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Shalek, A. K.

Publications and source records attributed to Shalek, A. K..

At least 73 records · Page 4Linked to original sources
bioRxiv2020

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice

Existing pre-clinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates using an autochthonous small cell lung cancer model, we extrapolated half-life times in the circulation of 50-100 seconds and intravasation rates between 4,000 and 27,000 CTCs/hour - an average daily shedding rate equivalent to [~]0.07% of the total number of primary tumor cells in the lung. Additionally, transfer of 1-2% of daily-shed CTCs from late-stage tumor-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

cancer biology↗
bioRxiv2020

Single-cell profiling of tuberculosis lung granulomas reveals functional lymphocyte signatures of bacterial control

Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. There is also a temporal aspect to bacterial control, in that granulomas that arise later in infection (in the context of an established immune response) share the functional characteristics of restrictive granulomas and are more capable of killing Mtb. Taken together, our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB. One-Sentence SummaryBacterial control in TB lung granulomas correlates with distinct cellular immune microenvironments and time of formation after infection.

immunology↗
bioRxiv2020

GM-CSF differentiation of human monocytes stabilizes macrophage state via oxidative signaling

Macrophages are central mediators of immunity that integrate diverse signals derived from differentiation cues, tissue location, and disease. Controlling macrophage state and function is an appealing therapeutic objective across many diseases including cancer, atherosclerosis, and tuberculosis. Despite the growing appreciation for the in vivo complexity of macrophage state, existing in vitro models of human monocyte-derived macrophages have used a limited number of individual perturbations to explore the complex phenotypic space that macrophages can occupy. Here, we leverage a tiered differentiation, activation, and stimulation strategy to generate libraries of in vitro monocyte-derived macrophages and examine the in vitro state space of macrophage function using high-dimensional technologies. Our tiered experimental approach further revealed a striking relationship between GM-CSF differentiation and IL-10 production. Cells that were differentiated with GM-CSF produced very low or undetectable levels of IL-10 independent of activation or stimulation condition. To nominate candidate regulators of this IL-10 response, we leverage unbiased single-cell mRNA sequencing to identify transcriptional networks associated with GM-CSF-derived cells. Using these data, we identify oxidative signaling pathways as upregulated in GM-CSF derived cells and demonstrate that scavenging of oxidative radicals can enhance IL-10 production in these cells. Collectively, these data underscore the complexity of monocyte-derived macrophage state over time and highlight a dominant role for GM-CSF in tuning macrophage inflammatory phenotype, metabolic state, and plasticity.

immunology↗
bioRxiv2020

Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer

Bulk transcriptomic studies have defined classical and basal-like gene expression subtypes in pancreatic ductal adenocarcinoma (PDAC) that correlate with survival and response to chemotherapy; however, the underlying mechanisms that govern these subtypes and their heterogeneity remain elusive. Here, we performed single-cell RNA-sequencing of 23 metastatic PDAC needle biopsies and matched organoid models to understand how tumor cell-intrinsic features and extrinsic factors in the tumor microenvironment (TME) shape PDAC cancer cell phenotypes. We identify a novel cancer cell state that co-expresses basal-like and classical signatures, demonstrates upregulation of developmental and KRAS-driven gene expression programs, and represents a transitional intermediate between the basal-like and classical poles. Further, we observe structure to the metastatic TME supporting a model whereby reciprocal intercellular signaling shapes the local microenvironment and influences cancer cell transcriptional subtypes. In organoid culture, we find that transcriptional phenotypes are plastic and strongly skew toward the classical expression state, irrespective of genotype. Moreover, we show that patient-relevant transcriptional heterogeneity can be rescued by supplementing organoid media with factors found in the TME in a subtype-specific manner. Collectively, our study demonstrates that distinct microenvironmental signals are critical regulators of clinically relevant PDAC transcriptional states and their plasticity, identifies the necessity for considering the TME in cancer modeling efforts, and provides a generalizable approach for delineating the cell-intrinsic versus -extrinsic factors that govern tumor cell phenotypes.

cancer biology↗
bioRxiv2020

Single-cell profiling of Ebola virus infection in vivo reveals viral and host transcriptional dynamics

Ebola virus (EBOV) causes epidemics with high case fatality rates, yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. To better understand EBOV infection in vivo, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cell activity during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, providing insight into pathogenesis. We find that immature, proliferative monocyte-lineage cells with reduced antigen presentation capacity replace conventional circulating monocyte subsets within days of infection, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying viral RNA abundance in individual cells, we identify molecular determinants of tropism and examine temporal dynamics in viral and host gene expression. Within infected cells, we observe that EBOV down-regulates STAT1 mRNA and interferon signaling, and up-regulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating cellular pathways the virus manipulates for its replication. Overall, this study sheds light on EBOV tropism, replication dynamics, and elicited immune response, and provides a framework for characterizing interactions between hosts and emerging viruses in a maximum containment setting.

microbiology↗
bioRxiv2020

Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

systems biology↗
bioRxiv2020

Single-cell analysis reveals mechanisms of plasticity of leukemia initiating cells

Leukemia initiating cells (LICs) fuel leukemic growth and spark relapse. Previously thought to be primitive and rare, the LIC state may actually be heterogeneous and dynamic, enabling evasion of therapies. Here, we use single-cell transcriptomics to track LIC multipotency within the cellular ontogeny of MLL-rearranged B-lymphoblastic leukemia (MLL-r B-ALL). Although we identify rare transcriptionally and phenotypically primitive LICs, we also observe LICs emerging from more differentiated populations with the capability to replenish the full leukemic cellular diversity. We find that activation of MYC-driven oxidative phosphorylation controls this process of facultative state conversion in LICs.

cancer biology↗
bioRxiv2020

High-throughput organoid screening enables engineering of intestinal epithelial composition

Barrier tissue epithelia play an essential role in maintaining organismal homeostasis, and changes in their cellular composition have been observed in multiple human diseases. Within the small intestinal epithelium, adult stem cells integrate diverse signals to regulate regeneration and differentiation, thereby establishing overall cellularity. Accordingly, directing stem cell differentiation could provide a tractable approach to alter the abundance or quality of specialized cells of the small intestinal epithelium, including the secretory Paneth, goblet, and enteroendocrine populations. Yet, to date, there has been a lack of suitable tools and rigorous approaches to identify biological targets and pharmacological agents that can modify epithelial composition to enable causal testing of disease-associated changes with novel therapeutic candidates. To empower the search for epithelia-modifying agents, we establish a first-of-its-kind high-throughput phenotypic organoid screen. We demonstrate the ability to screen thousands of samples and uncover biological targets and associated small molecule inhibitors which translate to in vivo. This approach is enabled by employing a functional, cell-type specific, scalable assay on an organoid model designed to represent the physiological cues of in vivo Paneth cell differentiation from adult intestinal stem cells. Further, we miniaturize and adapt the organoid culture system to enable automated plating and screening, thereby providing the ability to test thousands of samples. Strikingly, in our screen we identify inhibitors of the nuclear exporter Xpo1 modulate stem cell fate commitment by inducing a pan-epithelial stress response combined with an interruption of mitogen signaling in cycling intestinal progenitors, thereby significantly increasing the abundance of Paneth cells independent of known WNT and Notch differentiation cues. We extend our observation in vivo, demonstrating that oral administration of Xpo1 inhibitor KPT-330 at doses 1,000-fold lower than conventionally used in hematologic malignancies increases Paneth cell abundance. In total, we provide a framework to identify novel biological cues and therapeutic leads to rebalance intestinal stem cell differentiation and modulate epithelial tissue composition via high-throughput phenotypic screening in rationally-designed organoid model of differentiation.

bioengineering↗
bioRxiv2020

Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.

genomics↗
bioRxiv2020

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Whether cultured in vitro or part of a complex tissue in vivo, a cells phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cells spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT--Spatially PhotoActivatable Color Encoded Cell Address Tags--to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and single-cell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental insights into the connections between tissue microenvironments and biological (dys)function.

systems biology↗
bioRxiv2019

Sensory Neurons Innervate Peripheral Lymph Nodes and Locally Regulate Gene Expression in Postsynaptic Endothelium, Stromal Cells, and Innate Leukocytes

Immune responses within barrier tissues are regulated, in part, by nociceptors, specialized peripheral sensory neurons that detect noxious stimuli. Previous work has shown that nociceptor ablation not only alters local responses to immune challenge at peripheral sites, but also within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Indeed, although sympathetic innervation of LNs is well documented, it has been unclear whether the LN parenchyma itself is innervated by sensory neurons. Here, using a combination of high-resolution imaging, retrograde viral tracing, single-cell transcriptomics (scRNA-seq), and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is preferentially located in the outermost cortex of skin-draining LNs. Transcriptomic profiling revealed that there are at least four discrete subsets of sensory neurons that innervate LNs with a predominance of peptidergic nociceptors, and an innervation pattern that is distinct from that in the surrounding skin. To uncover potential LN-resident communication partners for LN-innervating sensory neurons, we employed scRNA-seq to generate a draft atlas of all murine LN cells and, based on receptor-ligand expression patterns, nominated candidate target populations among stromal and immune cells. Using selective optogenetic stimulation of LN-innervating sensory axons, we directly experimentally tested our inferred connections. Acute neuronal activation triggered rapid transcriptional changes preferentially within our top-ranked putative interacting partners, principally endothelium and other nodal stroma cells, as well as several innate leukocyte populations. Thus, LNs are monitored by a unique population of sensory neurons that possesses immunomodulatory potential.

immunology↗
bioRxiv2019

Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology

The development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts - such as transcription factors, affinity receptors, and signaling molecules - by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (\"Second-Strand Synthesis\") that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commercial analogs. We first characterized the performance of Seq-Well S^3 in cell lines and PBMCs, and then examined five different inflammatory skin diseases, illustrative of distinct types of inflammation, to explore the breadth of potential immune and parenchymal cell states. Our work presents an essential methodological advance as well as a valuable resource for studying the cellular and molecular features that inform human skin inflammation.

immunology↗
bioRxiv2019

Identification of a master regulator of differentiation in Toxoplasma

Toxoplasma gondii chronically infects a quarter of the worlds population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Chronic stages are established by differentiation of rapidly replicating tachyzoites into slow-growing bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. Despite its central role in infection, the molecular basis of chronic differentiation is not understood. Through Cas9-mediated genetic screening and single-cell transcriptional profiling, we identify and characterize a putative transcription factor (BFD1) as necessary and sufficient for differentiation. Translation of BFD1 appears to be stress regulated, and its constitutive expression elicits differentiation in the absence of stress. As a Myb-like factor, BFD1 provides a counterpoint to the ApiAP2 factors which dominate our current view of parasite gene regulation. Overall, BFD1 provides a genetic switch to study and control Toxoplasma differentiation, and will inform prevention and treatment of chronic infection.

microbiology↗
bioRxiv2019

Integrated Single-Cell Analysis of Multicellular Immune Dynamics during Hyper-Acute HIV-1 Infection

Cellular immunity is critical for controlling intracellular pathogens, but the dynamics and cooperativity of the evolving host response to infection are not well defined. Here, we apply single-cell RNA-sequencing to longitudinally profile pre- and immediately post-HIV infection peripheral immune responses of multiple cell types in four untreated individuals. Onset of viremia induces a strong transcriptional interferon response integrated across most cell types, with subsequent pro-inflammatory T cell differentiation, monocyte MHC-II upregulation, and cytolytic killing. With longitudinal sampling, we nominate key intra- and extracellular drivers that induce these programs, and assign their multi-cellular targets, temporal ordering, and duration in acute infection. Two individuals studied developed spontaneous viral control, associated with initial elevated frequencies of proliferating cytotoxic cells, inclusive of a previously unappreciated proliferating natural killer (NK) cell subset. Our study presents a unified framework for characterizing immune evolution during a persistent human viral infection at single-cell resolution, and highlights programs that may drive response coordination and influence clinical trajectory.

immunology↗
bioRxiv2019

Systematic comparative analysis of single cell RNA-sequencing methods

A multitude of single-cell RNA sequencing methods have been developed in recent years, with dramatic advances in scale and power, and enabling major discoveries and large scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single cell and/or single nucleus profiling from three types of samples - cell lines, peripheral blood mononuclear cells and brain tissue - generating 36 libraries in six separate experiments in a single center. To analyze these datasets, we developed and applied scumi, a flexible computational pipeline that can be used for any scRNA-seq method. We evaluated the methods for both basic performance and for their ability to recover known biological information in the samples. Our study will help guide experiments with the methods in this study as well as serve as a benchmark for future studies and for computational algorithm development.

genomics↗
bioRxiv2019

IL-5Rα marks nasal polyp IgG4 and IgE-secreting cells in aspirin-exacerbated respiratory disease

BackgroundThe cause of nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but the upstream drivers and cellular mechanisms of local antibody production in AERD remain to be investigated. ObjectiveWe sought to identify the upstream drivers and phenotypic properties of local antibody-secreting cells in nasal polyps and to understand their clinical relevance in AERD. MethodsSinus tissue was obtained from subjects with AERD, aspirin-tolerant chronic rhinosinusitis with nasal polyps (CRSwNP), aspirin-tolerant chronic rhinosinusitis without nasal polyps (CRSsNP), and healthy controls. Tissue antibody levels were quantified via ELISA and immunohistochemistry, and were correlated with clinical markers of disease severity. Tissue cytokine mRNA levels were measured with quantitative PCR (qPCR). Antibody-secreting cells were profiled with a combination of single-cell RNA-sequencing (scRNA-seq), flow cytometry and immunofluorescence. ResultsTissue IgE and IgG4 were elevated in AERD compared to controls (p<0.01 for IgE and p<0.001 for IgG4, vs. CRSwNP). Total IgG and IgG4 positively correlated with the number of polyp surgeries per subject (r=0.48, p=0.011 and r=0.58, p=0.0003, respectively). Polyp IL-10 mRNA expression was higher in AERD vs. CRSwNP (p<0.05), but there were no differences in mRNA expression of type 2 cytokines. ScRNA-seq revealed increased IL5RA, IGHG4, and IGHE in the antibody-associated cells of subjects with AERD compared to CRSwNP. Total plasma cells and IL-5R+ plasma cell numbers in the polyp tissue from AERD exceeded those in polyps from CRSwNP (p=0.0051 and p=0.026, respectively) by flow cytometry. With immunofluorescence, we determined that IL-5R and IgG4 are co-expressed in antibody-secreting cells in AERD. ConclusionsOur study identifies unique clusters of antibody-secreting cells in AERD defined by enrichment of transcripts encoding IL5RA, IGHG4 and IGHE. We confirm surface expression of IL-5R on these cells, and identify T cells as a unique transcriptional source of IL-5. Tissue antibody levels are elevated in AERD and correlate with disease severity. Our findings suggest a role for IL-5 in facilitating local antibody production that may drive features of severe sinus disease. Key MessagesO_LIIgG4 and IgE levels are markedly increased in nasal polyp tissue from subjects with AERD compared to aspirin-tolerant CRSwNP. C_LIO_LITissue IgG4 levels positively correlate with disease recurrence. C_LIO_LIIL-10 mRNA levels are significantly higher in AERD polyp tissue compared to CRSwNP tissue, but differences were not noted for type 2 cytokines or cytokines involved in class switch recombination. C_LIO_LIIL-5R transcript and protein surface expression is elevated in antibody-secreting cells from subjects with AERD and may play a role in facilitating class switching and/or survival of antibody-secreting cells. C_LI Capsule SummarySingle-cell RNA-sequencing (scRNA-seq) of whole nasal polyp tissue identified increased IL5RA, IGHE, and IGHG4 expression in the antibody-secreting cell compartment of subjects with aspirin-exacerbated respiratory disease (AERD) compared to aspirin-tolerant chronic rhinosinusitis with nasal polyps (CRSwNP). IgE and IgG4 levels are elevated in nasal polyp tissue from subjects with AERD compared to CRSwNP and correlate with disease recurrence.

immunology↗
bioRxiv2018

Linking single-cell measurements of mass, growth rate, and gene expression

We introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-of-principle, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts.

bioengineering↗