Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality.

Regulator of G protein signaling (RGS) proteins, and notably members of the RGS-R4 subfamily, control vasocontractility by accelerating the inactivation of Gα-dependent signaling. RGS5 is the most highly and differently expressed RGS-R4 subfamily member in arterial smooth muscle. Expression of RGS5 first appears in pericytes during development of the afferent vascular tree, suggesting that RGS5 is a good candidate for a regulator of arterial contractility and, perhaps, for determining the mass of the smooth muscle coats required to regulate blood flow in the branches of the arterial tree. Consistent with this hypothesis, using cultured vascular smooth muscle cells (VSMCs), we demonstrate RGS5 overexpression inhibits G protein-coupled receptor (GPCR)-mediated hypertrophic responses. The next objective was to determine which physiological agonists directly control RGS5 expression in VSMCs. GPCR agonists failed to directly regulate RGS5 mRNA expression; however, platelet-derived growth factor (PDGF) acutely represses expression. Downregulation of RGS5 results in the induction of migration and the activation of the GPCR-mediated signaling pathways. This stimulation leads to the activation of mitogen-activated protein kinases directly downstream of receptor stimulation, and ultimately VSMC hypertrophy. These results demonstrate that RGS5 expression is a critical mediator of both VSMC contraction and potentially, arterial remodeling.

Development of an adaptive electroporation system for intratumoral plasmid DNA delivery.

Intratumoral electroporation of plasmid DNA encoding the proinflammatory cytokine interleukin 12 promotes innate and adaptive immune responses correlating with anti-tumor effects. Clinical electroporation conditions are fixed parameters optimized in preclinical tumors, which consist of cells implanted into skin. These conditions have little translatability to clinically relevant tumors, as implanted models cannot capture the heterogeneity encountered in genetically engineered mouse models or clinical tumors. Variables affecting treatment outcome include tumor size, degree of vascularization, fibrosis, and necrosis, which can result in suboptimal gene transfer and variable therapeutic outcomes. To address this, a feedback controlled electroporation generator was developed, which is capable of assessing the electrochemical properties of tissue in real time. Determination of these properties is accomplished by impedance spectroscopy and equivalent circuit model parameter estimation. Model parameters that estimate electrical properties of cell membranes are used to adjust electroporation parameters for each applied pulse. Studies performed in syngeneic colon carcinoma tumors (MC38) and spontaneous mammary tumors (MMTV-PyVT) demonstrated feedback-based electroporation is capable of achieving maximum expression of reporter genes with significantly less variability and applied energy. These findings represent an advancement to the practice of gene electro-transfer, as reducing variability and retaining transfected cell viability is paramount to treatment success.

Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress.

MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments.