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 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.

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.

The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella.

BACKGROUND: Salmonella have potential as anticancer therapeutic because of their innate tumor specificity. In clinical studies, this specificity has been hampered by heterogeneous responses. Understanding the mechanisms that control tumor colonization would enable the design of more robust therapeutic strains. Two mechanisms that could affect tumor colonization are intracellular accumulation and intratumoral motility. Both of these mechanisms have elements that are controlled by the master motility regulator flhDC. We hypothesized that 1) overexpressing flhDC in Salmonella increases intracellular bacterial accumulation in tumor cell masses, and 2) intracellular accumulation of Salmonella drives tumor colonization in vitro. METHODS: To test these hypotheses, we transformed Salmonella with genetic circuits that induce flhDC and express green fluorescent protein after intracellular invasion. The genetically modified Salmonella was perfused into an in vitro tumor-on-a-chip device. Time-lapse fluorescence microscopy was used to quantify intracellular and colonization dynamics within tumor masses. A mathematical model was used to determine how these mechanisms are related to each other. RESULTS: Overexpression of flhDC increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively (P < 0.05). Non-motile Salmonella accumulated in cancer cells 26 times less than controls (P < 0.001). Minimally invasive, ΔsipB, Salmonella colonized tumor masses 2.5 times less than controls (P < 0.05). When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls (P < 0.05). Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both. CONCLUSIONS: This study demonstrates a mechanistic link between motility, intracellular accumulation and tumor colonization. Based on our results, we envision that therapeutic strains of Salmonella could use inducible flhDC to drive tumor colonization. More intratumoral bacteria would enable delivery of higher therapeutic payloads into tumors and would improve treatment efficacy.

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.

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.

Potent and tumor specific: arming bacteria with therapeutic proteins.

Bacteria are perfect vessels for targeted cancer therapy. Conventional chemotherapy is limited by passive diffusion, and systemic administration causes severe side effects. Bacteria can overcome these obstacles by delivering therapeutic proteins specifically to tumors. Bacteria have been modified to produce proteins that directly kill cells, induce apoptosis via signaling pathways, and stimulate the immune system. These three modes of bacterial treatment have all been shown to reduce tumor growth in animal models. Bacteria have also been designed to convert nontoxic prodrugs to active therapeutic compounds. The ease of genetic manipulation enables creation of arrays of bacteria that release many new protein drugs. This versatility will allow targeting of multiple cancer pathways and will establish a platform for individualized cancer medicine.

Protein phosphatase PP1-NIPP1 activates mesenchymal genes in HeLa cells.

The deletion of the protein phosphatase-1 (PP1) regulator known as Nuclear Inhibitor of PP1 (NIPP1) is embryonic lethal during gastrulation, hinting at a key role of PP1-NIPP1 in lineage specification. Consistent with this notion we show here that a mild, stable overexpression of NIPP1 in HeLa cells caused a massive induction of genes of the mesenchymal lineage, in particular smooth/cardiac-muscle and matrix markers. This reprogramming was associated with the formation of actin-based stress fibers and retracting filopodia, and a reduced proliferation potential. The NIPP1-induced mesenchymal transition required functional substrate and PP1-binding domains, suggesting that it involves the selective dephosphorylation of substrates of PP1-NIPP1.

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.

Genetically modified bacteria as a tool to detect microscopic solid tumor masses with triggered release of a recombinant biomarker.

Current tomographic methods of cancer detection have limited sensitivity and are unable to detect malignant masses smaller than half a centimeter in diameter. Mortality from tumor recurrence and metastatic disease would be reduced if small lesions could be detected earlier. To overcome this limitation, we created a detection system that combines the specificity of tumor-targeting bacteria with the sensitivity of a synthetic biomarker. Bacteria, specifically Salmonella, preferentially accumulate in tumors and microscopic metastases as small as five cell layers thick. To create tumor detecting bacteria, an attenuated strain of Salmonella was engineered to express and release the fluorescent protein ZsGreen. A single-layer antibody method was developed to measure low concentrations of ZsGreen. Engineered bacteria were administered to a microfluidic tumor-on-a-chip device to measure protein production. In culture, half of produced ZsGreen was released by viable bacteria at a rate of 87.6 fg bacterium(-1) h(-1). Single-layer antibody dots were able to detect bacterially produced ZsGreen at concentrations down to 4.5 ng ml(-1). Bacteria colonized in 0.12 mm(3) of tumor tissue in the microfluidic device released ZsGreen at a rate of 23.9 µg h(-1). This release demonstrates that ZsGreen readily diffuses through tissue and accumulates at detectable concentrations. Based on a mathematical pharmacokinetic model, the measured rate of release would enable detection of 0.043 mm(3) tumor masses, which is 2600 times smaller than the current limit of tomographic techniques. Tumor-detecting bacteria would provide a sensitive, minimally invasive method to detect tumor recurrence, monitor treatment efficacy, and identify the onset of metastatic disease.

NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes.

The histone methyltransferase EZH2 regulates cell proliferation and differentiation by silencing Polycomb group target genes. NIPP1, a nuclear regulator of serine/threonine protein phosphatase 1 (PP1), has been implicated in the regulation of EZH2 occupancy at target loci, but the underlying mechanism is not understood. Here, we demonstrate that the phosphorylation of EZH2 by cyclin-dependent kinases at Thr416 creates a docking site for the ForkHead-associated domain of NIPP1. Recruited NIPP1 enables the net phosphorylation of EZH2 by inhibiting its dephosphorylation by PP1. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated, and the knockdown of NIPP1 results in a reduced phosphorylation of endogenous EZH2. Conversely, the loss of PP1 is associated with a hyperphosphorylation of EZH2. A genome-wide promoter-binding profiling in HeLa cells revealed that the NIPP1-binding mutant shows a deficient association with about a third of the Polycomb target genes, and these are enriched for functions in proliferation. Our data identify PP1 as an EZH2 phosphatase and demonstrate that the phosphorylation-regulated association of EZH2 with proliferation-related targets depends on associated NIPP1.

A role for PP1/NIPP1 in steering migration of human cancer cells.

Electrical gradients are present in many developing and regenerating tissues and around tumours. Mimicking endogenous electric fields in vitro has profound effects on the behaviour of many cell types. Intriguingly, specific cell types migrate cathodally, others anodally and some polarise with their long axis perpendicular to the electric vector. These striking phenomena are likely to have in vivo relevance since one of the determining factors during cancer metastasis is the ability to switch between attractive and repulsive migration in response to extracellular guidance stimuli. We present evidence that the cervical cancer cell line HeLa migrates cathodally in a direct current electric field of physiological intensity, while the strongly metastatic prostate cancer cell line PC-3-M migrates anodally. Notably, genetic disruption of protein serine/threonine phosphatase-1 (PP1) and its regulator NIPP1 decrease directional migration in these cell lines. Conversely, the inducible expression of NIPP1 switched the directional response of HeLa cells from cathodal to slightly anodal in a PP1-dependent manner. Remarkably, induction of a hyperactive PP1/NIPP1 holoenzyme, further shifted directional migration towards the anode. We show that PP1 association with NIPP1 upregulates signalling by the GTPase Cdc42 and demonstrate that pharmacological inhibition of Cdc42 in cells overexpressing NIPP1 recovered cathodal migration. Taken together, we provide the first evidence for regulation of directional cell migration by NIPP1. In addition, we identify PP1/NIPP1 as a novel molecular compass that controls directed cell migration via upregulation of Cdc42 signalling and suggest a way by which PP1/NIPP1 may contribute to the migratory properties of cancer cells.

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets.

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex.