yano S, Uehara F, Yamamoto M, et al. (2014) Fluorescence-guided surgery of prostate cancer bone metastasis. J. Surgical Research 192:124–133.
  41. 41. Miwa S, Zhang Y, Baek K-E, Uehara F, Yano S, Yamamoto M, et al. (2014) Inhibition of spontaneous and experimental lung metastasis of soft-tissue sarcoma by tumor-targeting Salmonella typhimurium A1-R. Oncotarget 5:12849–12861. pmid:25528763
  42. 42. Uehara F, Hiroshima Y, Miwa S, Tome Y, Yano S, Yamamoto M, et al. (2015) Fluorescence-guided surgery of retroperitoneal-implanted human fibrosarcoma in nude mice delays or eliminates tumor recurrence and increases survival compared to bright-light surgery. PLoS One 10:e0116865. pmid:25710463
  43. 43. Holland-Frei Cancer Medicine, 6th edition. Kufe, DW, Pollock, RE, Weichselbaum, RR, Bast, Jr, RC, Gansler, TS, Holland, JF, Frei, III, E, eds. Hamilton (ON), BC Decker, 2003.
  44. 44. Pawelek JM, Low KB, Bermudes D. (2003) Bacteria as tumour-targeting vectors. Lancet Oncol 4:548–556. pmid:12965276
  45. 45. Forbes NS (2010). Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer 10:785–794. pmid:20944664
  46. 46. Kasinskas RW, Forbes NS (2006). Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro. Biotechnology and Bioengineering 94:710–721. pmid:16470601
  47. 47. Kasinskas RW, Forbes NS (2007). Salmonella typhimurim lacking ribose chemoreceptors localize in tumor quiescence and induce apoptosis. Cancer Res 67:3201–3209. pmid:17409428
  48. 48. Ganal S, Arenas RB, Sauer JP, Bentley B, Forbes NS (2011). In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis. Cancer Gene Ther 18:457–466. pmid:21436868
  49. 49. Forbes NS, Munn LL, Fukumura D, Jain RK (2003). Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors. Cancer Res 63:5188–5193. pmid:14500342
  50. 50. Pawelek JM, Low KB, Bermudes D (1997). Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57:4537–4544. pmid:9377566
  51. 51. Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M, et al. (1999) Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nature Biotechnol 17:37–41.
  52. 52. Zheng LM, Luo X, Feng M, Li ZJ, Le T, Ittensohn M, et al. (2000) Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector. Oncology Res 12:127–135.
  53. 53. Platt J, Sodi S, Kelley M, Rockwell S, Bermudes D, Low KB, et al. (2000) Antitumour effects of genetically engineered Salmonella in combination with radiation. Euro J Cancer 36:2397–2402.
  54. 54. Clairmont C, Lee KC, Pike J, Ittensohn M, Low KB, Pawelek J, et al. (2000) Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181:1996–2002. pmid:10837181
  55. 55. Luo X, Li ZJ, Lin S, Le T, Ittensohn M, Bermudes D, et al. (2001) Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models. Oncol Res 12:501–508. pmid:11939414
  56. 56. Li YH, Xie YM, Guo KY, Chen H, Wei Y, Huang JS, et al. (2001) Treatment of tumor in mice by oral administration of cytosine deaminase gene carried in live attenuated Salmonella. Acta Biochim Biophys Sinica 33:233–237.
  57. 57. Yuhua L, Kunyuan G, Hui C, Yongmei X, Chaoang S, Xun T, et al. (2001) Oral cytokine gene therapy against murine tumor using attenuated Salminella typhiurium. Int J Cancer 94:438–443. pmid:11745427
  58. 58. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, et al. (2002) Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20:142–152. pmid:11773163
  59. 59. Zhao M, Suetsugu A, Ma H, Zhang L, Liu F, Zhang Y, et al. (2012) Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice. Cell Cycle 11:187–193. pmid:22186786
  60. 60. Tome Y, Zhang Y, Momiyama M, Maehara H, Kanaya F, Tomita K, et al. (2013) Primer dosing of S. typhimurium A1-R potentiates tumor-targeting and efficacy in immunocompetent mice. Anticancer Res 33:97–102. pmid:23267132