Tumor targeting Salmonella typhimurium A1-R in combination with gemcitabine (GEM) regresses partially GEM-resistant pancreatic cancer patient-derived orthotopic xenograft (PDOX) nude mouse models

ABSTRACT

Gemcitabine (GEM) is first-line therapy for pancreatic cancer but has limited efficacy in most cases. Nanoparticle-albumin bound (nab)-paclitaxel is becoming first-line therapy for pancreatic cancer, but also has limited efficacy for pancreatic cancer. Our goal was to improve the treatment outcome in patient-like models of pancreatic cancer. We previously established patient-derived orthotopic xenografts (PDOX) pancreatic cancers from two patients. The pancreatic tumor was implanted orthotopically in the pancreatic tail of nude mice to establish the PDOX models. Five weeks after implantation, 50 PDOX mouse models were randomized into five groups of 10 mice for each pancreatic cancer PDOX: untreated control; GEM (100 mg/kg, i.p., once a week for 2 weeks); GEM + nab-PTX (GEM: 100 mg/kg, i.p., once a week for 2 weeks, nab-PTX: 10 mg/kg, i.v., twice a week for 2 weeks); S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks); GEM + S. typhimurium A1-R (GEM: 100 mg/kg, i.p., once a week for 2 weeks, S. typhimurium A1-R; 5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks). GEM + nab-PTX was significantly more effective than GEM alone in one PDOX model (p = 0.0004), but there was no significant difference in the other PDOX model. The combination of GEM + S. typhimurium A1-R regressed both PDOX models. These results show S. typhimurium A1-R can overcome the ineffectiveness or partial effectiveness of GEM in patient-like models of pancreatic cancer and demonstrate clinical potential for this combination.

KEYWORDS:

• Salmonella typhimurium A1-R
• pancreatic cancer
• PDOX
• nude mice
• orthotopic
• gemcitabine
• combination
• precision therapy

Introduction

Gemcitabine (GEM) is first-line chemotherapy for pancreatic cancer [1] with a response rate of < 10% [2,3]. A Phase III clinical trial showed nab-paclitaxel (nab-PTX) plus GEM had significant improvements over GEM alone in patients with metastatic pancreatic cancer [4]. GEM + nab-PTX improved the response rate to approximately 20% but still with almost all patients dying within 5 years. Transformative individualized treatment is necessary for this disease.

Our laboratory has developed clinically-relevant mouse models of cancer for discovery of transformative therapy for pancreatic cancer, the patient-derived orthotopic xenograft (PDOX) nude mouse model, with the technique of surgical orthotopic implantation (SOI). These models include breast [5], ovarian [6], lung [7], cervical [8], colon [9–11], and stomach cancer [12], as well as sarcoma [13–30] and melanoma [31–38] and pancreatic cancer [33,39–42]. The PDOX model, developed by our laboratory over the past 30 years, has many advantages over subcutaneous-transplant models which grow ectopically under the skin and rarely metastasize [43].

A candidate for transformative therapy is tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), also developed by our laboratory [44]. S. typhimurium A1-R is auxotrophic for Leu-Arg, which prevents it from mounting a continuous infection in normal tissues. Tumor-targeting S. typhimurium A1-R was effective against many types of PDOX models including melanoma [36–38], pancreatic cancer [39,45] and cases of osteosarcoma [20,46,47] and Ewing’s [21] and follicular dendritic cell sarcoma [15].

We preivously demonstrated the efficacy of S. typhimurium A1-R in combination with anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer including a PDOX model [39].

We preiovusly showed that tumor-targeting S. typhimurium A1-R had comparable efficacy with gemcitaine (GEM), cisplatinum (CDDP), and 5-fluorouracil (5-FU) on a pancreatic cncer PDOX models [45].

It has been reported for at least two hundred years that bacterial infection can regress cancer in patients. In the late 19th and early 20th centuries, cancer patients were treated with bacteria by Dr. William B. Coley. However, after Coley’s death, bacterial therapy of cancer stopped. At the present time, there has been a resurgence of bacterial therapy of cancer and the PDOX model has been shown to be very appropriate to develop of new combination to inhibitors or regression of recalcitrant cancer [48].

In the present study, we utilized two PDOX nude mouse models of pancreatic cancer from two different patients and determined whether S. typhimurium A1-R in combination with GEM could overcome GEM-resistance.

Results and discussion

For pancreatic cancer PDOX model #1, all treatments inhibited tumor growth compared to untreated control to a different degree: GEM: p < 0.0001; GEM + nab-PTX: p < 0.0001; S. typhimurium A1-R: p < 0.0001; GEM + S. typhimurium A1-R: p < 0.0001 on day 14 after treatment initiation. GEM + nab-PTX and GEM + S. typhimurium A1-R significantly inhibited tumor growth more than GEM alone (p = 0.0004 and p < 0.0001, respectively). However, GEM + S. typhimurium A1-R significantly inhibited tumor growth more compared to other treatments (GEM: p < 0.0001; GEM + nab-PTX: p = 0.0372; S. typhimurium A1-R: p < 0.0001). Only the GEM + S. typhimurium A1-R combination could regress the tumor (Figure 1(a)).

Figure 1. Quantitative treatment efficacy.

Bar graphs show relative tumor volume before and after treatment for each treatment condition. (a) Pancreatic cancer PDOX #1. (b) Pancreatic cancer PDOX #2. Error bars: ± SD. Relative tumor volume is the ratio of tumor volume after treatment to the tumor volume before the start of the treatment period.

For PDOX model #2, all treatments inhibited tumor growth compared to untreated control to a different degree: GEM: p < 0.0001; GEM + nab-PTX: p < 0.0001; S. typhimurium A1-R: p < 0.0001; GEM + S. typhimurium A1-R: p < 0.0001 on day 14. GEM + S. typhimurium A1-R significantly inhibited tumor growth more than GEM alone (p = 0.0003). However, there was no significant difference between GEM + nab-PTX and GEM alone, unlike pancreatic cancer PDOX #1. GEM + S. typhimurium A1-R significantly inhibited tumor growth more compared to other treatments (GEM: p = 0.0003; GEM + nab-PTX: p = 0.0022; S. typhimurium A1-R: p = 0.0006). Only the GEM + S. typhimurium A1-R combination therapy could regress the tumor (Figure 1(b)).

Body weight on day 14 of treatment, compared with day 0, did not significantly differ between any treatment group (Figure 2(a,b)). There were no animal deaths in any group.

Figure 2. Effect of each treatment on mouse body weight. Bar graphs show mouse body weight in each treatment group at pre- and post-treatment timepoints. (a) Pancreatic cancer PDOX #1. (b) Pancreatic cancer PDOX #2. Error bars: ± SD.

Histologically, the untreated control pancreatic cancer PDOX tumors were mainly comprised of viable cells. In contrast, the pancreatic cancer PDOX tumors treated with the combination therapy of GEM + S. typhimurium A1-R showed extensive necrosis (Figure 3).

Figure 3. Tumor histology. (a). Untreated control. (b). Tumor treated with GEM+ S. typhimurium A1-R). Scale bars: 100 μm.

In the present study, first-line therapy for pancreatic cancer, GEM, could not arrest or regress tumor growth in either of the 2 PDOX models. GEM showed some tumor inhibition compared to the untreated control, similar to the situation in the clinic.

Recently, a randomized Phase III clinical trial showed promising efficacy in metastatic pancreatic cancer treated with the combination of GEM + nab-PTX [4]. In the present study, GEM + nab-PTX was significantly more effective than GEM alone in pancreatic cancer PDOX #1 (Figure 1(a)), but there was no significantly difference in pancreatic cancer PDOX #2 (Figure 1(b)). Neither pancreatic cancer PDOX #1 or #2 could be regressed by GEM + nab-PTX. In contrast, the combination of S. typhimurium A1-R + GEM could regress both PDOX models.

The tumor-targeting S. typhimurium A1-R, developed by our laboratory [44], is auxotrophic for Leu-Arg, which prevents it from mounting a continuous infection in normal tissues. In the present study, S. typhimurium A1-R could increase the partial response of GEM of the pancreatic cancer PDOX #1 and #2 to cause regression, suggesting the generality of this treatment.

The present study has important implications since it showed the strong additional efficacy of S. typhimurium A1-R combined with GEM, in contrast to nab-PTX which showed benefit with GEM in only one of the two PDOX models, suggesting clinical potential of the GEM+ S. typhimurium A1-R combination. The PDOX model enables precise, individualized therapy, especially for recalcitrant disease such as pancreatic cancer [35].

S. typhimurium A1-R can directly target and kill cancer cells [49] as well as decoy the cancer cells from the chemoresistant G₀/G₁ phase to the chemosensitive S-phase of the cell cycle, making them more susceptible to GEM [50–52]. A similar type of cell-cycle decoy occurred when cancer cells were injected with an adenovirus [53]. The cell cycle decoy from G₀/G₁ to S may be a general response to cancer-cell infection. The decoy has important implications since the majority of cancer cells in a mature tumor are in G₀/G₁ and chemoresistant and may account for drug resistance found so often in solid tumors in the clinic [50,51,54].

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 [55–60].

Materials and methods

Mice

Athymic nu/nu male nude mice (AntiCancer, Inc., San Diego, CA), 4–6 weeks old, were used in this study. All mice were kept 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. All animal experiments were performed 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. Anesthesia and analgesics were used for all surgical experiments to avoid unnecessary suffering of the mice. Subcutaneous injection of a ketamine mixture (a 0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate) was used for mice. The response of animals during surgery was monitored carefully to maintain adequate depth of anesthesia. The animals were observed daily and humanely sacrificed by CO₂ inhalation when they met the following criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant body weight loss, difficulty breathing, rotational motion and body-temperature drop.

Patient-derived tumor

The tumor from a pancreatic cancer patient #1 was previously resected in the MD Anderson Cancer Center. Written informed consent was provided by the patient and the Institutional Review Board (IRB) of MD Anderson Cancer Center approved this experiment.

The tumor from pancreatic cancer patient #2 was previously resected in the University of California, San Diego (UCSD). Written informed consent was provided by the patient, and the Institutional Review Board (IRB #140046CX) of UCSD approved this experiment.

Establishment of PDOX models of pancreatic cancer by surgical orthotopic implantation (SOI)

After nude mice were anesthetized with the ketamine solution described above, a 1–1.5 cm skin incision was made on the left-side abdomen through the skin, fascia and peritoneum and pancreas was exposed. Surgical sutures (8–0 nylon) were used to implant tumor fragments onto the tail of the pancreas to establish the PDOX model. The wound was closed with a 6–0 nylon suture (Ethilon, Ethicon, Inc., NJ) [33,39,41,42,45,61].

Preparation and administration of S. typhimurium A1-R

GFP-expressing S. typhimurium A1-R (AntiCancer Inc., San Diego, CA) was grown overnight on LB medium (Fisher Sci., Hanover Park, IL) and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then diluted in PBS. S. typhimurium A1-R was injected intravenously. A total of 5 × 10⁷ CFU S. typhimurium A1-R in 100 μl PBS was administered to each mouse [49,62,63].

Treatment study design for the PDOX models of pancreatic cancer

Pancreatic cancer PDOX mouse models were randomized into five groups of 10 mice each: untreated control; GEM (100 mg/kg, i.p., once a week for 2 weeks); GEM + nab-PTX (GEM: 100 mg/kg, i.p., once a week for 2 weeks, nab-PTX: 10 mg/kg, i.v., twice a week for 2 weeks); S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks); GEM + S. typhimurium A1-R (GEM: 100 mg/kg, i.p., once a week for 2 weeks; S. typhimurium A1-R, 5 × 10⁷ CFU/100 μl, i.v., once a week for 2 weeks). Treatments started when the PDOX tumors reached 50 mm³. Tumor length and width were measured on day 0 and 14. Tumor volume was calculated with the following formula: Tumor volume (mm³) = length (mm) × width (mm) × width (mm) × 1/2. Data are presented as mean ± SD. The tumor volume ratio is defined at the tumor volume at day 14 to day 0. Relative tumor volume enables tumor growth or inhibition to be visualized more clearly. Tumor size at the initiation and end of treatment was measured with calipers during laparotomy.

Histological examination

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 protocols. Histological examination was performed with a BHS System Microscope (Olympus Corporation, Tokyo, Japan). Images were acquired with INFINITY ANALYZE software (Lumenera Corporation, Ottawa, Canada) [33].

Statistical analysis

JMP version 11.0 was used for all statistical analyzes. Significant differences for continuous variables were determined using the Mann-Whitney U test. Line graphs expressed average values and error bar show SD. A probability value of P ≤ 0.05 is considered statistically significant.

Disclosure statement

No potential conflict of interest was reported by the authors.