Bacteria-based immune therapies for cancer treatment.

Engineered bacterial therapies that target the tumor immune landscape offer a new class of cancer immunotherapy. Salmonella enterica and Listeria monocytogenes are two species of bacteria that have been engineered to specifically target tumors and serve as delivery vessels for immunotherapies. Therapeutic bacteria have been engineered to deliver cytokines, gene silencing shRNA, and tumor associated antigens that increase immune activation. Bacterial therapies stimulate both the innate and adaptive immune system, change the immune dynamics of the tumor microenvironment, and offer unique strategies for targeting tumors. Bacteria have innate adjuvant properties, which enable both the delivered molecules and the bacteria themselves to stimulate immune responses. Bacterial immunotherapies that deliver cytokines and tumor-associated antigens have demonstrated clinical efficacy. Harnessing the diverse set of mechanisms that Salmonella and Listeria use to alter the tumor-immune landscape has the potential to generate many new and effective immunotherapies.

Bacterial delivery of therapeutic proteins to the nuclei of cancer cells.

Targeting nucleic targets with therapeutic proteins would enhance the treatment of hard-to-treat cancers. However, exogenous proteins are excluded from the nucleus by both the cellular and nuclear membranes. We have recently developed Salmonella that deliver active proteins into the cytoplasm of cancer cells. Here, we hypothesized that bacterially delivered proteins accumulate within nuclei, nuclear localization sequences (NLSs) increase delivery, and bacterially delivered proteins kill cancer cells. To test this hypothesis, we developed intranuclear delivering (IND) Salmonella and quantified the delivery of three model proteins. IND Salmonella delivered both ovalbumin and green fluorescent protein to nuclei of MCF7 cancer cells. The amount of protein in nuclei was linearly dependent on the amount delivered to the cytoplasm. The addition of a NLSs increased both the amount of protein in each nucleus and the number of nuclei that received protein. Delivery of Omomyc, a protein inhibitor of the nuclear transcript factor, Myc, altered cell physiology, and significantly induced cell death. These results show that IND Salmonella deliver functional proteins to the nucleus of cancerous cells. Extending this method to other transcription factors will increase the number of accessible targets for cancer therapy.

The Effects of Critical Care Nutrition on Weight Gain in African Penguin (Spheniscus demersus) Chicks.

Nutritional support in malnourished animals is an essential aspect of wildlife rehabilitation; this support is especially relevant when providing lifesaving nutrition to endangered species such as the African penguin (Spheniscus demersus). This study investigated the short-term effects of a commercially available, semi-elemental, critical care diet compared with a hand-made fish formula. Twenty-one African penguin chicks were selected on admission to the Southern African Foundation for the Conservation of Costal Birds in Cape Town, South Africa, in November 2015. Initial assessment included body weight, a full clinical exam, white blood cell count, packed cell volume, and total plasma protein. Ten animals received the commercial critical care diet, whereas a control group of 11 animals were fed hand-made formula for the 2-week study period. All animals were weighed daily and blood sampling was repeated after 14 days. The median weight of both groups increased significantly over 14 days (critical care diet χ2 = 10.1, P = 0.002; control χ2 = 7.4, P = 0.006). The difference was not significant between the groups for start weight (χ2 = 0.1, P = 0.725) or end weight (χ2 = 0, P = 1.000) and was not significantly different in the change over time for either absolute numbers (χ2 = 1.7, P = 0.193) or percent gain (χ2 = 0.8, P = 0.36). The values for packed cell volume, total plasma protein, and white blood cell count increased in all animals after the 14-day study period was complete. On the basis of the results of this study, it was determined that the differing diets led to similar weight gain.

Targeting circulating monocytes with CCL2-loaded liposomes armed with an oncolytic adenovirus.

Oncolytic viruses (OVs) selectively replicate in and destroy cancer cells resulting in anti-tumor immunity. However, clinical use remains a challenge because of virus clearance upon intravenous delivery. OV packaging using a nanomedicine approach could overcome this. Here we encapsulate an oncolytic adenovirus (Ad[I/PPT-E1A]) into CCL2-coated liposomes in order to exploit recruitment of CCR2-expressing circulating monocytes into tumors. We demonstrate successful encapsulation of Ad[I/PPT-E1A] into CCL2-coated liposomes that were preferentially taken up by CCR2-expressing monocytes. No complex-related toxicities were observed following incubation with prostate tumor cells and the encapsulation did not affect virus oncolytic activity in vitro. Furthermore, intravenous administration of our nanomedicine resulted in a significant reduction in tumor size and pulmonary metastasis in prostate cancer-bearing mice whereby a 1000-fold less virus was needed compared to Ad[I/PPT-E1A] alone. Taken together our data provide an opportunity to target OVs via circulation to inaccessible tumors using liposome-assisted drug delivery.

Detection of tumors with fluoromarker-releasing bacteria.

Combining the specificity of tumor-targeting bacteria with the sensitivity of biomarker detection would create a screening method able to detect small tumors and metastases. To create this system, we genetically modified an attenuated strain of Salmonella enterica to release a recombinant fluorescent biomarker (or fluoromarker). Salmonella expressing ZsGreen were intravenously administered to tumor-bearing mice and fluoromarker production was induced after 48 hr. The quantities and locations of bacteria and ZsGreen were measured in tumors, livers and spleens by immunofluorescence, and the plasma concentration of ZsGreen was measured using single-layer ELISA. In the plasma, the ZsGreen concentration was in the range of 0.5-1.5 ng/ml and was dependent on tumor mass (with a proportion of 0.81 ± 0.32 ng·ml-1 ·g-1 ). No adverse reaction to ZsGreen or bacteria was observed in any mice. ZsGreen was released at an average rate of 4.3 fg·CFU-1 ·hr-1 and cleared from the plasma with a rate constant of 0.259 hr-1 . ZsGreen production was highest in viable tissue (7.6 fg·CFU-1 ·hr-1 ) and lowest in necrotic tissue (0.47 fg·CFU-1 ·hr-1 ). The mass transfer rate constant from tumor to blood was 0.0125 hr-1 . Based on these measurements, this system has the capability to detect tumors as small as 0.12 g. These results demonstrate four essential mechanisms of this method: (i) preferential tumor colonization by bacteria, (ii) fluoromarker release in vivo, (iii) fluoromarker transport through tumor tissue and (iv) slow enough systemic clearance to enable measurement. This bacteria-based blood test would be minimally invasive and has the potential to identify previously undetectable microscopic tumors.

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion.

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host-bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.

Tissue transport affects how treatment scheduling increases the efficacy of chemotherapeutic drugs.

A method to predict the effect of tissue transport on the scheduling of chemotherapeutic treatment could increase efficacy. Many drugs with desirable pharmacokinetic properties fail in vivo due to poor transport through tissue. To predict the effect of treatment schedule on drug efficacy we developed an in silico method that integrates diffusion through tissue and cell binding into a pharmacokinetic model. The model was evaluated with an array of theoretical drugs that had different rates of diffusivity, binding, and clearance. The efficacy of each drug, quantified as the fraction of cells killed, was calculated for twenty dosage schedules. Simulations showed that efficacy strongly depended on tissue transport, with a range of 0.00 to 99.99%, despite each drug having equal plasma areas under the curve (AUC). For most drugs, schedules that increased exposure also increased efficacy. Drugs with fast clearance benefited the most from increasing the number of doses and this was most effective for those with intermediary binding. All drugs with slow diffusivity were ineffective. For a subset of drugs, increasing the number of doses decreased efficacy. This phenomenon was unexpected because, when considering uptake into tissue, sustained plasma levels from multiple doses are generally assumed to be more effective. This counterintuitive decrease in efficacy was caused by drug retention within tumor tissue. These results established a set of rules that suggests how transport parameters affect the efficacy of drugs at different schedules. The two most predominant rules are (1) multiple doses improve efficacy for drugs with fast clearance, fast diffusivity and low to intermediate cell binding; and (2) one dose is most effective for drugs with slow clearance, slow diffusivity or strong cell binding. Understanding the role of tissue transport when determining drug treatment schedules would improve the outcome of preclinical animal experiments and early clinical trials.

Quorum-sensing Salmonella selectively trigger protein expression within tumors.

Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 10(10) cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 10(10) cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 10(10) cfu/g) was less than the critical density (0.11 × 10(10) cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue.

Superficial Chronic Ulcerative Dermatitis (SCUD) in Psittacine Birds: Review of 11 Cases (2008-2016).

We reviewed 11 cases of superficial chronic ulcerative dermatitis (SCUD) in psittacine birds that presented to an exotic animal practice over an 8-year period. African grey parrots ( Psittacus erithacus) were overrepresented, accounting for 55% of SCUD-affected birds. All affected birds were hand-reared pets and bonded strongly to their owners. In all cases, fungal culture results were negative. The most commonly cultured bacteria were Enterobacter cloacae (27%), followed by Escherichia coli, and Staphylococcus aureus (both 18%). An underlying cause of behavioral self-mutilation with secondary infection of dermatitis lesions by opportunistic bacteria was suspected in 10 of 11 cases. The axillae were the most common anatomic sites affected (45%). In two cases, more than one site was affected concurrently. Mean age of birds affected was 11.4 years. Two birds had suffered from SCUD previously, with 1 bird having suffered from 3 previous episodes and was euthanized as a result. All birds were treated with systemic antibiotics, topical antibacterial preparations, and systemic nonsteroidal anti-inflammatory drugs. A neck collar was applied in all cases. Median time to clinical resolution was 2 months (range, 1-21 months). Excluding the bird that was euthanized, all birds showed a positive response to treatment.

Engineered bacteria detect spatial profiles in glucose concentration within solid tumor cell masses.

Tumor heterogeneity makes cancer difficult to treat. Many small molecule cancer drugs target rapidly dividing cells on the periphery of tumors but have difficulty in penetrating deep into tumors and are ineffective at treating entire tumors. Targeting both rapidly dividing and slower growing regions of tumors is essential to effectively treat cancer. A cancer drug carrier that penetrates deep into tumors and identifies metabolically activity could supply treatment to those areas based on the local microenvironment. We hypothesized that glucose sensing bacteria could identify sugar gradients in solid tumors. To test this hypothesis, a genetic circuit was designed to trigger expression of a green fluorescent protein (GFP) reporter through the chemotaxis-osmoporin fusion protein, Trz1, a receptor for sensing glucose and ribose sugars. E. coli equipped with the Trz1-GFP expression system, were administered to an in vitro model of a continuously perfused tumor tissue that mimics systemic delivery and clearance of bacteria through a blood vessel adjacent to a solid tumor. The level of GFP expressed, per bacterium, was time independent and indicated the glucose concentration as a function of penetration depth within the microfluidic tumors. The measured glucose concentration, correlated (P-value = 2.6 × 10(-5) ) with tumor cell viability as a function of depth. Mathematical analysis predicted drug delivery by glucose-sensing bacteria would eliminate a higher percentage of the viable tumor cell population than a systemically administered drug. Glucose-sensing bacteria could deliver cancer therapies with increased drug penetration and nutrient-dependent dosing to continuously treat viable regions of cancer tissue that have a higher prevalence for metastatic dissemination. Biotechnol. Bioeng. 2016;113: 2474-2484. © 2016 Wiley Periodicals, Inc.

Cloacal Prolapse in Raptors: Review of 16 Cases.

Sixteen cases of cloacal prolapse in raptors were reviewed in this study. Colonic prolapse was the most common presentation (56% of cases). Red-tailed hawks ( Buteo jamaicensis ) were overrepresented, comprising 66% of colonic prolapse cases. In cases of colonic prolapse, postsurgical stricture formation was a commonly identified complication after resection and anastomosis of the colon. A novel technique was used in 2 cases of colonic prolapse, in which sterile, semirigid rubber tubing was placed in the distal colon and removed per-cloaca at the end of the procedure; this facilitated a secure, fluid-tight anastomosis while maintaining sufficient intestinal lumen. Oviductal prolapse (31% of cases) was associated with the most guarded prognosis (40% treatment success). Cloacoliths were treated successfully in 2 birds (13% of cases) by minimally invasive per-cloacal manual removal.

Distraction Osteogenesis Correction of Mandibular Ramis Fracture Malunion in a Juvenile Mute Swan ( Cygnus olor ).

A juvenile mute swan (Cygnus olor) was presented with right lateral deviation of the mandible. Radiographs demonstrated a healed fracture of the right mandibular ramis, which had compromised osteogenesis. A corrective osteotomy was performed and an osteogenic distractor was inserted over the lateral aspect of the right mandible. Dental acrylic implants were fixed to the rhinotheca to correct rotational alignment. A pharyngostomy tube was placed to facilitate administration of nutrition and medication. Postoperative images confirmed correct alignment of the mandible in relation to the maxilla. Implants were removed and postoperative complications were not reported. This is the first report of an osteogenic distractor used to correct mandibular deviation in an avian species. Distraction osteogenesis should be considered as a valid surgical option in juvenile or adult avian patients with pathologic bone shortening.

Calcinosis Circumscripta in the Digital Extensor Tendon of a Tawny Eagle (Aquila rapax).

A 9-month-old, captive-bred, female tawny eagle (Aquila rapax) presented with a nonpainful, firm, nodular structure attached to the digital extensor tendon. The mass was surgically resected without complications and was submitted for histopathologic examination. Grossly, cut surfaces of the lesion had chalky-white deposits. Histologically, the resected tissue was identified as calcinosis circumscripta. No recurrence was observed in follow-up after 6 and 12 months. To our knowledge, this is the first case of calcinosis circumscripta in a bird involving a limb extremity, similar to the presentation recognized more commonly in domestic animals. Calcinosis circumscripta should be included in the differential diagnosis list for nodular masses attached to the tendons in birds.

Persistent enhancement of bacterial motility increases tumor penetration.

Motile bacteria can overcome the transport limitations that hinder many cancer therapies. Active bacteria can penetrate through tissue to deliver treatment to resistant tumor regions. Bacterial therapy has had limited success, however, because this motility is heterogeneous, and within a population many individuals are non-motile. In human trials, heterogeneity led to poor dispersion and incomplete tumor colonization. To address these problems, a swarm-plate selection method was developed to increase swimming velocity. Video microscopy was used to measure the velocity distribution of selected bacteria and a microfluidic tumor-on-a-chip device was used to measure penetration through tumor cell masses. Selection on swarm plates increased average velocity fourfold, from 4.9 to 18.7 µm/s (P < 0.05) and decreased the number of non-motile individuals from 51% to 3% (P < 0.05). The selected phenotype was both robust and stable. Repeating the selection process consistently increased velocity and eliminated non-motile individuals. When selected strains were cryopreserved and subcultured for 30.1 doublings, the high-motility phenotype was preserved. In the microfluidic device, selected Salmonella penetrated deeper into cell masses than unselected controls. By 10 h after inoculation, control bacteria accumulated in the front 30% of cell masses, closest to the flow channel. In contrast, selected Salmonella accumulated in the back 30% of cell masses, farthest from the channel. Selection increased the average penetration distance from 150 to 400 µm (P < 0.05). This technique provides a simple and rapid method to generate high-motility Salmonella that has increased penetration and potential for greater tumor dispersion and clinical efficacy.

Bacterial delivery of Staphylococcus aureus α-hemolysin causes regression and necrosis in murine tumors.

Bacterial therapies, designed to manufacture therapeutic proteins directly within tumors, could eliminate cancers that are resistant to other therapies. To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells. To date, these properties have not been shown for a single protein. The gene for Staphylococcus aureus α-hemolysin (SAH), a pore-forming protein, was cloned into Escherichia coli. These bacteria were injected into tumor-bearing mice and volume was measured over time. The location of SAH relative to necrosis and bacterial colonies was determined by immunohistochemistry. In culture, SAH was released and killed 93% of cancer cells in 24 hours. Injection of SAH-producing bacteria reduced viable tissue to 9% of the original tumor volume. By inducing cell death, SAH moved the boundary of necrosis toward the tumor edge. SAH diffused 6.8 ± 0.3 µm into tissue, which increased the volume of affected tissue from 48.6 to 3,120 µm(3). A mathematical model of molecular transport predicted that SAH efficacy is primarily dependent on colony size and the rate of protein production. As a payload protein, SAH will enable effective bacterial therapy because of its ability to diffuse in tissue, kill cells, and expand tumor necrosis.

Ileo-ceco-rectal Intussusception Requiring Intestinal Resection and Anastomosis in a Tawny Eagle (Aquila rapax).

A 23-year-old male tawny eagle (Aquila rapax) was examined because of sudden onset of lethargy, regurgitation, and hematochezia. An intestinal obstruction was suspected based on radiographic findings, and an ileo-ceco-rectal intussusception was confirmed by coelioscopy. A 14.3-cm section of intestine was resected before an intestinal anastomosis was done. Coelomic endoscopic examination confirmed a postsurgical complication of adhesions between the intestinal anastomosis and the dorsal coelomic wall, resulting in a partial luminal stricture and requiring surgical removal of the adhesions. Rectoscopy was useful in diagnosing a mild luminal stricture related to the second surgery. Complete recovery was observed 2 months after surgery. Lack of further complications in the 2 years after surgery demonstrates good tolerance of intestinal resection and anastomosis of a large segment of bowel in an eagle. This report is the first reported case of intussusception in an eagle and emphasizes the potential use of endoscopic examination in the diagnosis as well as in the management of complications.

Evaluation of the phenol red thread tear test in falconiformes.

Falconiformes are active searchers and hunters that require visual precision to catch their prey and survive in the wild. Despite this, ocular disease is likely to be underdiagnosed in these species, at least in part because of limited proven diagnostic methods and lack of published scientific data. Tear film deficiency is recognized as an important ocular surface disease in dogs but has not been well evaluated in birds. To evaluate the phenol red thread (PRT) tear test as an alternative method to the Schirmer tear test (STT) for determining tear production in Falconiformes, we assessed the PRT tear test twice for each eye in 21 birds from the genus Falco. The mean PRT test values for the right (OD) and left (OS) eyes were OD=30.2±4.6 mm/15 s and OS=29.1±3.7 mm/15 s, with an overall PRT test value for both eyes (OU) of 30.6±4.2 mm/15 s. Good reproducibility was seen with the PRT test as shown by the strong positive correlation between the first and second tests in the birds. The PRT test method provides repeatable results that are easy to obtain, easy to read, and achieved under the same conditions as the STT.

Lipid A controls the robustness of intratumoral accumulation of attenuated Salmonella in mice.

Engineered Salmonella have the potential to treat cancers that are not responsive to standard molecular therapies. This potential has not been realized because colonization in human tumors is insufficient and variable as shown in preliminary phase I trials. Recent studies have shown that Salmonella colonization is associated with an inflammatory response mediated by tumor necrosis factor (TNF). An injectable agent, molecular lipid A, could be used to control bacterial accumulation because it induces TNF production and is rapidly cleared. We hypothesized that concurrently administrating lipid A with attenuated Salmonella would increase intratumoral accumulation, improve the robustness of tumor-targeting and be nontoxic. To test this hypothesis, Salmonella and lipid A were injected into mice with 4T1 mammary tumors. Colonization was quantified after 48 hr using anti-Salmonella immunofluorescence. A 2 µg/mouse dose of lipid A increased the area of colonized tissue fourfold, reduced variance 50% and ensured colonization in all mice. Comparatively, Salmonella failed to colonize some control mice, similar to human trials. No toxicity was observed in any treated mice. The fraction of tumor tissue with more than 25% bacterial coverage was eight times greater for treated mice compared to controls. Lipid A treatment also reduced the maximum average distance of tissue to Salmonella colonies from 1348 to 260 µm. A mathematical model of bacterial drug production predicted that 2 µg lipid A would increase tumor cell death by 82%. These results suggest that lipid A could solve the clinical challenges of Salmonella therapy and enable safe and robust treatment of cancer with bacteria.

Identification of Staphylococcus aureus α-hemolysin as a protein drug that is secreted by anticancer bacteria and rapidly kills cancer cells.

Targeted bacterial delivery of anticancer proteins has the ability to overcome therapeutic resistance in tumors that limits the efficacy of chemotherapeutics. The ability of bacteria to specifically target tumors allows for delivery of aggressive proteins that directly kill cancer cells and cannot be administered systemically. However, few proteins have been tested for this purpose. To identify effective molecules, we systematically sorted proteins that have been shown to cause mammalian cell death. The genes for five proteins were selected and cloned into Escherichia coli and Salmonella. Supernatant from cultures of the transformed bacteria was applied to flasks of MCF-7 mammary carcinoma cells to identify proteins that (1) were expressed, (2) secreted, and (3) rapidly killed cancer cells. Time-lapse images were taken to visualize mammalian cell morphology. Of the investigated proteins, α-hemolysin from Staphylococcus aureus (SAH) was the most promising because it was secreted, caused trauma to cellular membranes, and induced oncosis in 18 min. After exposure for 6 h, SAH decreased cell viability by 90%. In comparison, the positive control, Pseudomonas aeruginosa exotoxin A (PEA), required 11 days to achieve a similar effect, when administered at 3,000 times its LC50 . The maximum death rate induced by SAH was calculated to be a reduction in cell viability of 7.1% per min, which was 200-fold faster than the PEA control. Two proteins, Dermonecrotic Toxin and Phospholipase C were active when extracted from the bacterial cytoplasm but were not secreted. This investigation revealed for the first time SAH as a potent anticancer drug for delivery by bacteria because of its ability to be secreted in a fully functional form and aggressively kill cancer cells.