Salmonella enterica Typhimurium engineered for nontoxic systemic colonization of autochthonous tumors.

Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium (S. Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S. Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 106 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.

Virulence-attenuated Salmonella engineered to secrete immunomodulators reduce tumour growth and increase survival in an autochthonous mouse model of breast cancer.

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli.

Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.

Antioxidant oils and Salmonella enterica Typhimurium reduce tumor in an experimental model of hepatic metastasis.

Fruit seeds high in antioxidants have been shown to have anticancer properties and enhance host protection against microbial infection. Recently we showed that a single oral dose of Salmonella enterica serovar Typhimurium expressing a truncated human interleukin-2 gene (SalpIL2) is avirulent, immunogenic, and reduces hepatic metastases through increased natural killer cell populations in mice. To determine whether antioxidant compounds enhance the antitumor effect seen in SalpIL2-treated animals, we assayed black cumin (BC), black raspberry (BR), and milk thistle (MT) seed oils for the ability to reduce experimental hepatic metastases in mice. In animals without tumor, BC and BR oil diets altered the kinetics of the splenic lymphocyte response to SalpIL2. Consistent with previous reports, BR and BC seed oils demonstrated independent antitumor properties and moderate adjuvant potential with SalpIL2. MT oil, however, inhibited the efficacy of SalpIL2 in our model. Based on these data, we conclude that a diet high in antioxidant oils promoted a more robust immune response to SalpIL2, thus enhancing its antitumor efficacy.

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

PURPOSE: The current management of osteosarcoma (OS) entails an aggressive preoperative and postoperative chemotherapeutic regimen with limb salvage surgery. Despite these efforts, relapse-free survival is less than 60% in patients with classic OS, whereas most patients relapse with pulmonary metastases. In these studies, we sought to prevent the establishment of pulmonary metastases from OS with a single oral dose of SalpIL2. METHODS: Mice were administered attenuated Salmonella typhimurium with (SalpIL2) and without a gene for human interleukin 2 (Sal-NG) 7 days before challenge with 2 x 10(5) OS cells via tail vein. Three weeks after injection, mice were harvested for splenic lymphocytes and tumor enumeration. RESULTS: Prophylaxis with attenuated SalpIL2 significantly reduces pulmonary metastases in number and volume (P < .0001 and P < .0001) with respect to saline controls. Furthermore, splenic natural killer cell populations were increased 396% with SalpIL2 (P < .0007) and 426% with Sal-NG (P < .0003) compared to nontreated groups. CONCLUSIONS: Host natural killer response is greatly amplified and maybe partially responsible for the effective immune response against the formation of pulmonary metastases. A single oral dose of SalpIL2 may be a novel form of adjuvant therapy for patients after early detection of primary OS.

Attenuated Salmonella typhimurium with IL-2 gene reduces pulmonary metastases in murine osteosarcoma.

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 x 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 invades and decreases tumor burden in neuroblastoma.

INTRODUCTION: We have previously shown that Salmonella elicits an antitumor response against hepatic adenocarcinomatous metastases. In vitro studies have demonstrated both intracellular invasion and proliferation of Salmonella within cultured neuroblastoma cells. We sought to demonstrate in vivo invasion, proliferation, and a potential antitumor response. METHODS: A murine model for retroperitoneal neuroblastoma was established with viable neuroblastoma cells. A green fluorescent protein (GFP--Clontech, Palo Alto, CA) gene was inserted into our attenuated Salmonella species via electroporation. Fourteen days after retroperitoneal injection, the Salmonella typhimurium-pGFP construct was administered and studied. In separate experiments, the antitumor effect against neuroblastoma was studied in controls, Salmonella lacking an interleukin 2 (IL-2) gene (Sal-NG), and Salmonella containing an IL-2 gene (Salmonella-pIL2 ). RESULTS: Consistent with previous reports, 74% of mice injected were found to have recognizable tumors. Salmonella was present within tumor cells. Average tumor volumes for control, Sal-NG, and Salmonella-pIL2 mice were 2024.3, 749.5, and 332.4 mm3 , respectively (P < .0001). Tumor weights for control, Sal-NG, and Salmonella-pIL2 mice were 2.218, 0.880, and 0.377 g, respectively (P < .0001). CONCLUSIONS: Attenuated Salmonella species can now be tracked via fluorescent microscopy within tumor cells. Furthermore, an 84% reduction in tumor burden was observed in those animals gavage fed Salmonella with the IL-2 gene.

Preferential proliferation of attenuated Salmonella typhimurium within neuroblastoma.

BACKGROUND/PURPOSE: Attenuated Salmonella typhimurium, a facultative intracellular parasite that colonizes the liver, has been shown to accumulate within extrahepatic malignancies. The authors sought to define a mechanism for attenuated Salmonella accumulation within cancer cells compared with hepatocytes. METHODS: Invasion and intracellular proliferation of attenuated Salmonella was assessed through an in vitro assay performed on neuroblasoma, osteosarcoma, hepatoma, and colon adenocarcinoma cell lines and compared with freshly isolated mouse hepatocytes. RESULTS: The efficiency of attenuated S typhimurium invasion into hepatocytes was greater than any malignant cell line (3.8 v 0.46; P <.04). However, the intracellular proliferation of the bacteria was most abundant within neuroblastoma, exceeding the proliferation within hepatocytes (14.3 v 6.2; P <.01). CONCLUSIONS: Attenuated S typhimurium may prove to be an effective in vivo immunotherapy for the local delivery of therapeutic proteins to neuroblastoma.

Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model.

PURPOSE: The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model. METHODS: A single, oral 10(8) cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases. Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival. RESULTS: Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls. The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes. Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days. All long-term survivors were devoid of hepatic tumor. CONCLUSIONS: Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit. Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer.

Cyclooxygenase-2 inhibition augments the hepatic antitumor effect of oral Salmonella typhimurium in a model of mouse metastatic colon cancer.

INTRODUCTION: Oral inoculation with a nontoxic, attenuated strain of Salmonella typhimurium reduces tumor burden and improves survival in a mouse model of metastatic colon cancer. These effects are likely mediated by S. typhimurium-induced increases in hepatic natural killer leukocytes. Cyclooxygenase-2 inhibitors may mediate antitumor effects through antiangiogenic, immune, or proapoptotic pathways. We hypothesized that cyclooxygenase-2 inhibitors would act synergistically with S. typhimurium, resulting in additional antitumor effects. METHODS: Four groups of mice were studied: control, S. typhimurium alone, cyclooxygenase-2 inhibitor alone, and S. typhimurium plus cyclooxygenase-2 inhibitor. Mice were given normal drinking water (control, S. typhimurium alone) or water with 1,600 parts per million cyclooxygenase-2 inhibitor (cyclooxygenase-2 inhibitor alone, and S. typhimurium plus cyclooxygenase-2 inhibitor) and orally inoculated with saline (control, cyclooxygenase-2 inhibitor alone) or 10(9) S. typhimurium (S. typhimurium alone, S. typhimurium plus cyclooxygenase-2 inhibitor). Twenty-four hours later, all mice underwent laparotomy, and 5 x 10(4) MCA38 murine adenocarcinoma cells were injected into the spleen. On Day 14, hepatic tumor number and tumor volume was quantitated and hepatic leukocytes were analyzed by flow cytometry. RESULTS: Compared with control mice orally inoculated with saline, S. typhimurium-treated mice had fewer and smaller tumors; mice treated with cyclooxygenase-2 inhibitor alone had tumor burden similar to control mice, and mice treated with S. typhimurium plus cyclooxygenase-2 inhibitor had fewer and smaller tumors compared with all other groups. Increased liver natural killer cells and decreased CD4+ and CD8+ T cells were observed in both S. typhimurium-treated groups. No alterations in hepatic leukocyte phenotype were observed in mice receiving cyclooxygenase-2 inhibitor alone. CONCLUSION: Oral cyclooxygenase-2 inhibitor appeared to act synergistically with S. typhimurium to reduce tumor burden. This combination therapy may have clinical application in the treatment or prevention of hepatic metastases associated with colorectal cancer.

Surgical experience in patients with cystic fibrosis: a 25-year perspective.

The spectrum of surgical diseases in patients with cystic fibrosis (CF) has not been comprehensively studied. A retrospective review of 792 consecutive patients with CF presenting over a 25 year period (1970-1994) was made to determine the incidence of operations, procedures performed, complications encountered, and impact on physical development and pulmonary function tests (PFTs). A total of 191 operations were performed on 130 (16%) of the 792 patients; 98 operations (51%) were abdominal, 58 (30%) thoracic, and 31 (16%) hernias; 64 were male, and 66 female; average age was 14 +/- 10 years. Complications occurred in 12 (16%); 9 deaths were from progressive respiratory failure, 2 from superficial wound infections, and 1 from an episode of line sepsis. In the first 15 years, 9 complications occurred in 126 operations vs. 3 in 73 operations during the last 10 years. Operations were classified as emergent, urgent, or elective. Of the 9 deaths, 8 occurred after emergent or urgent operations (4 abdominal and 4 thoracic), while 1 death occurred following elective herniorrhaphy. For each subgroup, (abdominal, thoracic, and hernia), there was no difference in height/weight indicies, peak flow, forced vital capacity (FVC), forced expired volume in 1 sec (FEV(1)), or FEV(1)/FVC ratio when comparing 1 year preoperation and 1 year postoperation. In conclusion, patients in this high-risk population were operated on with few complications, but when a complication occurred it tended to be pulmonary and fatal (4.7% of all operations). Furthermore, operations did not cause significant deteriorations in PFTs and they did not cause these children to fall off their expected age-adjusted growth curves.