Transcriptional regulation of living materials via extracellular electron transfer.

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials.

Tackling the tumor microenvironment - how can complex tumor models in vitro aid oncology drug development?

INTRODUCTION: Identifying effective cancer drugs remains an inefficient process. Drug efficacy in traditional preclinical cancer models translates poorly into therapy in the clinic. Implementation of preclinical models that incorporate the tumor microenvironment (TME) is needed to improve selection of active drugs prior to clinical trials. AREAS COVERED: Progression of cancer results from the behavior of cancer cells in concert with the host's histopathological background. Nonetheless, complex preclinical models with a relevant microenvironment have yet to become an integral part of drug development. This review discusses existing models and provides a synopsis of active areas of cancer drug development where implementation would be of value. Their contribution to finding therapeutics in immune oncology, angiogenesis, regulated cell death and targeting tumor fibroblasts as well as optimization of drug delivery, combination therapy, and biomarkers of efficacy is considered. EXPERT OPINION: Complex tumor models in vitro (CTMIVs) that mimic the organotypic architecture of neoplastic tumors have boosted research into TME influence on traditional cytoreductive chemotherapy as well as the detection of specific TME targets. Despite advances in technical prowess, CTMIVs can only address specific aspects of cancer pathophysiology.

A strategy to quantify myofibroblast activation on a continuous spectrum.

Myofibroblasts are a highly secretory and contractile cell phenotype that are predominant in wound healing and fibrotic disease. Traditionally, myofibroblasts are identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers, leading to a binary classification: "activated" or "quiescent (non-activated)". More recently, however, myofibroblast activation has been considered on a continuous spectrum, but there is no established method to quantify the position of a cell on this spectrum. To this end, we developed a strategy based on microscopy imaging and machine learning methods to quantify myofibroblast activation in vitro on a continuous scale. We first measured morphological features of over 1000 individual cardiac fibroblasts and found that these features provide sufficient information to predict activation state. We next used dimensionality reduction techniques and self-supervised machine learning to create a continuous scale of activation based on features extracted from microscopy images. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of myofibroblast activation and provide an imaging-based strategy to quantify the position of a cell on that spectrum.

Rapid hydrogel formation via tandem visible light photouncaging and bioorthogonal ligation.

The formation of benign polymer scaffolds in water using green-light-reactive photocages is described. These efforts pave an avenue toward the fabrication of synthetic scaffolds that can facilitate the study of cellular events for disease diagnosis and treatment. First, a series of boron dipyrromethene (BODIPY) photocages with nitrogen-containing nucleophiles were examined to determine structure-reactivity relationships, which resulted in a >1,000× increase in uncaging yield. Subsequently, photoinduced hydrogel formation in 90 wt % water was accomplished via biorthogonal carbonyl condensation using hydrophilic polymer scaffolds separately containing BODIPY photocages and ortho-phthalaldehyde (OPA) moieties. Spatiotemporal control is demonstrated with light on/off experiments to modulate gel stiffness and masking to provide <100 µm features. Biocompatability of the method was shown through pre-/post-crosslinking cell viability studies. Short term, these studies are anticipated to guide translation to emergent additive manufacturing technology, which, longer term, will enable the development of 3D cell cultures for tissue engineering applications.