Attenuated Salmonella typhimurium with IL-2 Gene Reduces Pulmonary Metastases in Murine Osteosarcoma

Abstract

Historically, osteosarcoma has been a problematic metastatic disease, with 40–80% of patients developing pulmonary metastasis after primary tumor resection. Recent treatment advancements have reduced the occurrence of metastatic lesions to less than 30%. Using attenuated Salmonella typhimurium, we previously demonstrated regression in tumor burden in murine solid tumor and metastatic models. We established a murine model for metastatic osteosarcoma to determine the effect of treatment with a single oral dose of attenuated S. typhimurium with (SalpIL2) and without (Sal-NG) a gene for a truncated human interleukin-2. Female balb/c mice were administered 2 × 10⁵ (ATCC K7M2) osteosarcoma cells via tail vein injection from culture and treated by oral gavage of Salmonella species 3 days later. Mice were harvested for splenic lymphocytes and tumor enumeration by intratracheal injection with India ink 21 days after injection. Treatment with attenuated SalpIL2 reduced pulmonary metastases in number and volume compared to saline controls. Furthermore, splenic natural killer cell populations were increased 93% with SalpIL2 and 114% with Sal-NG compared to nontreated groups. This pulmonary metastasis model demonstrates attenuated Salmonella typhimurium with human interleukin-2 reduced metastatic osteosarcoma in mice and confirm the need for further investigation into the immunologic properties of SalpIL2 as a possible treatment for metastatic osteosarcoma.

Introduction

Osteosarcoma is the most common primary bone cancer, with approximately 900 new cases annually in the United States [14]. There is a peak incidence in early adolescence correlated with pubertal bone growth and a second peak after age 50 [5]. Primary tumors develop in the distal femur and proximal tibia and humerus. Current management of primary osteosarcoma involves surgical resection with wide margins or limb amputation in conjunction with pre- and postoperative neoadjuvant chemotherapy. Survival from local disease has improved from 20% in 1970 [10] to approximately 70% at 3 years [16, 18] with the advent of current treatment with high-dose methotrexate, cisplatin, ifosfamide, and doxorubicin [20]. Despite the dramatic enhancement in patients’ event free survival, toxicity affects nearly all patients treated with these therapies [20]. However, in patients who present with metastatic disease detectable by CT, less than 30% disease-free survival has been achieved [9, 21]. In some cases, intravenous interleukin-2 treatment has resulted in complete regression of the primary tumor, though severe side effects have been noted, including fever, nausea, capillary leak syndrome, and death [25, 26].

We recently demonstrated a single oral dose of an attenuated Salmonella typhimurium genetically engineered with a gene for a truncated human interleukin-2 (SalpIL2) substantially reduces unresectable adenocarcinoma metastases to the liver in experimental treatment and prophylactic mouse models [7, 8, 27, 29, 33]. In addition, SalpIL2 reduces the volume and mass of retroperitoneal neuroblastoma tumors in an experimental murine treatment model [1]. Interestingly, the Salmonella species of bacteria also have a unique propensity to colonize tumor cells [2]. In vitro experiments have demonstrated the ability of SalpIL2 to invade and divide preferentially within K7M2 osteosarcoma cells with respect to primary murine hepatocytes [34]. Thus SalpIL2 may be able to persist for long periods in malignant tissues providing a prolonged antigen presentation state and enhanced immune response in the region.

Based on our previous observations, we hypothesize SalpIL2 would substantially reduce osteosarcoma pulmonary metastases by increasing splenic and local NK cell populations in this newly developed experimental model.

Materials and Methods

In triplicate experiments, 45 balb/c mice were administered murine K7M2 osteosarcoma cells by tail vein injection. Three days later, animals were orally gavaged saline or attenuated Salmonella species; they were then euthanized on day 21 for tumor enumeration, volume, and assessment of systemic NK and T cell populations. In an additional experiment, animals were harvested for pulmonary lymphocyte analysis.

Attenuated S. typhimurium χ4550 and plasmid pYA292 were a gift from Dr. Roy Curtiss III, Washington University, St. Louis, MO. χ4550 was attenuated by Tn10 transposon mutagenesis to remove adenylate cyclase (cya), cyclin adenosine monophosphate receptor protein (crp), and aspartate semialdehyde dehydrogenase (asd) genes from the bacterial genome [21]. These mutants have virulence factors removed, but retain immunogenic properties [4]. Plasmid constructs with and without a truncated gene for human interleukin-2 were electroporated into χ4550 using well-described techniques and renamed SalpIL2 and Sal-NG [28]. Standardized glycerol stocks of approximately 10⁸ CFU/mL were prepared by creating growth curves for overnight cultures in Luria broth (Difco Laboratories, Detroit, MI) and freezing aliquots with an O.D.₆₀₀ of 0.160 in liquid nitrogen. For experiments, cryovials were thawed to room temperature, serially diluted, and plated on MacConkey agar plates to verify CFU concentration. Use of S. typhimurium with a gene for a truncated human interleukin-2 was approved by the University of Minnesota Institutional Biosafety Committee (numbers 541 and 542).

Female balb/c mice 6 to 8 weeks old were acquired from Harlan Sprague Dawley (Indianapolis, IN) and housed in microisolator cages, fed standard mouse chow and water ad libitum, and given 12 hours light/dark cycles under the strict care of the University of Minnesota Research Animal Resources.

The murine osteosarcoma cell line K7M2 was acquired from the American Type Culture Collection and maintained in 25 mL DMEM, 10% fetal bovine serum, 1% penicillin, streptomycin, and L-glutamine (Sigma Chemical, St. Louis, MO) at 37°C at 5% CO₂. Media was changed twice weekly and cells were not allowed to become confluent. Tumor cells were incubated with 0.3% trypsin EDTA (Invitrogen, Carlsbad, CA) at 37°C at 5% CO₂ for 3 minutes or until nonadherent. Cells were serially washed in Hanks’ balanced salt solution (HBSS, Invitrogen) before enumeration via trypan blue exclusion (Sigma Chemical) on a phase contrast hemocytometer (Hausser Scientific, Horsham, PA). The suspension was diluted to a concentration of 2 × 10⁶ cells per mL and placed on ice prior to injection. All tumor preparations were more than 90% viable and used within 1 hour of preparation.

We developed a model for pulmonary metastases for these experiments. Similar techniques have been implemented for quantifying the metastatic potential of the K7M2 cell line [12]. In triplicate experiments, animals were anesthetized by intraperitoneal injection of 2:1 xylazine 20 mg/mL (Phoenix Pharmaceuticals, St. Joseph, MO) and ketamine 100 mg/mL (Abbot Laboratories, North Chicago, IL). The animals’ eyes were swabbed with Betadine ophthalmic eye ointment (Purdue Pharma LP, Stamford, CT) and the animals were placed in a Broome Rodent Holder (Kent Scientific, Torrington, CT). Tails were incubated for one minute in 47°C Betadine solution (Purdue Pharma LP) to allow for vasodilatation of the left lateral tail vein and scrubbed with a 70% ethanol swab before 200,000 K7M2 cells were injected into the left lateral tail vein. Mice were placed at random in cages with microisolators and placed on a warming pad for 2 hours or until animals were walking. On Day 3, mice were orally gavaged with their respective treatments (n = 5), 200 μL HBSS for controls or 3 × 10⁷ CFU of either Sal-NG or SalpIL2. In all experiments the mice were evaluated for presence of metastases 3 weeks after injection by euthanasia followed by an intratracheal injection of 1.5 mL of 15% India-ink solution via a blunt-ended needle. The stained lungs were carefully resected and rinsed in Fekete’s solution (300 mL 70% ethanol, 30 mL 37% formaldehyde, 5 mL glacial acetic acid) and then placed in fresh Fekete’s solution overnight in a 60 × 15 mm tissue culture dish. Tumors were enumerated, their diameters were measured and volume was calculated by 4/3πr³, assuming the metastases were spherical. Spleens were aseptically removed and placed in 60 × 15 mm culture dishes for FACS analysis of splenic lymphocytes. Due to inability to collect pulmonary lymphocytes or perform histopathological analysis from Fekete stained lungs, two additional experiments with 25 mice were conducted. Lungs for histopathological analysis were aseptically removed and placed in 10% formalin and sent to the University of Minnesota’s Histopathological Core for slide preparation.

Spleens and lungs for FACS analysis were incubated with 37°C DMEM containing 10% fetal goat serum and crushed with sterile glass stoppers. Homogenates were filtered through a 150-μm nitex mesh (Sefar American, Kansas City, MO) and transferred onto 5-mL lymphocyte separation medium (Ficol, Mediatech Inc., Hendon, VA). The cell suspension was centrifuged for 1 hour and the lymphocyte layer was carefully collected. Cells were serially washed with PBS with 1% bovine serum albumin (BSA) and 0.1% NaN₃ (Sigma Chemical) and split for monoclonal antibody staining. Cells were stained with DX5/CD 49a PE and CD 3 FITC for NK cell analysis and CD 8 PerCp and CD4 FITC (Pharminogen, San Diego, CA) for T lymphocyte populations. Cells were cold incubated for 30 minutes at 4°C before a final wash with PSB/BSA/NaN₃ and stored under foil at 4°C until FACS analysis.

Splenic and pulmonary lymphocytes collected from experimental mice were analyzed with a FACScalibur (Becton Dickenson, Grenoble, France) and analyzed with Cell Quest Pro Software (Becton Dickenson, San Jose, CA). Lymphocyte populations were identified using forward-scatter versus side-scatter profiles and gated for mononuclear lymphocytes. Natural killer cell populations then were identified by DX5/CD 49b⁺/Cd 3⁻, T_H and T_C cells by single positive populations based on 10,000 gated events.

Number of tumors, volume, and lymphocyte populations were entered for each mouse at the experimental endpoint to calculate the total mean values for each treatment group. All differences between two groups were determined by Fisher’s exact test. Graphs were constructed using Microsoft Excel (Microsoft, Redmond, WA). Statistical tests were performed using StatView software v. 5.0.1 (SAS Institute, Cary, NC).

Results

Attenuated S. typhimurium with and without a gene for human interleukin-2 had fewer total tumors (20.93 and 33, respectively; p < 0.0175 and 0.0006, respectively) compared to saline controls (58.42). SalpIL2 reduced (p < 0.0037) overall volume of pulmonary metastatic nodules by 78% with respect to saline controls (Fig. 1). There was no discernable difference in the reduction of tumor number and volume between the two Salmonella treatments. NK cell populations increased (p < 0.0163 and p < 0.0407, respectively) in Sal-NG- and SalpIL2-treated groups (18.5% and 16.8%, respectively) with respect to saline controls (8.6) (Fig. 2). Cytotoxic T lymphocyte populations were not noticeably affected by oral administration of Sal-NG and SalpIL2 (p = 0.270 and p = 0.237) compared to saline controls. T helper cell populations were reduced in the SalpIL2 group (14.3%; p < 0.0077) compared to saline controls(20.7%) (Fig. 2). Local pulmonary lymphocytes collected were elevated in SalpIL2 compared to control and Sal-NG treated animals (p < 0.0196 and p < 0.0070 respectively) (Fig. 3). Gross examination of the harvested pulmonary tissues demonstrate the reduction in the mean number of metastatic tumors by SalpIL2 with respect to saline controls (Fig. 4). Histological analysis of the tissues treated with SalpIL2 show a decreased invasion of the metastases into the subpleural space and an increase of mononuclear cells in the area (Fig. 5).

Fig. 1.

A single oral dose of SalpIL2 decreased pulmonary tumor volume by 78% with respect to saline controls (17.07 and 79.76 mm³ respectively; p < 0.0037). Sal-NG tended (p = 0.25) to reduce pulmonary metastatic tumor volume 50% from a mean of 80.0 in control animals to 39.8 mm³. * Indicates statistically significance.

Fig. 2.

SalpIL2 (right column) and Sal-NG (middle column) considerably increase natural killer cells by 93% and 113% (p = 0.0407 and p = 0.0163) with respect to saline-treated (left column) animals. CD 8⁺ T lymphocytes were not substantially increased by a single oral dose of Salmonella spp. CD4⁺ T lymphocytes were decreased (p < 0.0077) by 34% following SalpIL2 treatment. * Indicates statistically significance.

Fig. 3.

In an additional experiment, pulmonary lymphocytes populations in animals treated with SalpIL2 increased local NK cell populations 154% and 224% as compared to control and Sal-NG groups (p < 0.0196 and p < 0.0070, respectively). CD 8+ and CD 4+ T cell populations were not sufficiently detectable for analysis by flow cytometry. * Indicates statistically significance.

Fig. 4A–B.

The photographs represent mean samples from (A) control and (B) SalpIL2 orally treated mice. SalpIL2 substantially reduced the number and volume of surface detectable pulmonary metastases in the treatment model.

Fig. 5A–B.

Histopathological analysis of representative samples displays increased mononuclear infiltrate and decreased structure of tumor. (A) Histological representation of 20x control lung tissues displaying subpleural nodules (arrows) and linear aggregates and (B) 20x SalpIL2 treated tissues. Considerably less invasion into the subpleural space by osteosarcoma metastases may be observed in SalpIL2-treated lungs.

Discussion

While the prognosis for primary osteosarcoma has improved in the last three decades, no such survival advantage has been conferred upon patients with metastatic disease. Survival for patients diagnosed with osteosarcoma dramatically improved after the implementation of neoadjuvant chemotherapy for primary tumors nearly three decades ago. However, the benefits of such treatments have seemingly reached a plateau over the last decade [13, 19]. Individual drugs have been ineffective in treating pulmonary metastases [31], and combined therapy has not substantially altered the course of disease in patients who present with detectable pulmonary osteosarcoma metastases, as the 5-year survival rate continues to hover around 30% [9, 11, 37]. Our previous research demonstrates an antitumor effect in a variety of animal cancer models associated with oral dosing of attenuated Salmonella typhimurium with and without a truncated gene for human IL-2 [1, 7, 8, 27, 29, 33]. This project represents the early assessment of S. typhimurium in an osteosarcoma pulmonary metastatic model. We hypothesized SalpIL2 may substantially reduce osteosarcoma pulmonary metastases and increase splenic and local NK cell populations in a newly developed experimental model.

Our model’s most notable limitation is the absence of a primary tumor. By directly injecting highly metastatic tumor cells into the venous system, we are not allowing for the usual selective pressures normally exhibited on primary tumors in which only truly metastatic cells metastasize. Our model allows for a more timely investigation of an antitumor agent against pulmonary metastatic osteosarcoma. Despite this shortcoming, tumors in this model are grossly and microscopically similar to tumors produced by intratibial injection metastases models and mimic tumors produced in spontaneous orthotopic models [12]. Other limitations include our evaluation of the combined effect of two immunogenic particles (Salmonella and a truncated human IL2) simultaneously within a single species. In addition, we are evaluating tumor responses in an immunocompetent mouse strain, adding to the complexity of the data from which we draw conclusions, but also allowing for extrapolative interpretation for fully functioning biologic systems. The current plasmid construct of SalpIL2 produces a truncated IL-2 within the Salmonella spp. and exports recombinant peptide from the intracellular environment by an unknown mechanism. We do not currently know the fate of the truncated recombinant IL-2 after its synthesis; it may form inclusion bodies or be released upon bacterial lysis. Also, we do not fully understand whether the observed antitumor response is due to the biologic activity of the truncated IL-2 or its ability to initiate an antigenic response within our animal models. Further optimization of Salmonella as a delivery vector will involve utilizing prokaryotic excretion systems for localization of human and mouse homologs of truncated and full-length IL-2 into the local tumor environment.

Interleukin-2, a 15-kDa cytokine produced by activated CD4⁺ T cells and other immune cells, is involved in the activation of T lymphocytes and NK cells. Intravenous injections of liposomes containing IL-2 cDNA are effective in treating pulmonary metastases in canine spontaneous osteosarcomas, and have produced complete remission in some pediatric cases [6, 30, 32]. However, the highly toxic effects of intravenous IL-2 therapy has required investigators to alter the frequency, method of delivery and dosing, limiting the benefits of and response to the therapy [22, 30, 32, 36]. Intravenous interleukin-2 therapy requires multiple injections for results similar to those seen with a single oral dose of attenuated SalpIL2. Other investigators have used other cytokines in S. typhimurium in murine experimental models of pulmonary metastases. In those experiments S. typhimurium was able to reduce breast carcinoma metastases, however treatments required multiple intravenous injections and tissues were evaluated after 50 days [15]. Conversely, the intravenous administration of 2 × 10⁵ K7M2 cells results in mortality of the animal subject by day 28 (data not shown). Other Salmonella strains using secretion mechanisms to deliver tumor specific antigens have been fairly successful when administered orally. However, these constructs were only effective when the tumor also produced the antigen, because tumors without the antigen were resistant to oral administration of S. typhimurium [23].

SalpIL2 appears a safe and effective delivery system that has similar antitumor efficacy compared to other methods of IL2 treatment, but without creating systemic toxicity. S. typhimurium is a facultative intracellular organism that preferentially tracks to and divides within osteosarcoma cells in vitro as well as reduces tumor burden in vivo in our treatment model of osteosarcoma pulmonary metastasis. In an attempt to establish a local delivery system for IL-2 that may diminish or prevent side effects associated with intravenous delivery, an attenuated strain of S. typhimurium was genetically engineered to carry a gene for a truncated human IL-2. Various strains of attenuated S. typhimurium have been investigated by other researchers as biologic vectors for antitumor therapy with limited success when given intravenously [2, 3, 17, 24, 35, 38]. By implementing an oral delivery system, SalpIL2 is able to colonize the Peyer’s patches, liver, lung, and spleen [28], and thus may be used to target primary and metastatic tumor in these tissues.

Attenuated S. typhimurium with a gene for a truncated human IL-2 limits the metastatic tumor burden in experimental models of neuroblastoma and metastatic colorectal adenocarcinoma. In these studies, NK cell populations appear largely responsible for the decrease in tumor burden in salmonella-treated animals [7, 8, 27, 29, 33]. We sought to determine whether previous antitumor mechanisms of SalpIL2 could be applied to an experimental model of metastatic osteosarcoma.

Our data suggest both strains of attenuated S. typhimurium elicited an antitumor response through proliferation of NK cells peripherally as demonstrated by our surrogate measure of splenic lymphocytes. In addition, mice gavaged with SalpIL2 also exhibited local expansion of NK cells in pulmonary tissues likely secondary to the locally produced truncated IL-2 carried by the bacterium. Other subtypes of lymphocytes were also evaluated. Interestingly, CD 8⁺ and CD 4⁺ T cells were either not involved in the local or systemic response or their expansion was suppressed by concomitant salmonella infection. Despite their absence, tumor growth was inhibited. This suggests nonspecific immune killing of tumor is sufficient to reduce metastatic disease associated with osteosarcoma.

Other experimental metastasis models utilizing SalpIL2 demonstrate NK expansion and involvement [7, 8, 27, 29, 33], implicating NK cell receptor involvement in the antitumor mechanism. Future studies within this model are focusing on the prophylactic use of SalpIL2 in metastatic osteosarcoma, local tumor/NK cell interaction, and the systemic cytokine response to tumor and SalpIL2. Further in vitro studies will focus on protein expression of metastatic determinants and endotoxin effects as seen with other S. typhimurium mutants in endothelial cells.

Based on the experimental results, SalpIL2 is effective at eliciting local and systemic NK cell proliferation and reducing murine pulmonary metastases. The addition of SalpIL2 to current treatment regimens may potentially result in improved prognosis and disease-free survival for osteosarcoma patients in the future.