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The irony of highly-effective bacterial therapy of a patient-derived orthotopic xenograft (PDOX) model of Ewing's sarcoma, which was blocked by Ewing himself 80 years ago

Takashi MurakamiAntiCancer, Inc., San Diego, CA, USA;Department of Surgery, University of California, San Diego, CA, USA;Department of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
,
Tasuku KiyunaAntiCancer, Inc., San Diego, CA, USA
,
Kei KawaguchiAntiCancer, Inc., San Diego, CA, USA
,
Kentaro IgarashiAntiCancer, Inc., San Diego, CA, USA
,
Arun S. SinghDivision of Hematology-Oncology, University of California, Los Angeles, CA, USA
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Yukihiko HiroshimaDepartment of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
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Yong ZhangAntiCancer, Inc., San Diego, CA, USA
,
Ming ZhaoAntiCancer, Inc., San Diego, CA, USA
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Kentaro MiyakeAntiCancer, Inc., San Diego, CA, USA;Department of Surgery, University of California, San Diego, CA, USA;Department of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, JapanORCID Iconhttp://orcid.org/0000-0002-4680-4317
ORCID Icon,
Scott D. NelsonDepartment of Pathology, University of California, Los Angeles, CA, USA
,
Sarah M. DryDepartment of Pathology, University of California, Los Angeles, CA, USA
,
Yunfeng LiDepartment of Pathology, University of California, Los Angeles, CA, USA
,
Jonathan C. DeLongDepartment of Surgery, University of California, San Diego, CA, USA
,
Thinzar M. LwinDepartment of Surgery, University of California, San Diego, CA, USA
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Takashi ChishimaDepartment of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
,
Kuniya TanakaDepartment of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan
,
Michael BouvetDepartment of Surgery, University of California, San Diego, CA, USA
,
Itaru EndoDepartment of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, JapanORCID Iconhttp://orcid.org/0000-0001-5520-8114
ORCID Icon,
Fritz C. EilberDivision of Surgical Oncology, University of California, Los Angeles, CA, USACorrespondence[email protected]
ORCID Iconhttp://orcid.org/0000-0003-3336-9333
ORCID Icon &
Robert M. HoffmanAntiCancer, Inc., San Diego, CA, USA;Department of Surgery, University of California, San Diego, CA, USACorrespondence[email protected]
 show all
Pages 1046-1052 | Received 19 Jan 2017, Accepted 06 Mar 2017, Published online: 11 May 2017

ABSTRACT

William B. Coley developed bacterial therapy of cancer more than 100 years ago and had clinical success. James Ewing, a very famous cancer pathologist for whom the Ewing sarcoma is named, was Coley's boss at Memorial Hospital in New York and terminated Coley's bacterial therapy of cancer. A tumor from a patient with soft-tissue Ewing's sarcoma, who failed doxorubicin (DOX) therapy, was previously implanted in nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In the present study, the Ewing's sarcoma PDOX was treated with tumor-targeting S. typhimurium A1-R expressing green fluorescent (GFP), alone and in combination with DOX. S. typhimurium A1-R-GFP was detected in the tumors after intratumor (i.t.) or intravenous (i.v.) injection. The combination of S. typhimurium A1-R and DOX significantly reduced tumor weight (37.8 ± 15.6 mg) compared to the untreated control (73.8 ± 10.1 mg, P < 0.01). S. typhimurium A1-R monotherapy-treated tumors tended to be smaller (50.9 ± 17.8 mg, P = 0.051). DOX monotherapy did not show efficacy (66.3 ± 26.4 mg, P = 0.82), as was the case with the patient. The PDOX model faithfully replicated the DOX resistance the Ewing's sarcoma had in the patient. S. typhimurium A1-R converted the Ewing's sarcoma from DOX resistant to sensitive. One can only wonder how bacterial therapy and immunotherapy of cancer would have developed over the past 80 years if Ewing did not stop Coley.

Introduction

Ewing's sarcoma is a recalcitrant malignancy that usually occurs in adolescence and young adulthood. Multimodal treatment for localized Ewing's sarcoma has improved the 5-year survival to 65–70%. However, patients with metastatic disease have a 30% of 5-year survival rate.Citation1 Therefore, development of effective treatment for Ewing's sarcoma is needed.

William B. Coley developed bacterial therapy of cancer more than 100-years ago and had clinical success.Citation2,3

James Ewing was a very famous cancer pathologist for whom the Ewing sarcoma is named, was a bitter opponent of Coley's work at Memorial Hospital in New York and finally ended Coley's career and bacterial therapy of cancer.

In the current century, bacterial therapy is making a strong come back to enhance immunotherapy of cancer.Citation3 The tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), developed by our laboratoryCitation4,5 is auxotrophic for Leu-Arg, which prevents it from mounting a continuous infection in normal tissues. S. typhimurium A1-R was effective against primary and metastatic tumors as monotherapy in nude mouse models of major cancers,Citation3 including prostate,Citation4,6 breast,Citation7-9 lung,Citation10,11 pancreatic,Citation12-16 ovarian,Citation17-18 stomach,Citation19 and cervical cancer,Citation20 as well as sarcoma cell linesCitation21-24 and glioma,Citation3,25 all of which are highly aggressive tumor models. In addition, S. typhimurium A1-R was effective against patient-derived orthotopic models of pancreatic cancer,Citation15,16 sarcomaCitation24,26-29 and melanoma.Citation30-32

A tumor from a patient with soft-tissue Ewing's sarcoma was previously established as a patient-derived orthotopic xenograft (PDOX) model.Citation28 In the present study, we observed that S. typhimurium A1-R converted the Ewing's sarcoma from doxorubicin (DOX)-resistant to DOX-sensitive.

Results and discussion

Ewing's sarcoma PDOX tumor recapitulates the original patient tumor

Four weeks after orthotopic implantation, the Ewing's sarcoma PDOX tumor grew to more than 60 mm3 in the right chest wall of nude mice (). Histologically, the patient's tumor demonstrated an infiltrative proliferation of small round blue cells. Cancer cells showed round-to-ovoid hyperchromatic nuclei, and scanty eosinophilic-to-clear cytoplasm (). Histology of the PDOX tumor had a similar morphologic appearance to the original patient tumor ().

Figure 1. Established Ewing's sarcoma PDOX model. (A) Four weeks after tumor implantation in the space between the pectoral muscle and intercostal muscle, the Ewing's sarcoma PDOX tumors grew in the chest wall (arrow). (B) Resected tumor from the same mouse. Scale bars: 5 mm.

Figure 1. Established Ewing's sarcoma PDOX model. (A) Four weeks after tumor implantation in the space between the pectoral muscle and intercostal muscle, the Ewing's sarcoma PDOX tumors grew in the chest wall (arrow). (B) Resected tumor from the same mouse. Scale bars: 5 mm.

Figure 2. Histological comparison between patient original tumors and a PDOX tumor. (A) H&E staining of the patient original tumor and (B) untreated PDOX tumor. Scale bars: 100 μm.

Figure 2. Histological comparison between patient original tumors and a PDOX tumor. (A) H&E staining of the patient original tumor and (B) untreated PDOX tumor. Scale bars: 100 μm.

S. typhimurium A1-R-GFP was detected in treated PDOX tumors

GFP-expressing S. typhimurium A1-R was detected in tumors on the next day after the 2nd i.t. injection () or i.v. injection of S. typhimurium A1-R-GFP (). More S. typhimurium A1-R-GFP was detected in the tumor after i.t. injection than i.v. injection, perhaps because the bacteria were directly injected. However, even with i.v. injection, a large amount of bacteria targeted the tumor from the circulation.

Figure 3. Bacterial culture from S. typhimurium A1-R-treated Ewing's sarcoma PDOX. Bright field (A, C) and fluorescence (B, D) images of GFP-expressing S. typhimurium A1-R colonies cultured in LB-agar. Administered S. typhimurium either by i.t. (A, B) or i.v. (C, D) was detected in treated tumors 24 hours after the second injection of S. typhimurium A1-R (day 9). Scale bars: 10 mm. GFP, green fluorescent protein. OV100 imaging.

Figure 3. Bacterial culture from S. typhimurium A1-R-treated Ewing's sarcoma PDOX. Bright field (A, C) and fluorescence (B, D) images of GFP-expressing S. typhimurium A1-R colonies cultured in LB-agar. Administered S. typhimurium either by i.t. (A, B) or i.v. (C, D) was detected in treated tumors 24 hours after the second injection of S. typhimurium A1-R (day 9). Scale bars: 10 mm. GFP, green fluorescent protein. OV100 imaging.

S. typhimurium A1-R (i.t. and i.v.) inhibited tumor growth in the Ewing's sarcoma PDOX

The tumor doubling time of the Ewing's sarcoma PDOX was 12.7 days. Both administrative routes (i.t. and i.v.) of S. typhimurium A1-R inhibited tumor growth (). A PDOX was previously established in the right chest wall of nude mice which corresponded to the origin of the tumor in the patient. Eighteen PDOX mice were randomized into three groups when tumor volume reached 60 mm3: untreated control; S. typhimurium A1-R intratumor administration (i.t., 5 × 107 CFU/25 µl, day 1 and 8), and S. typhimurium A1-R intravenous administration (i.v., 5 × 107 CFU/100 µl, day 1 and 8). Tumor growth was significantly suppressed in both of the S. typhimurium-treated groups. However, S. typhimurium A1-R i.v. was more effective than i.t. injection.

Figure 4. S. typhimurium A1-R administered i.v. or i.t. on the Ewing's sarcoma PDOX Line graph shows tumor volume ratio. S. typhimurium A1-R was administered i.v. or i.t. Please see the Materials and Methods for doses and schedules. *P < 0.05 and **P < 0.01 compared to control. Error bars: ± 1 SD. i.t., intratumoral injection; i.v., intravenous injection.

Figure 4. S. typhimurium A1-R administered i.v. or i.t. on the Ewing's sarcoma PDOX Line graph shows tumor volume ratio. S. typhimurium A1-R was administered i.v. or i.t. Please see the Materials and Methods for doses and schedules. *P < 0.05 and **P < 0.01 compared to control. Error bars: ± 1 SD. i.t., intratumoral injection; i.v., intravenous injection.

S. typhimurium A1-R i.v. increased the efficacy of doxorubicin in the Ewing's sarcoma PDOX model

S. typhimurium A1-R i.v. in combination with DOX, was then tested. Twenty PDOX mice were prepared as described above and randomized into the following groups; G1, untreated control; G2, DOX (i.v., 3 mg/kg, day 1 and 8); G3, S. typhimurium A1-R (i.v., 5 × 107 CFU/100 µl, day 1 and 8); G4, combination of S. typhimurium A1-R (i.v., 5 × 107 CFU/100 µl, day 1 and 8); and DOX (i.v., 3 mg/kg, day 4 and 11).

Only the combination of S. typhimurium A1-R and DOX significantly reduced tumor weight (37.8 μ ± 15.6 mg) compared to the untreated control (73.8 ± 10.1 mg, P < 0.01). S. typhimurium A1-R monotherapy-treated tumors tended to be smaller (50.9 ± 17.8 mg, P = 0.051). DOX monotherapy did not show efficacy (66.3 ± 26.4 mg, P = 0.82), as was the case with the patient (). Mouse body weight was not significantly different between groups at any time point.

Figure 5. Combination therapy of intravenous S. typhimurium A1-R (i.v.) combined with doxorubicin in the Ewing's sarcoma PDOX model. Bar graphs show resected tumor weight in each group. Please see the Materials and Methods for doses and schedules.

Figure 5. Combination therapy of intravenous S. typhimurium A1-R (i.v.) combined with doxorubicin in the Ewing's sarcoma PDOX model. Bar graphs show resected tumor weight in each group. Please see the Materials and Methods for doses and schedules.

Bacterial therapy has potential side effects, even though they were not observed in the present study. Such side effects could include infection outside the tumor and potential unwanted immunological reaction.

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.Citation33-38

The aim of the present study was to develop S. typhimurium A1-R treatment for an Ewing's sarcoma using different administrative routes and in combination with standard first-line DOX.

Conclusions

The present study has demonstrated the potential of S. typhimurium A1-R to be an effective treatment as both monotherapy and combination with first-line therapy for Ewing's sarcoma. Ironically, bacterial therapy was highly effective on a PDOX model of Ewing's sarcoma, demonstrating that the very treatment that was halted by Ewing was found more than 80 years later to be highly effective on the sarcoma named for him. One can only wonder how bacterial therapy and immunotherapy of cancer would have developed over the past 80 years if Ewing did not stop Coley.

Materials and methods

Mice

Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4–6 weeks old, were used in this study under Assurance Number A3873-1. The animals were fed an autoclaved laboratory rodent diet.Citation28 Animals were anesthetized by subcutaneous injection of a 0.02 ml solution of 25 mg/kg ketamine, 19 mg/kg xylazine, and 0.60 mg/kg acepromazine maleate. Animals were housed with no more than 5 per cage and housed in a barrier facility on a high efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles.

Patient-derived Ewing's sarcoma tumor

The female 46-year-old patient was previously diagnosed with Ewing's sarcoma in the right chest wall and received neoadjuvant chemotherapy including DOX. Then the tumor was resected by JY in the Department of Surgery, University of California, Los Angeles (UCLA). Written informed consent was obtained from the patient, and the Institutional Review Board (IRB) of UCLA approved this experiment.Citation28

Establishment of a PDOX model of Ewing's sarcoma

Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI), including pancreatic,Citation16,39-41 breast,Citation42 ovarian,Citation43 lung,Citation44 cervical,Citation45 colon,Citation46-48 stomach,Citation49 sarcoma,Citation26-28,50 and melanoma.Citation30-32

A Ewing's sarcoma PDOX was previously established on the right chest wall where a single tumor fragment was implanted orthotopically into the layer between pectoral muscle and intercostal muscle in nude mice.Citation28

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.Citation4,6,7

Efficacy of S. typhimurium A1-R i.v. vs. i.t. on the Ewing's sarcoma PDOX

PDOX tumors (n = 18) were divided into 3 groups when tumor volume reached 60 mm3: untreated control (n = 6); S. typhimurium A1-R (n = 6, i.t., 5 × 107 CFU/25 µl, day 1 and 8); S. typhimurium A1-R (n = 6, i.v., 5 × 107 CFU/100 µl, day 1 and 8). The mice were followed up until day 15. Tumor length, width, and mouse body weight were measured twice a week. Tumor volume was calculated by the following formula: Tumor volume (mm3) = length (mm) × width (mm) × width (mm) × 1/2. Tumor volume ratio was defined as the ratio of volume on each measurement day relative to day 0.

Efficacy of S. typhimurium A1-R in combination with DOX on the Ewing's sarcoma PDOX

PDOX tumors (n = 20) were divided into following 4 groups when the tumor volume reached 60 mm3: G1, untreated control (n = 5); G2; DOX (n = 5; i.v., 3 mg/kg, day 1 and 8); G3, S. typhimurium A1-R (n = 5, i.v., 5 × 107 CFU/100 µl, day 1 and 8); and G4; combination of S. typhimurium A1-R (n = 5, i.v., 5 × 107 CFU/100 µl, day 1 and 8) and DOX (n = 5, i.v., 3 mg/kg, day 4 and 11). Mice were sacrificed and tumors were removed on day 15 for weight determination. Tumor growth measurement and evaluation was performed as described above.

Histological analysis

Fresh tumor 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 protocol. Histological examination was performed with a BHS system microscope (Olympus Corp., Tokyo, Japan). Images were acquired with INFINITY ANALYZE software (Lumenera Corporation, Ottawa, Canada).Citation28

Bacterial culture from tumor tissue

To identify S. typhimurium A1-R in treated tumors, two Ewing's sarcoma PDOX mice were treated with S. typhimurium A1-R i.t. or i.v. These mice were sacrificed and tumors were resected on day 9 to culture bacteria. After removal of tumors from PDOX mice, the tumor specimens were homogenized and suspended in PBS (phosphate-buffered saline, Corning, New York, NY). The suspension was serially diluted, then cultured in LB agar for 12 hours. GFP-expressing colonies of S. typhimurium A1-R were detected with the OV100 Small Animal Imaging System (Olympus, Tokyo, Japan).Citation51

Statistical analysis

SPSS statistics version 21.0 was used for all statistical analyses (IBM, New York City, NY, USA). Significant differences for continuous variables were determined using the Student's t-test. Both line graphs and bar graph express average and error bar show standard deviation (SD). A probability value of P was calculated between control groups and each treatment group. P ≤ 0.05 was considered statistically significant.

Dedication

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

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by National Cancer Institute grant number CA213649 and JSPS KAKENHI grant numbers 26830081 to YH, 26462070 to IE and 24592009 to KT.

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