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.

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.

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.

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.

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.

Controlled production of lipopolysaccharides increases immune activation in Salmonella treatments of cancer.

Immunotherapies have revolutionized cancer treatment. These treatments rely on immune cell activation in tumours, which limits the number of patients that respond. Inflammatory molecules, like lipopolysaccharides (LPS), can activate innate immune cells, which convert tumour microenvironments from cold to hot, and increase therapeutic efficacy. However, systemic delivery of lipopolysaccharides (LPS) can induce cytokine storm. In this work, we developed immune-controlling Salmonella (ICS) that only produce LPS in tumours after colonization and systemic clearance. We tuned the expression of msbB, which controls production of immunogenic LPS, by optimizing its ribosomal binding sites and protein degradation tags. This genetic system induced a controllable inflammatory response and increased dendritic cell cross-presentation in vitro. The strong off state did not induce TNFα production and prevented adverse events when injected into mice. The accumulation of ICS in tumours after intravenous injection focused immune responses specifically to tumours. Tumour-specific expression of msbB increased infiltration of immune cells, activated monocytes and neutrophils, increased tumour levels of IL-6, and activated CD8 T cells in draining lymph nodes. These immune responses reduced tumour growth and increased mouse survival. By increasing the efficacy of bacterial anti-cancer therapy, localized production of LPS could provide increased options to patients with immune-resistant cancers.

Intracellular delivery of oncolytic viruses with engineered Salmonella causes viral replication and cell death.

As therapies, oncolytic viruses regress tumors and have the potential to induce antitumor immune responses that clear hard-to-treat and late-stage cancers. Despite this promise, clearance from the blood prevents treatment of internal solid tumors. To address this issue, we developed virus-delivering Salmonella (VDS) to carry oncolytic viruses into cancer cells. The VDS strain contains the PsseJ-lysE delivery circuit and has deletions in four homologous recombination genes (ΔrecB, ΔsbcB, ΔsbcCD, and ΔrecF) to preserve essential hairpins in the viral genome required for replication and infectivity. VDS delivered the genome for minute virus of mice (MVMp) to multiple cancers, including breast, pancreatic, and osteosarcoma. Viral delivery produced functional viral particles that are cytotoxic and infective to neighboring cells. The release of mature virions initiated new rounds of infection and amplified the infection. Using Salmonella for delivery will circumvent the limitations of oncolytic viruses and will provide a new therapy for many cancers.

Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue.

Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity. The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cell communication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the PBAD promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies.

Build-a-bug workshop: Using microbial-host interactions and synthetic biology tools to create cancer therapies.

Many systemically administered cancer therapies exhibit dose-limiting toxicities that reduce their effectiveness. To increase efficacy, bacterial delivery platforms have been developed that improve safety and prolong treatment. Bacteria are a unique class of therapy that selectively colonizes most solid tumors. As delivery vehicles, bacteria have been genetically modified to express a range of therapies that match multiple cancer indications. In this review, we describe a modular "build-a-bug" method that focuses on five design characteristics: bacterial strain (chassis), therapeutic compound, delivery method, immune-modulating features, and genetic control circuits. We emphasize how fundamental research into gut microbe pathogenesis has created safe bacterial therapies, some of which have entered clinical trials. The genomes of gut microbes are fertile grounds for discovery of components to improve delivery and modulate host immune responses. Future work coupling these delivery vehicles with insights from gut microbes could lead to the next generation of microbial cancer therapy.

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Introduction: Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. Methods: To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. Results: We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. Discussion: This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

Microbial Imbalance Induces Inflammation by Promoting Salmonella Penetration through the Mucosal Barrier.

The balance of microbial species in the intestine must be maintained to prevent inflammation and disease. Healthy bacteria suppress infection by pathogens and prevent disorders such as inflammatory bowel diseases (IBDs). The role of mucus in the relation between pathogens and the intestinal microbiota is poorly understood. Here, we hypothesized that healthy bacteria inhibit infection by preventing pathogens from penetrating the mucus layer and that microbial imbalance leads to inflammation by promoting the penetration of the mucosal barrier. We tested this hypothesis with an in vitro model that contains mucus, an epithelial cell layer, and resident immune cells. We found that, unlike probiotic VSL#3 bacteria, Salmonella penetrated the mucosal layers and induced the production of interleukin-8 (IL-8) and tumor necrosis factor (TNF)-α. At ratios greater than 104:1, probiotic bacteria suppressed the growth and penetration of Salmonella and reduced the production of inflammatory cytokines. Counterintuitively, low densities of healthy bacteria increased both pathogen penetration and cytokine production. In all cases, mucus increased Salmonella penetration and the production of cytokines. These results suggest that mucus lessens the protective effect of probiotic bacteria by promoting barrier penetration. In this model, a more imbalanced microbial population caused infection and inflammation by selecting pathogens that are more invasive and immunogenic. Combined, the results suggest that the depletion of commensal bacteria or an insufficient dosage of probiotics could worsen an infection and cause increased inflammation. A better understanding of the interactions between pathogens, healthy microbes, and the mucosal barrier will improve the treatment of infections and inflammatory diseases.

Escherichia coli Swimming back Toward Stiffer Polyetheylene Glycol Coatings, Increasing Contact in Flow.

Bacterial swimming in flow near surfaces is critical to the spread of infection and device colonization. Understanding how material properties affect flagella- and motility-dependent bacteria-surface interactions is a first step in designing new medical devices that mitigate the risk of infection. We report that, on biomaterial coatings such as polyethylene glycol (PEG) hydrogels and end-tethered layers that prevent adhesive bacteria accumulation, the coating mechanics and hydration control the near-surface travel and dynamic surface contact of E. coli cells in gentle shear flow (order 10 s-1). Along relatively stiff (order 1 MPa) PEG hydrogels or end-tethered layers of PEG chains of similar polymer correlation length, run-and-tumble E. coli travel nanometrically close to the coating's surface in the flow direction in distinguishable runs or "engagements" that persist for several seconds, after which cells leave the interface. The duration of these engagements was greater along stiff hydrogels and end-tethered layers compared with softer, more-hydrated hydrogels. Swimming cells that left stiff hydrogels or end-tethered layers proceeded out to distances of a few microns and then returned to engage the surface again and again, while cells engaging the soft hydrogel tended not to return after leaving. As a result of differences in the duration of engagements and tendency to return to stiff hydrogel and end-tethered layers, swimming E. coli experienced 3 times the integrated dynamic surface contact with stiff coatings compared with softer hydrogels. The striking similarity of swimming behaviors near 16-nm-thick end-tethered layers and 100-µm-thick stiff hydrogels argues that only the outermost several nanometers of a highly hydrated coating influence cell travel. The range of material stiffnesses, cell-surface distance during travel, and time scales of travel compared with run-and-tumble time scales suggests the influence of the coating derives from its interactions with flagella and its potential to alter flagellar bundling. Given that restriction of flagellar rotation is known to trigger increased virulence, bacteria influenced by surfaces in one region may become predisposed to form a biofilm downstream.

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases.

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.

In Vitro Reconstitution of an Intestinal Mucus Layer Shows That Cations and pH Control the Pore Structure That Regulates Its Permeability and Barrier Function.

Dysfunction of the intestinal mucus barrier causes disorders such as ulcerative colitis and Crohn's disease. The function of this essential barrier may be affected by the periodically changing luminal environment. We hypothesized that the pH and ion concentration in mucus control its porosity, molecular permeability, and the penetration of microbes. To test this hypothesis, we developed a scalable method to extract porcine small intestinal mucus (PSIM). The aggregation and porosity of PSIM were determined using rheometry, spectrophotometry, and microscopy. Aggregation of PSIM at low pH increased both the elastic (G') and viscous (G″) moduli, and it slowed the transmigration of pathogenic Salmonella. Molecular transport was dependent on ion concentration. At moderate concentrations, many microscopic aggregates (2-5 µm in diameter) impeded diffusion. At higher concentrations, PSIM formed aggregate islands, increasing both porosity and diffusion. This in vitro model could lead to a better understanding of mucus barrier functions and improve the treatment of intestinal diseases.