Kinome state is predictive of cell viability in pancreatic cancer tumor and cancer-associated fibroblast cell lines.

Numerous aspects of cellular signaling are regulated by the kinome-the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC.

TRANSCRIPTOMIC DIFFERENCES IN PERIPHERAL MONOCYTE POPULATIONS IN SEPTIC PATIENTS BASED ON OUTCOME.

ABSTRACT: Postsepsis early mortality is being replaced by survivors who experience either a rapid recovery and favorable hospital discharge or the development of chronic critical illness with suboptimal outcomes. The underlying immunological response that determines these clinical trajectories remains poorly defined at the transcriptomic level. As classical and nonclassical monocytes are key leukocytes in both the innate and adaptive immune systems, we sought to delineate the transcriptomic response of these cell types. Using single-cell RNA sequencing and pathway analyses, we identified gene expression patterns between these two groups that are consistent with differences in TNF-α production based on clinical outcome. This may provide therapeutic targets for those at risk for chronic critical illness in order to improve their phenotype/endotype, morbidity, and long-term mortality.

Data-driven selection of analysis decisions in single-cell RNA-seq trajectory inference.

Single-cell RNA sequencing (scRNA-seq) experiments have become instrumental in developmental and differentiation studies, enabling the profiling of cells at a single or multiple time-points to uncover subtle variations in expression profiles reflecting underlying biological processes. Benchmarking studies have compared many of the computational methods used to reconstruct cellular dynamics; however, researchers still encounter challenges in their analysis due to uncertainty with respect to selecting the most appropriate methods and parameters. Even among universal data processing steps used by trajectory inference methods such as feature selection and dimension reduction, trajectory methods' performances are highly dataset-specific. To address these challenges, we developed Escort, a novel framework for evaluating a dataset's suitability for trajectory inference and quantifying trajectory properties influenced by analysis decisions. Escort evaluates the suitability of trajectory analysis and the combined effects of processing choices using trajectory-specific metrics. Escort navigates single-cell trajectory analysis through these data-driven assessments, reducing uncertainty and much of the decision burden inherent to trajectory inference analyses. Escort is implemented in an accessible R package and R/Shiny application, providing researchers with the necessary tools to make informed decisions during trajectory analysis and enabling new insights into dynamic biological processes at single-cell resolution.

The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Sub-Cluster Identification through Semi-Supervised Optimization of Rare-Cell Silhouettes (SCISSORS) in single-cell RNA-sequencing.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) has enabled the molecular profiling of thousands to millions of cells simultaneously in biologically heterogenous samples. Currently, the common practice in scRNA-seq is to determine cell type labels through unsupervised clustering and the examination of cluster-specific genes. However, even small differences in analysis and parameter choosing can greatly alter clustering results and thus impose great influence on which cell types are identified. Existing methods largely focus on determining the optimal number of robust clusters, which can be problematic for identifying cells of extremely low abundance due to their subtle contributions toward overall patterns of gene expression. RESULTS: Here, we present a carefully designed framework, SCISSORS, which accurately profiles subclusters within broad cluster(s) for the identification of rare cell types in scRNA-seq data. SCISSORS employs silhouette scoring for the estimation of heterogeneity of clusters and reveals rare cells in heterogenous clusters by a multi-step semi-supervised reclustering process. Additionally, SCISSORS provides a method for the identification of marker genes of high specificity to the cell type. SCISSORS is wrapped around the popular Seurat R package and can be easily integrated into existing Seurat pipelines. AVAILABILITY AND IMPLEMENTATION: SCISSORS, including source code and vignettes, are freely available at https://github.com/jr-leary7/SCISSORS.

Interpretable trajectory inference with single-cell Linear Adaptive Negative-binomial Expression (scLANE) testing.

The rapid proliferation of trajectory inference methods for single-cell RNA-seq data has allowed researchers to investigate complex biological processes by examining underlying gene expression dynamics. After estimating a latent cell ordering, statistical models are used to determine which genes exhibit changes in expression that are significantly associated with progression through the biological trajectory. While a few techniques for performing trajectory differential expression exist, most rely on the flexibility of generalized additive models in order to account for the inherent nonlinearity of changes in gene expression. As such, the results can be difficult to interpret, and biological conclusions often rest on subjective visual inspections of the most dynamic genes. To address this challenge, we propose scLANE testing, which is built around an interpretable generalized linear model and handles nonlinearity with basis splines chosen empirically for each gene. In addition, extensions to estimating equations and mixed models allow for reliable trajectory testing under complex experimental designs. After validating the accuracy of scLANE under several different simulation scenarios, we apply it to a set of diverse biological datasets and display its ability to provide novel biological information when used downstream of both pseudotime and RNA velocity estimation methods.

Data-driven selection of analysis decisions in single-cell RNA-seq trajectory inference.

Single-cell RNA sequencing (scRNA-seq) experiments have become instrumental in developmental and differentiation studies, enabling the profiling of cells at a single or multiple time-points to uncover subtle variations in expression profiles reflecting underlying biological processes. Benchmarking studies have compared many of the computational methods used to reconstruct cellular dynamics, however researchers still encounter challenges in their analysis due to uncertainties in selecting the most appropriate methods and parameters. Even among universal data processing steps used by trajectory inference methods such as feature selection and dimension reduction, trajectory methods' performances are highly dataset-specific. To address these challenges, we developed Escort, a framework for evaluating a dataset's suitability for trajectory inference and quantifying trajectory properties influenced by analysis decisions. Escort navigates single-cell trajectory analysis through data-driven assessments, reducing uncertainty and much of the decision burden associated with trajectory inference. Escort is implemented in an accessible R package and R/Shiny application, providing researchers with the necessary tools to make informed decisions during trajectory analysis and enabling new insights into dynamic biological processes at single-cell resolution.