The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Interfacing gene circuits with microelectronics through engineered population dynamics.

While there has been impressive progress connecting bacterial behavior with electrodes, an attractive observation to facilitate advances in synthetic biology is that the growth of a bacterial colony can be determined from impedance changes over time. Here, we interface synthetic biology with microelectronics through engineered population dynamics that regulate the accumulation of charged metabolites. We demonstrate electrical detection of the bacterial response to heavy metals via a population control circuit. We then implement this approach to a synchronized genetic oscillator where we obtain an oscillatory impedance profile from engineered bacteria. We lastly miniaturize an array of electrodes to form "bacterial integrated circuits" and demonstrate its applicability as an interface with genetic circuits. This approach paves the way for new advances in synthetic biology, analytical chemistry, and microelectronic technologies.

Rock-paper-scissors: Engineered population dynamics increase genetic stability.

Advances in synthetic biology have led to an arsenal of proof-of-principle bacterial circuits that can be leveraged for applications ranging from therapeutics to bioproduction. A unifying challenge for most applications is the presence of selective pressures that lead to high mutation rates for engineered bacteria. A common strategy is to develop cloning technologies aimed at increasing the fixation time for deleterious mutations in single cells. We adopt a complementary approach that is guided by ecological interactions, whereby cyclical population control is engineered to stabilize the functionality of intracellular gene circuits. Three strains of Escherichia coli were designed such that each strain could kill or be killed by one of the other two strains. The resulting "rock-paper-scissors" dynamic demonstrates rapid cycling of strains in microfluidic devices and leads to an increase in the stability of gene circuit functionality in cell culture.

Rational engineering of synthetic microbial systems: from single cells to consortia.

One promise of synthetic biology is to provide solutions for biomedical and industrial problems by rational design of added functionality in living systems. Microbes are at the forefront of this biological engineering endeavor due to their general ease of handling and their relevance in many potential applications from fermentation to therapeutics. In recent years, the field has witnessed an explosion of novel regulatory tools, from synthetic orthogonal transcription factors to posttranslational mechanisms for increased control over the behavior of synthetic circuits. Tool development has been paralleled by the discovery of principles that enable increased modularity and the management of host-circuit interactions. Engineered cell-to-cell communication bridges the scales from intracellular to population-level coordination. These developments facilitate the translation of more than a decade of circuit design into applications.

A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis.

Microbial ecologists are increasingly turning to small, synthesized ecosystems1-5 as a reductionist tool to probe the complexity of native microbiomes6,7. Concurrently, synthetic biologists have gone from single-cell gene circuits8-11 to controlling whole populations using intercellular signalling12-16. The intersection of these fields is giving rise to new approaches in waste recycling17, industrial fermentation18, bioremediation19 and human health16,20. These applications share a common challenge7 well-known in classical ecology21,22-stability of an ecosystem cannot arise without mechanisms that prohibit the faster-growing species from eliminating the slower. Here, we combine orthogonal quorum-sensing systems and a population control circuit with diverse self-limiting growth dynamics to engineer two 'ortholysis' circuits capable of maintaining a stable co-culture of metabolically competitive Salmonella typhimurium strains in microfluidic devices. Although no successful co-cultures are observed in a two-strain ecology without synthetic population control, the 'ortholysis' design dramatically increases the co-culture rate from 0% to approximately 80%. Agent-based and deterministic modelling reveal that our system can be adjusted to yield different dynamics, including phase-shifted, antiphase or synchronized oscillations, as well as stable steady-state population densities. The 'ortholysis' approach establishes a paradigm for constructing synthetic ecologies by developing stable communities of competitive microorganisms without the need for engineered co-dependency.

Synchronized cycles of bacterial lysis for in vivo delivery.

The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body. 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 ata 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 used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. Asa proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. 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 administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour 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.