Programmable probiotics for detection of cancer in urine

Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine. Our microbial diagnostic generated a high-contrast urine signal through selective expansion in liver metastases (10[superscript 6]-fold enrichment) and high expression of a lacZ reporter maintained by engineering a stable plasmid system. The lacZ reporter cleaves a substrate to produce a small molecule that can be detected in urine. E. coli Nissle 1917 robustly colonized tumor tissue in rodent models of liver metastasis after oral delivery but did not colonize healthy organs or fibrotic liver tissue. We saw no deleterious health effects on the mice for more than 12 months after oral delivery. Our results demonstrate that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissue microenvironments in vivo.Ludwig Center for Molecular OncologyAmar G. Bose Research GrantSan Diego Center for Systems Biology (United States. National Institutes of Health Grant P50 GM085764)National Institute of General Medical Sciences (U.S.) (R01GM69811)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109)Misrock Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award)Burroughs Wellcome Fund (Career Award at the Scientific Interface

Programmable probiotics for detection of cancer in urine

Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine. Our microbial diagnostic generated a high-contrast urine signal through selective expansion in liver metastases (10 6 -fold enrichment) and high expression of a lacZ reporter maintained by engineering a stable plasmid system. The lacZ reporter cleaves a substrate to produce a small molecule that can be detected in urine. E. coli Nissle 1917 robustly colonized tumor tissue in rodent models of liver metastasis after oral delivery but did not colonize healthy organs or fibrotic liver tissue. We saw no deleterious health effects on the mice for more than 12 months after oral delivery. Our results demonstrate that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissue microenvironments in vivo

Synchronized cycles of bacterial lysis for in vivo delivery

The pervasive view of bacteria as strictly pathogenic has given way to an ppreciation of the widespread prevalence of beneficial microbes within the human body. Given this milieu, it is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbor disease and thus provide a natural platform for the development of engineered therapies. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ. Here, we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We use microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug deliver platform via co-culture with human cancer cells in vitro. As a proof of principle, we track the bacterial population dynamics in ectopic syngeneic colorectal tumors in mice. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies, we orally administer the lysis strain, alone or in combination with a clinical chemotherapeutic, to a syngeneic transplantation model of hepatic colorectal metastases. We find that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumor activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites.National Institute of General Medical Sciences (U.S.) (GM069811)San Diego Center for Systems Biology (P50 GM085764)National Cancer Institute (U.S.). Swanson Biotechnology Center (Koch Institute Support Grant (P30-CA14051))National Institute of Environmental Health Sciences (Core Center Grant (P30- ES002109))National Institutes of Health (U.S.) (NIH Pathway to Independence Award NIH (K99 CA197649-01))Misrock Postdoctoral fellowshipNational Defense Science and Engineering Graduate (NDSEG) Fellowshi

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC

In-Silico Patterning of Vascular Mesenchymal Cells in Three Dimensions

Cells organize in complex three-dimensional patterns by interacting with proteins along with the surrounding extracellular matrix. This organization provides the mechanical and chemical cues that ultimately influence a cell's differentiation and function. Here, we computationally investigate the pattern formation process of vascular mesenchymal cells arising from their interaction with Bone Morphogenic Protein-2 (BMP-2) and its inhibitor, Matrix Gla Protein (MGP). Using a first-principles approach, we derive a reaction-diffusion model based on the biochemical interactions of BMP-2, MGP and cells. Simulations of the model exhibit a wide variety of three-dimensional patterns not observed in a two-dimensional analysis. We demonstrate the emergence of three types of patterns: spheres, tubes, and sheets, and show that the patterns can be tuned by modifying parameters in the model such as the degradation rates of proteins and chemotactic coefficient of cells. Our model may be useful for improved engineering of three-dimensional tissue structures as well as for understanding three dimensional microenvironments in developmental processes.National Institutes of Health (U.S.) (GM69811)United States. Dept. of Energy (DOE CSGF fellowship

Testing Gait with Ankle-Foot Orthoses in Children with Cerebral Palsy by Using Functional Mixed-Effects Analysis of Variance

Dr Morrissey is part funded by the NIHR/HEE Senior Clinical Lecturer scheme. Tis report presents independent research part-funded by the National Institute for Health Research (NIHR) CAT SCL-2013-04-00

Synthetic gene oscillators and their applications

Synthetic biology seeks to understand and engineer biological networks that perform a quantitative dynamic function in organisms. Since the original toggle switch (Gardner et al., 2000a) and oscillator designs (Elowitz and Leibler, 2000a), genetic circuits have been constructed that control cellular population growth (You et al., 2004b), detect edges in an image (Friedland et al., 2009), and count discrete cellular events (Friedland et al., 2009). In this thesis, we focus on synthetic gene circuits that produce oscillations. Oscillations are important in a vast range of natural contexts such as circadian rhythms, cardiac function, cell division, and hormonal regulation, as well as key to building synthetic control systems that rely on precise timing. Here we discuss modeling, designing, constructing, and characterizing synthetic gene oscillators. In Chapter One, we give an overview and introduction to the field of synthetic biology and how our research area fits into this discipline. In Chapter Two, I discuss a network design which produces synchronized oscillations in a growing population of cells. In Chapter Three, I discuss an introduction to modeling simple genetic networks. In Chapter Four, we look at a scenario where synthetic gene circuits that produce an overabundance of tagged components lead to unexpected correlations. In Chapter Five, I discuss modeling genetic networks and further go into detail about spatial modeling of networks that produce patterns in mammalian cells. These parts combine to illustrate how to design, model, construct, and characterize synthetic gene networks in bacteria and mammalian system

A synchronized quorum of genetic clocks

The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic biology. This focus was elegantly set in motion a decade ago with the design and construction of a genetic toggle switch and an oscillator, with subsequent highlights that have included circuits capable of pattern generation, noise shaping, edge detection, and event counting. Here, we describe an engineered gene network with global intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Using microfluidic devices tailored for cellular populations at differing length scales, we investigate the collective synchronization properties along with spatiotemporal waves occurring on millimeter scales. We use computational modeling to quantitatively describe the observed dependence of the period and amplitude of the bulk oscillations on the flow rate. The synchronized genetic clock sets the stage for the use of microbes in the creation of a macroscopic biosensor with an oscillatory output. In addition, it provides a specific model system for the generation of a mechanistic description of emergent coordinated behavior at the colony level. Synchronized clocks are of fundamental importance in the coordination of rhythmi