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McFaline-Figueroa, J. L.

Publications and source records attributed to McFaline-Figueroa, J. L..

17 recordsLinked to original sources

SHERLOCK: Structured representation learning and causal inference of downstream perturbation effects

Understanding the effects of genetic, molecular, and experimental perturbations is essential for decoding cellular mechanisms and guiding biomedical interventions. Existing computational approaches are typically designed for individual tasks, such as predicting perturbation responses, characterizing downstream transcriptional effects, or modeling perturbation combinations, and therefore do not provide a unified framework for learning interpretable perturbation representations while enabling causal analysis of downstream effects and characterization of condition-dependent responses. We present SHERLOCK, an interpretable deep generative framework for single-cell perturbation analysis that represents perturbation effects as structured interventions on a latent baseline cellular state. SHERLOCK learns correlated and sparse perturbation representations that organize genetic and pharmacological perturbations according to shared transcriptional responses. % By formulating perturbations as interventions within a structural causal model, it enables counterfactual estimation of their downstream transcriptional effects under explicit identifiability assumptions. The same framework quantifies how perturbation responses vary across conditions and compositionally models combinatorial perturbations, enabling prediction of held-out combinations and classification of genetic interactions. Across genome-scale CRISPR, chemical, and spatial perturbation datasets, SHERLOCK recovers perturbation relationships concordant with known biological pathways and pharmacological properties, identifies condition-dependent responses, and predicts combinatorial perturbation effects. Together, SHERLOCK provides a unified framework for interpretable and causal analysis of perturbation effects across diverse single-cell perturbation experiments.

genomics↗

Supramolecular hydrogel viscoelasticity regulates in situ tertiary lymphoid neogenesis

Tertiary lymphoid structures (TLSs) are organized three-dimensional immune niches associated with improved antitumor immunity, which has galvanized efforts to induce them artificially using biomaterials. However, these strategies have largely focused on soluble cue delivery, leaving the role of scaffold physical properties poorly understood. Here, we developed injectable, liposome-crosslinked supramolecular hydrogels spanning soft and stiff formulations to determine how scaffold mechanical properties regulate in situ tertiary lymphoid neogenesis. The formulations differed in their viscoelastic properties while maintaining broadly comparable release of ovalbumin and LIGHT. Soft hydrogels underwent distributed cellular infiltration and material replacement, transitioning from an early myeloid-rich response to vascularized, lymphoid-dominant tissues containing B-cell-rich aggregates adjacent to T-cell regions, with B-cell organization peaking at day 14. Single-cell RNA sequencing revealed that transient interferon-associated neutrophil and macrophage states in the early niche preceded the emergence of TLS-associated transcriptional programs across lymphoid and myeloid populations. In contrast, stiff hydrogels resisted infiltration and perpetuated a niche dominated by activated myeloid cells with limited lymphoid organization. Prophylactically implanted soft hydrogels improved early melanoma control relative to stiff hydrogels under checkpoint blockade, and a single soft-hydrogel implantation restrained tumor growth even without checkpoint blockade. Together, these findings establish scaffold mechanics and remodeling as active regulators of engineered immune-tissue organization and provide design principles for directing the development of local TLS-like niches.

bioengineering↗

A modular transcript enrichment strategy for scalable, atlas-aligned, and clonotype-resolved single-cell transcriptomics

Existing targeted approaches either rely on per-gene barcoded probes and transcript tiling, which limit scalability, or forgo reverse transcription, precluding capture of highly variable transcripts. Here, we present targeted reverse transcription-linker (TRTL), a modular method that can be integrated into existing single-cell transcriptomic workflows to enable targeted readout of user-defined transcript panels, with minimal changes in protocol when scaling from tens to thousands of genes. Because TRTL retains reverse transcription, it also enables the capture of variable transcripts, such as TCR and BCR sequences. Applying TRTL and combinatorial indexing RNA-seq to the mouse brain, we show that carefully designed panels support robust alignment to existing reference atlases, enabling accurate cell type annotation and detection of cellular populations at low sequencing depths. Lastly, we combine TRTL with nuclear hashing-based multiplexing for a targeted-sci-Plex protocol and further demonstrate that targeted-sci-Plex can resolve dynamic T-cell fate trajectories following diverse activating exposures while concurrently profiling T-cell clonotypes.

genomics↗

A robust human airway organoid platform enables scalable expansion and trajectory mapping of pulmonary neuroendocrine cells

Pulmonary neuroendocrine cells (PNECs) are rare chemosensory epithelial cells, facultative stem cells, and a cell-of-origin for neuroendocrine lung cancers, yet the mechanisms governing their differentiation and heterogeneity are poorly understood. Here we establish NEr-fAOs, a human fetal airway organoid platform that robustly enriches PNECs, and identify a cooperative requirement for dual GSK3 and NOTCH inhibition to drive directed PNEC differentiation. This strategy yields stable cultures with up to 60-fold expansion of PNECs whose transcriptomes closely match fetal and adult PNECs. In addition to PNEC-enrichment, NEr-fAOs retain diverse airway epithelial cell types, preserving epithelial complexity. Time-resolved single-cell transcriptomics maps PNEC trajectories in NEr-fAOs, resolving precursor and mature states. Comparative analyses further reveal a distal airway bias in NEr-fAOs and enrichment for lower-airway progenitors. NEr-fAOs thus provide a scalable, tractable platform to dissect human PNEC biology and distal airway progenitor hierarchies relevant to lung development, cancer, and disease.

developmental biology↗

Mapping kinase-dependent tumor immune adaptation with multiplexed single-cell CRISPR screens

Immune dysfunction in cancer is enacted by multiple programs, including tumor cell-intrinsic responses to distinct immune subpopulations. A subset of these immune evasion programs can be systematically recapitulated through direct tumor-immune interactions in vitro. Here, we present an integrated, high-throughput single-cell CRISPR screening framework focused on the protein kinome for mapping the tumor-intrinsic regulation of T cell-driven immune pressure in glioblastoma (GBM). We combine pooled CRISPR interference and activation (CRISPRi/a) with immune-matched NY-ESO-1 antigen-specific allogeneic GBM-T cell co-culture and massively multiplexed single-cell transcriptomics to systematically quantify how genetic perturbation reshapes baseline tumor state and adaptive responses across graded effector-to-target ratios. We further leverage deep generative models for analyzing pooled CRISPR screens to decipher the effects of genetic perturbations on the mechanisms of tumor resistance. This framework resolves distinct modules of immune evasion and survival, including the regulation of the antigen-presentation machinery, interferon/NF-{kappa}B signaling, oxidative stress resilience, and checkpoint/cytokine programs, while identifying perturbations that reroute the continuous tumor transcriptional trajectory induced by T cell engagement. A secondary chemical screen in patient-derived GBM cultures identified putative kinase targets of immune evasion phenotypes (e.g., EPHA2 and PDGFRA), whose inhibition leads to the blockade of evasive programs and enhances T cell-mediated GBM killing. Together, this workflow provides a scalable blueprint for comprehensive charting of the genetic control of tumor-immune interactions.

genomics↗

Multiplexed single-cell transcriptomics reveals diverse phenotypic outcomes for pathogenic SHP2 variants

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is an important regulator of Ras/MAPK signaling that acts downstream of receptor tyrosine kinases and other transmembrane receptors. Germline PTPN11 mutations cause developmental disorders such as Noonan Syndrome, whereas somatic mutations drive various cancers. While many pathogenic mutations enhance SHP2 catalytic activity, others are inactivating or affect protein interactions, confounding our understanding of SHP2-driven disease. Here, we combine single-cell transcriptional profiling of cells expressing clinically diverse SHP2 variants with protein biochemistry, structural analysis, and cell biology to explain how pathogenic mutations dysregulate signaling. Our analyses reveal that loss of catalytic activity does not phenocopy SHP2 knock-out at the gene expression level, that some mechanistically distinct mutations have convergent phenotypic effects, and that different mutations at the same hotspot residue can yield divergent cell states. These findings provide a framework for understanding the connection between SHP2 structural perturbations, cellular outcomes, and human diseases.

molecular biology↗

Brain-wide cell-type-specific noradrenergic modulation of the transcriptome

Neuromodulatory systems such as the locus coeruleus-norepinephrine (LC-NE) system exert a widespread influence on brain function, yet the transcriptional consequences of such neuromodulatory perturbations remain largely unknown across the many unique cell types in the brain. In this study, we establish a generalizable framework to map brain-wide, cell-type-specific gene expression changes in mice following in vivo chemogenetic activation or inhibition of LC neurons. Single-nucleus RNA sequencing revealed that LC perturbation induces widespread but highly cell type- and region-specific transcriptional program changes, shaped by the distribution of adrenergic receptor subtypes. These findings support a model in which a shared global signal of neuromodulatory tone can produce discrete, context-dependent cellular outcomes through distinct molecular gating mechanisms of cell-type-specific adrenergic receptor subtype combinations. By establishing gene expression as a quantifiable metric of neuromodulatory control, this study lays the foundation for transcriptionally informed interventions capable of modulating brain functions with cellular precision.

neuroscience↗

Lineage-specific proteome remodeling of diverse lung cancer cells by targeted epigenetic inhibitors

Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogeneous and dependent on factors such as the genetic background and metabolic state of cells, as well as on-/off-target engagement of individual small-molecule compounds. The molecular study of the extent of this heterogeneity often measures changes in a single cell line. To more comprehensively profile the effects of small-molecule perturbations and their influence on heterogeneous cellular responses, we present a molecular resource based on the quantification of chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitors (HDACi) in non-isogenic cell lines. Through quantitative molecular profiling of 10,621 proteins, these data reveal coordinated molecular remodeling of HDACi treated cancer cells. HDACi-regulated proteins differ greatly across cell lines with consistent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) cell-state effectors. Together these data provide valuable insight into cell-type driven and heterogeneous responses that must be taken into consideration when monitoring molecular perturbations in culture models. We have also built a web interface for the extensive amount of data to allow users to explore the data as a resource for understanding chemical perturbation of diverse cell types.

cancer biology↗

A heterogenous pharmaco-transcriptomic landscape induced by targeting a single oncogenic kinase

Over-activation of the epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR-targeted therapies have led to minimal clinical response. While delivery of EGFR inhibitors (EGFRis) to the brain constitutes a major challenge, how additional drug-specific features alter efficacy remains poorly understood. We introduce SCHEMATIC, which integrates multiplex single-cell chemical transcriptomics with deep-generative classification to resolve chemotype-specific and shared programs and apply it to to define the molecular response of glioblastoma to EGFRis. We identify programs that differ by the chemical properties of EGFRis, including induction of adaptive transcription and modulation of immunogenic gene expression. We find that induction of an adaptive transcriptional program is associated with persistence of surviving cells after EGFR inhibition, and that concurrent EGFR/PI3K inhibition attenuates this program. We also find that pro-immunogenic expression changes associated with a subset of tyrphostin-family EGFR inhibitors are accompanied by enhanced antigen-specific cytotoxic T-cell killing in vitro. Our study provides a framework that considers each agents unique and often unknown poly-pharmacology to prioritize compounds pre-clinically that induce favorable molecular responses.

genomics↗

Mitosis exit followed by death in interphase prevents the development of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells can escape death in mitosis and exit mitosis, and become malignant polyploid giant cancer cells (PGCC). Considering the low number of malignant cells undergoing mitosis in tumor tissue, killing these cells in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule assembly, preferentially kills cancer cells in interphase as opposed to mitosis, and avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces an integrated stress response and promotes mitochondria fission accompanied by a reduction in energy metabolism. This cell response may underly death in interphase and avoid the development of PGCC.

cell biology↗

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Chemical genetic screens are a powerful tool for exploring how cancer cells response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

genomics↗

High-Capacity Sample Multiplexing for Single Cell Chromatin Accessibility Profiling

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

molecular biology↗

Starfysh reveals heterogeneous spatial dynamics in the breast tumor microenvironment

Spatially-resolved gene expression profiling provides valuable insight into tissue organization and cell-cell crosstalk; however, spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for a rigorous interpretation of cell states and do not utilize associated histology images. Significant sample variation further complicates the integration of ST datasets, which is essential for identifying commonalities across tissues or altered cellular wiring in disease. Here, we present Starfysh, the first comprehensive computational toolbox for joint modeling of ST and histology data, dissection of refined cell states, and systematic integration of multiple ST datasets from complex tissues. Starfysh uses an auxiliary deep generative model that incorporates archetypal analysis and any known cell state markers to avoid the need for a single-cell-resolution reference in characterizing known or novel tissue-specific cell states. Additionally, Starfysh improves the characterization of spatial dynamics in complex tissues by leveraging histology images and enables the comparison of niches as spatial "hubs" across tissues. Integrative analysis of primary estrogen receptor-positive (ER+) breast cancer, triple-negative breast cancer (TNBC), and metaplastic breast cancer (MBC) tumors using Starfysh led to the identification of heterogeneous patient- and disease-specific hubs as well as a shared stromal hub with varying spatial orientation. Our results show the ability to delineate the spatial co-evolution of tumor and immune cell states and their crosstalk underlying intratumoral heterogeneity in TNBC and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC. Starfysh is publicly available (https://github.com/azizilab/starfysh).

bioinformatics↗

Developmental and conditional dynamics of gene expression in single root cells of A. thaliana

Single-cell RNA-seq can yield high-resolution cell-type-specific expression signatures that reveal new cell types and the developmental trajectories of cell lineages. Here, we apply this approach to A. thaliana root cells to capture gene expression in 3,121 root cells. We analyze these data with Monocle 3, which orders single cell transcriptomes in an unsupervised manner and uses machine learning to reconstruct single-cell developmental trajectories along pseudotime. We identify hundreds of genes with cell-type-specific expression, with pseudotime analysis of several cell lineages revealing both known and novel genes that are expressed along a developmental trajectory. We identify transcription factor motifs that are enriched in early and late cells, together with the corresponding candidate transcription factors that likely drive the observed expression patterns. We assess and interpret changes in total RNA expression along developmental trajectories and show that trajectory branch points mark developmental decisions. Finally, by applying heat stress to whole seedlings, we address the longstanding question of possible heterogeneity among cell types in the response to an abiotic stress. Although the response of canonical heat shock genes dominates expression across cell types, subtle but significant differences in other genes can be detected among cell types. Taken together, our results demonstrate that single-cell transcriptomics holds promise for studying plant development and plant physiology with unprecedented resolution.

genetics↗

Towards inferring causal gene regulatory networks from single cell expression measurements

Single-cell transcriptome sequencing now routinely samples thousands of cells, potentially providing enough data to reconstruct causal gene regulatory networks from observational data. Here, we present Scribe, a toolkit for detecting and visualizing causal regulatory interactions between genes and explore the potential for single-cell experiments to power network reconstruction. Scribe employs Restricted Directed Information to determine causality by estimating the strength of information transferred from a potential regulator to its downstream target. We apply Scribe and other leading approaches for causal network reconstruction to several types of single-cell measurements and show that there is a dramatic drop in performance for \"pseudotime\" ordered single-cell data compared to true time series data. We demonstrate that performing causal inference requires temporal coupling between measurements. We show that methods such as \"RNA velocity\" restore some degree of coupling through an analysis of chromaffin cell fate commitment. These analyses therefore highlight an important shortcoming in experimental and computational methods for analyzing gene regulation at single-cell resolution and point the way towards overcoming it.

genomics↗

crisprQTL mapping as a genome-wide association framework for cellular genetic screens

Expression quantitative trait locus (eQTL) and genome-wide association studies (GWAS) are powerful paradigms for mapping the determinants of gene expression and organismal phenotypes, respectively. However, eQTL mapping and GWAS are limited in scope (to naturally occurring, common genetic variants) and resolution (by linkage disequilibrium). Here, we present crisprQTL mapping, a framework in which large numbers of CRISPR/Cas9 perturbations are introduced to each cell on an isogenic background, followed by single-cell RNA-seq (scRNA-seq). crisprQTL mapping is analogous to conventional human eQTL studies, but with individual humans replaced by individual cells; genetic variants replaced by unique combinations of unlinked guide RNA (gRNA)-programmed perturbations per cell; and tissue-level RNA-seq of many individuals replaced by scRNA-seq of many cells. By randomly introducing gRNAs, a single population of cells can be leveraged to test for association between each perturbation and the expression of any potential target gene, analogous to how eQTL studies leverage populations of humans to test millions of genetic variants for associations with expression in a genome-wide manner. However, crisprQTL mapping is neither limited to naturally occurring, common genetic variants nor by linkage disequilibrium. As a proof-of-concept, we applied crisprQTL mapping to evaluate 1,119 candidate enhancers with no strong a priori hypothesis as to their target gene(s). Perturbations were made by a nuclease-dead Cas9 (dCas9) tethered to KRAB, and introduced at a mean allele frequency of 1.1% into a population of 47,650 profiled human K562 cells (median of 15 gRNAs identified per cell). We tested for differential expression of all genes within 1 megabase (Mb) of each candidate enhancer, effectively evaluating 17,584 potential enhancer-target gene relationships within a single experiment. At an empirical false discovery rate (FDR) of 10%, we identify 128 cis crisprQTLs (11%) whose targeting resulted in downregulation of 105 nearby genes. crisprQTLs were strongly enriched for proximity to their target genes (median 34.3 kilobases (Kb)) and the strength of H3K27ac, p300, and lineage-specific transcription factor (TF) ChIP-seq peaks. Our results establish the power of the eQTL mapping paradigm as applied to programmed variation in populations of cells, rather than natural variation in populations of individuals. We anticipate that crisprQTL mapping will facilitate the comprehensive elucidation of the cis-regulatory architecture of the human genome.

genomics↗

On the design of CRISPR-based single cell molecular screens

Several groups recently reported coupling CRISPR/Cas9 perturbations and single cell RNA-seq as a potentially powerful approach for forward genetics. Here we demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guide-barcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity. We optimize a published strategy, CROP-seq, that instead uses a Pol II transcribed copy of the sgRNA sequence itself, doubling the rate at which guides are assigned to cells to 94%. We confirm this strategy performs robustly and further explore experimental best practices for CRISPR/Cas9-based single cell molecular screens.

genomics↗