Celen, C. | bioRxiv Science

bioRxiv2026

Linking live-cell behavior to transcriptional responses across perturbations using dynamic caging

Single-cell technologies, encompassing molecular, morphological, and functional assays, have emerged as cornerstones of modern biological research and discovery. However, current experimental methods often fail to explicitly link these omic modalities, especially in live cells or longitudinally through time, impeding the study of multi-scale interactions and mechanisms of regulation. CellCage Enclosure (CCE) technology overcomes these limitations by dynamically compartmentalizing cells, allowing for scalable, live-cell, longitudinal exploration and simultaneous analysis of transcriptomic, proteomic, and morphological profiles. Using this novel technology, we generate previously inaccessible insights across various in vitro cellular systems under a diverse set of perturbations, including the discovery of morphological and proteomic features linked to immune suppressive gene set expression in human primary regulatory T cells (Tregs), as well as direct association of morphological and proteomic features with inflammatory gene modules in human colonic fibroblasts. We then develop a novel pooled CRISPR genetic screening technology using CCEs, PERTURB-LINK (PERTURBational LINKage of transcriptomics and imaging in single cells via enclosure-based screening) and apply this approach in murine bone marrow derived macrophages (BMDMs), enabling multiomic dissection of NF-{kappa}B pathway regulation in response to lipopolysaccharide (LPS) stimulation. Together, these findings demonstrate the broad impact that advancements in live-cell, paired multimodal technologies, especially upon perturbation, may offer in deepening our understanding of cellular biology.

cell biology↗

bioRxiv2023

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF{kappa}B signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

systems biology↗