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 9 10 5 (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.

Attenuated Salmonella typhimurium with interleukin 2 gene prevents the establishment of pulmonary metastases in a model of osteosarcoma

“…Previously, we reported that a single oral dose of SalpIL2 significantly increased pulmonary and splenic NK cells in a murine model of OS. In both our treatment and prevention models, we were also unable to detect significant differences in splenic NK or CD 8 + T-cell populations but did find a significant increase of local NK cells in the SalpIL2 group with respect to saline and Sal-NG in the treatment model animals [15]. The presence of an increased local NK cell population suggests functionality of the recombinant IL-2 locally within the pulmonary tissue; however, the fate of the recombinant IL-2 is currently unknown.…”

“…We found the peak NK cell response on day 14 postgavage; thus, tumor cells implanting on day 7 will encounter the highest rate of NK cell proliferation during the infection of SalpIL2. In our prophylactic model, a more dramatic reduction in tumor volume was observed than in our previous treatment model of metastatic OS [15]. Tumor volume was reduced more than 6-fold with SalpIL2 prophylaxis with respect to saline controls.…”

“…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, Ill). On day 0, animals were prepared and administered 2 × 10 5 K7M2 mouse OS cells using welldescribed techniques [15]. Mice were selected at random from their respective groups for IV tail vein injection.…”

“…The gene for human IL-2 was ligated into the plasmid pYA292, renamed pIL2, and electroporated into χ4550, and the resulting bacteria were renamed SalpIL2. In previous experiments, SalpIL2 has been shown to reduce hepatic metastases of colon adenocarcinoma, the volume and mass of primary neuroblastoma, and OS pulmonary metastases [10][11][12][13][14][15]. Experiments described in these studies attempt to determine the antitumor capabilities of SalpIL2 in a prophylactic model of pulmonary metastasis in OS.…”

Safety and immunogenicity of Salmonella typhimurium expressing C-terminal truncated human IL-2 in a murine model

“…We engineered an attenuated strain of S. enterica Typhimurium, χ4550, to express a truncated human interleukin-2 (IL-2) protein and named it SalpIL2 . A single oral dose of SalpIL2, in animals with established pulmonary osteosarcoma metastases, significantly reduced tumor burden while increasing pulmonary natural killer (NK) cell populations as compared to non-IL2-expressing S. enterica and saline controls 7. Furthermore, SalpIL2 oral administration prior to tumor injection prevented the establishment of metastatic tumors and increased splenic and hepatic NK cell populations 8,9.…”