Learning single-cell perturbation responses using neural optimal transport.

Understanding and predicting molecular responses in single cells upon chemical, genetic or mechanical perturbations is a core question in biology. Obtaining single-cell measurements typically requires the cells to be destroyed. This makes learning heterogeneous perturbation responses challenging as we only observe unpaired distributions of perturbed or non-perturbed cells. Here we leverage the theory of optimal transport and the recent advent of input convex neural architectures to present CellOT, a framework for learning the response of individual cells to a given perturbation by mapping these unpaired distributions. CellOT outperforms current methods at predicting single-cell drug responses, as profiled by scRNA-seq and a multiplexed protein-imaging technology. Further, we illustrate that CellOT generalizes well on unseen settings by (1) predicting the scRNA-seq responses of holdout patients with lupus exposed to interferon-ß and patients with glioblastoma to panobinostat; (2) inferring lipopolysaccharide responses across different species; and (3) modeling the hematopoietic developmental trajectories of different subpopulations.

Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections.

Highly multiplexed protein measurements from multiple spatial scales using fluorescence microscopy recently emerged as a powerful way to investigate tumor microenvironments in biomedicine and the multivariate nature of complex systems' interactions. A range of methods for this exist, which either rely on directly labeling the primary antibody with oligonucleotides/rare metals or employing methods to remove fluorescence for cyclic acquisition. Here, we describe a protocol that uses off-the-shelf primary and secondary antibodies without further need for modification and only commonly available chemical reagents. The method harnesses the observation that antibodies can crosslink to bound epitopes during light exposure, thus preventing elution. By utilizing a simple oxygen radical scavenging buffer during imaging and by blocking free sulfhydryl groups before antibody incubation, the presented method can employ comparably mild conditions to remove bound antibodies from epitopes, which preserves sample integrity. Thus, with the stated minor modifications, it allows for a standard immunofluorescence imaging protocol in cyclic fashion, currently permitting staining of up to ~80 unique epitopes.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Characterization of the neurogenic niche in the aging dentate gyrus using iterative immunofluorescence imaging.

Advancing age causes reduced hippocampal neurogenesis, associated with age-related cognitive decline. The spatial relationship of age-induced alterations in neural stem cells (NSCs) and surrounding cells within the hippocampal niche remains poorly understood due to limitations of antibody-based cellular phenotyping. We established iterative indirect immunofluorescence imaging (4i) in tissue sections, allowing for simultaneous detection of 18 proteins to characterize NSCs and surrounding cells in 2-, 6-, and 12-month-old mice. We show that reorganization of the dentate gyrus (DG) niche already occurs in middle-aged mice, paralleling the decline in neurogenesis. 4i-based tissue analysis of the DG identifies changes in cell-type contributions to the blood-brain barrier and microenvironments surrounding NSCs to play a pivotal role to preserve neurogenic permissiveness. The data provided represent a resource to characterize the principles causing alterations of stem cell-associated plasticity within the aging DG and provide a blueprint to analyze somatic stem cell niches across lifespan in complex tissues.

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

Multiplexed protein maps link subcellular organization to cellular states.

Obtaining highly multiplexed protein measurements across multiple length scales has enormous potential for biomedicine. Here, we measured, by iterative indirect immunofluorescence imaging (4i), 40-plex protein readouts from biological samples at high-throughput from the millimeter to the nanometer scale. This approach simultaneously captures properties apparent at the population, cellular, and subcellular levels, including microenvironment, cell shape, and cell cycle state. It also captures the detailed morphology of organelles, cytoskeletal structures, nuclear subcompartments, and the fate of signaling receptors in thousands of single cells in situ. We used computer vision and systems biology approaches to achieve unsupervised comprehensive quantification of protein subcompartmentalization within various multicellular, cellular, and pharmacological contexts. Thus, highly multiplexed subcellular protein maps can be used to identify functionally relevant single-cell states.

Trajectories of cell-cycle progression from fixed cell populations.

An accurate dissection of sources of cell-to-cell variability is crucial for quantitative biology at the single-cell level but has been challenging for the cell cycle. We present Cycler, a robust method that constructs a continuous trajectory of cell-cycle progression from images of fixed cells. Cycler handles heterogeneous microenvironments and does not require perturbations or genetic markers, making it generally applicable to quantifying multiple sources of cell-to-cell variability in mammalian cells.