Publications by authors named "Linas Mazutis"

37 Publications

Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer's disease.

Nat Neurosci 2021 03 8;24(3):343-354. Epub 2021 Feb 8.

The Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY, USA.

Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APP+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APP+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.
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http://dx.doi.org/10.1038/s41593-020-00796-zDOI Listing
March 2021

A gene-environment-induced epigenetic program initiates tumorigenesis.

Nature 2021 02 3;590(7847):642-648. Epub 2021 Feb 3.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Tissue damage increases the risk of cancer through poorly understood mechanisms. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
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http://dx.doi.org/10.1038/s41586-020-03147-xDOI Listing
February 2021

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Cell Rep 2020 10;33(2):108268

Faculty of Biological and Environmental Sciences, P.O. Box 56, 00014 University of Helsinki, Helsinki, Finland. Electronic address:

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.
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http://dx.doi.org/10.1016/j.celrep.2020.108268DOI Listing
October 2020

Multi-step processing of single cells using semi-permeable capsules.

Lab Chip 2020 10;20(21):4052-4062

Institute of Biotechnology, Life Science Centre, Vilnius University, 7 Sauletekio av., Vilnius, LT-10257, Lithuania.

Droplet microfluidics technology provides a powerful approach to isolate and process millions of single cells simultaneously. Despite many exciting applications that have emerged based on this technology, workflows based on multi-step operations, including molecular biology and cell-based phenotypic screening assays, cannot be easily adapted to droplet format. Here, we present a microfluidics-based technique to isolate single cells, or biological samples, into semi-permeable hydrogel capsules and perform multi-step biological workflows on thousands to millions of individual cells simultaneously. The biochemical reactions are performed by changing the aqueous buffer surrounding the capsules, without needing sophisticated equipment. The semi-permeable nature of the capsules' shell retains large encapsulated biomolecules (such as genome) while allowing smaller molecules (such as proteins) to passively diffuse. In contrast to conventional hydrogel bead assays, the approach presented here improves bacterial cell retention during multi-step procedures as well as the efficiency of biochemical reactions. We showcase two examples of capsule use for single genome amplification of bacteria, and expansion of individual clones into isogenic microcolonies for later screening for biodegradable plastic production.
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http://dx.doi.org/10.1039/d0lc00660bDOI Listing
October 2020

Emergence of a High-Plasticity Cell State during Lung Cancer Evolution.

Cancer Cell 2020 08 23;38(2):229-246.e13. Epub 2020 Jul 23.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Cell and Developmental Biology, Weill-Cornell Medical College, New York, NY 10065, USA. Electronic address:

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.
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http://dx.doi.org/10.1016/j.ccell.2020.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745838PMC
August 2020

Cancer cells deploy lipocalin-2 to collect limiting iron in leptomeningeal metastasis.

Science 2020 07;369(6501):276-282

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

The tumor microenvironment plays a critical regulatory role in cancer progression, especially in central nervous system metastases. Cancer cells within the cerebrospinal fluid (CSF)-filled leptomeninges face substantial microenvironmental challenges, including inflammation and sparse micronutrients. To investigate the mechanism by which cancer cells in these leptomeningeal metastases (LM) overcome these constraints, we subjected CSF from five patients with LM to single-cell RNA sequencing. We found that cancer cells, but not macrophages, within the CSF express the iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17. These macrophages generate inflammatory cytokines that induce cancer cell LCN2 expression but do not generate LCN2 themselves. In mouse models of LM, cancer cell growth is supported by the LCN2/SLC22A17 system and is inhibited by iron chelation therapy. Thus, cancer cells appear to survive in the CSF by outcompeting macrophages for iron.
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http://dx.doi.org/10.1126/science.aaz2193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816199PMC
July 2020

Regenerative potential of prostate luminal cells revealed by single-cell analysis.

Science 2020 05;368(6490):497-505

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Androgen deprivation is the cornerstone of prostate cancer treatment. It results in involution of the normal gland to ~90% of its original size because of the loss of luminal cells. The prostate regenerates when androgen is restored, a process postulated to involve stem cells. Using single-cell RNA sequencing, we identified a rare luminal population in the mouse prostate that expresses stemlike genes ( and ) and a large population of differentiated cells (, ). In organoids and in mice, both populations contribute equally to prostate regeneration, partly through androgen-driven expression of growth factors (Nrg2, Rspo3) by mesenchymal cells acting in a paracrine fashion on luminal cells. Analysis of human prostate tissue revealed similar differentiated and stemlike luminal subpopulations that likewise acquire enhanced regenerative potential after androgen ablation. We propose that prostate regeneration is driven by nearly all persisting luminal cells, not just by rare stem cells.
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http://dx.doi.org/10.1126/science.aay0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313621PMC
May 2020

Regenerative lineages and immune-mediated pruning in lung cancer metastasis.

Nat Med 2020 02 10;26(2):259-269. Epub 2020 Feb 10.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.
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http://dx.doi.org/10.1038/s41591-019-0750-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021003PMC
February 2020

Rapid non-uniform adaptation to conformation-specific KRAS(G12C) inhibition.

Nature 2020 01 8;577(7790):421-425. Epub 2020 Jan 8.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma. KRAS(G12C) inhibitors are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation, and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic.
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http://dx.doi.org/10.1038/s41586-019-1884-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308074PMC
January 2020

Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity.

Cell 2019 10 24;179(4):846-863.e24. Epub 2019 Oct 24.

Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Ludwig Center at Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Dendritic cells (DCs) play a critical role in orchestrating adaptive immune responses due to their unique ability to initiate T cell responses and direct their differentiation into effector lineages. Classical DCs have been divided into two subsets, cDC1 and cDC2, based on phenotypic markers and their distinct abilities to prime CD8 and CD4 T cells. While the transcriptional regulation of the cDC1 subset has been well characterized, cDC2 development and function remain poorly understood. By combining transcriptional and chromatin analyses with genetic reporter expression, we identified two principal cDC2 lineages defined by distinct developmental pathways and transcriptional regulators, including T-bet and RORγt, two key transcription factors known to define innate and adaptive lymphocyte subsets. These novel cDC2 lineages were characterized by distinct metabolic and functional programs. Extending our findings to humans revealed conserved DC heterogeneity and the presence of the newly defined cDC2 subsets in human cancer.
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http://dx.doi.org/10.1016/j.cell.2019.09.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838684PMC
October 2019

A chemical probe of CARM1 alters epigenetic plasticity against breast cancer cell invasion.

Elife 2019 10 28;8. Epub 2019 Oct 28.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States.

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here ( in this work) is presented as a CARM1 chemical probe with pro-drug properties. () can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of , and on-target engagement. () recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the ()-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, () and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.
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http://dx.doi.org/10.7554/eLife.47110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917500PMC
October 2019

Author Correction: Characterization of cell fate probabilities in single-cell data with Palantir.

Nat Biotechnol 2019 Oct;37(10):1237

Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41587-019-0282-0DOI Listing
October 2019

Natural Genetic Variation Reveals Key Features of Epigenetic and Transcriptional Memory in Virus-Specific CD8 T Cells.

Immunity 2019 05 23;50(5):1202-1217.e7. Epub 2019 Apr 23.

Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Ludwig Center at Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Stable changes in chromatin states and gene expression in cells of the immune system form the basis for memory of infections and other challenges. Here, we used naturally occurring cis-regulatory variation in wild-derived inbred mouse strains to explore the mechanisms underlying long-lasting versus transient gene regulation in CD8 T cells responding to acute viral infection. Stably responsive DNA elements were characterized by dramatic and congruent chromatin remodeling events affecting multiple neighboring sites and required distinct transcription factor (TF) binding motifs for their accessibility. Specifically, we found that cooperative recruitment of T-box and Runx family transcription factors to shared targets mediated stable chromatin remodeling upon T cell activation. Our observations provide insights into the molecular mechanisms driving virus-specific CD8 T cell responses and suggest a general mechanism for the formation of transcriptional and epigenetic memory applicable to other immune and non-immune cells.
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http://dx.doi.org/10.1016/j.immuni.2019.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7023907PMC
May 2019

Characterization of cell fate probabilities in single-cell data with Palantir.

Nat Biotechnol 2019 04 21;37(4):451-460. Epub 2019 Mar 21.

Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Single-cell RNA sequencing studies of differentiating systems have raised fundamental questions regarding the discrete versus continuous nature of both differentiation and cell fate. Here we present Palantir, an algorithm that models trajectories of differentiating cells by treating cell fate as a probabilistic process and leverages entropy to measure cell plasticity along the trajectory. Palantir generates a high-resolution pseudo-time ordering of cells and, for each cell state, assigns a probability of differentiating into each terminal state. We apply our algorithm to human bone marrow single-cell RNA sequencing data and detect important landmarks of hematopoietic differentiation. Palantir's resolution enables the identification of key transcription factors that drive lineage fate choice and closely track when cells lose plasticity. We show that Palantir outperforms existing algorithms in identifying cell lineages and recapitulating gene expression trends during differentiation, is generalizable to diverse tissue types, and is well-suited to resolving less-studied differentiating systems.
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http://dx.doi.org/10.1038/s41587-019-0068-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549125PMC
April 2019

Editorial overview: Current advances in analytical biotechnology: from single molecules to whole organisms.

Curr Opin Biotechnol 2019 02 7;55:iii-vi. Epub 2019 Jan 7.

Laboratory of Microtechnologies, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, Vilnius LT-10257, Lithuania. Electronic address:

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http://dx.doi.org/10.1016/j.copbio.2018.12.006DOI Listing
February 2019

Multi-omics at single-cell resolution: comparison of experimental and data fusion approaches.

Curr Opin Biotechnol 2019 02 24;55:159-166. Epub 2018 Oct 24.

Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio Av. 7, Vilnius LT-10257, Lithuania. Electronic address:

Biological samples are inherently heterogeneous and complex. Tackling this complexity requires innovative technological and analytical solutions. Recent advances in high-throughput single-cell isolation and nucleic acid barcoding methods are rapidly changing the technological landscape of biological sciences and now make it possible to measure the (epi)genomic, transcriptomic, or proteomic state of individual cells. In addition, few experimental approaches enable multi-omics measurements of the same cell. However, merging-omics data collected from different experiments remains a considerable challenge. Although several strategies for merging transcriptomics datasets have recently been introduced, cell-to-cell variability and heterogeneity remains one of the confounding factors limiting data fusion and integration. Here, we focus our discussion on the latest single-cell technological and analytical solutions to achieve high data dimensionality and resolution. Obtaining datasets with a wealth of multi-omics information will undoubtedly provide new avenues for researchers to unravel the complexity of biological samples encountered in modern biological research and molecular diagnostics.
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http://dx.doi.org/10.1016/j.copbio.2018.09.012DOI Listing
February 2019

CD49b defines functionally mature Treg cells that survey skin and vascular tissues.

J Exp Med 2018 11 24;215(11):2796-2814. Epub 2018 Oct 24.

Howard Hughes Medical Institute, Immunology Program, and Ludwig Center, Memorial Sloan Kettering Cancer Center, New York, NY

Regulatory T (Treg) cells prevent autoimmunity by limiting immune responses and inflammation in the secondary lymphoid organs and nonlymphoid tissues. While unique subsets of Treg cells have been described in some nonlymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. Furthermore, it is possible that Treg cells from similar tissue types share largely similar properties. We have identified a short-lived effector Treg cell subset that expresses the α integrin, CD49b, and exhibits a unique tissue distribution, being abundant in peripheral blood, vasculature, skin, and skin-draining lymph nodes, but uncommon in the intestines and in viscera-draining lymph nodes. CD49b Treg cells, which display superior functionality revealed by in vitro and in vivo assays, appear to develop after multiple rounds of cell division and TCR-dependent activation. Accordingly, single-cell RNA-seq analysis placed these cells at the apex of the Treg developmental trajectory. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues.
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http://dx.doi.org/10.1084/jem.20181442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219731PMC
November 2018

Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.

Cell 2018 08 28;174(5):1293-1308.e36. Epub 2018 Jun 28.

Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.
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http://dx.doi.org/10.1016/j.cell.2018.05.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348010PMC
August 2018

Recovering Gene Interactions from Single-Cell Data Using Data Diffusion.

Cell 2018 07 28;174(3):716-729.e27. Epub 2018 Jun 28.

Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed "dropout," which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations.
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http://dx.doi.org/10.1016/j.cell.2018.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771278PMC
July 2018

Tumor progression effects on drug vector access to tumor-associated capillary bed.

J Control Release 2017 09 31;261:216-222. Epub 2017 May 31.

The Houston Methodist Research Institute, Houston, TX, USA. Electronic address:

Over the last decade, the benefits of drug vectors to treat cancer have been well recognized. However, drug delivery and vector distribution differences in tumor-associated capillary bed at different stages of disease progression are not well understood. To obtain further insights into drug vector distribution changes in vasculature during tumor progression, we combined intra-vital imaging of metastatic tumors in mice, microfluidics-based artificial tumor capillary models, and Computational Fluid Dynamics (CFD) modeling. Microfluidic and CFD circulation models were designed to mimic tumor progression by escalating flow complexity and chaoticity. We examined flow of 0.5 and 2μm spherical particles, and tested the effects of hematocrit on particle local accessibility to flow area of capillary beds by co-circulating red blood cells (RBC). Results showed that tumor progression modulated drug vector distribution in tumor-associated capillaries. Both particles shared 80-90% common flow area, while 0.5 and 2μm particles had 2-9% and 1-2% specific flow area, respectively. Interestingly, the effects of hematocrit on specific circulation area was opposite for 0.5 and 2μm particles. Dysfunctional capillaries with no flow, a result of tumor progression, limited access to all particles, while diffusion was shown to be the only prevailing transport mechanism. In view of drug vector distribution in tumors, independent of formulation and other pharmacokinetic aspects, our results suggest that the evolution of tumor vasculature during progression may influence drug delivery efficiency. Therefore, optimized drug vectors will need to consider primary vs metastatic tumor setting, or early vs late stage metastatic disease, when undergoing vector design.
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http://dx.doi.org/10.1016/j.jconrel.2017.05.031DOI Listing
September 2017

Single-cell barcoding and sequencing using droplet microfluidics.

Nat Protoc 2017 Jan 8;12(1):44-73. Epub 2016 Dec 8.

Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.
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http://dx.doi.org/10.1038/nprot.2016.154DOI Listing
January 2017

Statistical Mechanics of Allosteric Enzymes.

J Phys Chem B 2016 07 29;120(26):6021-37. Epub 2016 Apr 29.

Department of Applied Physics and Division of Biology, California Institute of Technology , Pasadena, California 91125, United States.

The concept of allostery in which macromolecules switch between two different conformations is a central theme in biological processes ranging from gene regulation to cell signaling to enzymology. Allosteric enzymes pervade metabolic processes, yet a simple and unified treatment of the effects of allostery in enzymes has been lacking. In this work, we take a step toward this goal by modeling allosteric enzymes and their interaction with two key molecular players-allosteric regulators and competitive inhibitors. We then apply this model to characterize existing data on enzyme activity, comment on how enzyme parameters (such as substrate binding affinity) can be experimentally tuned, and make novel predictions on how to control phenomena such as substrate inhibition.
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http://dx.doi.org/10.1021/acs.jpcb.6b01911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452729PMC
July 2016

DNA Nanoparticles for Improved Protein Synthesis In Vitro.

Angew Chem Int Ed Engl 2016 Feb 28;55(9):3120-3. Epub 2016 Jan 28.

Institute of Biotechnology Vilnius University, 8 Graiciuno street, 02241, Vilnius, Lithuania.

The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4)  clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.
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http://dx.doi.org/10.1002/anie.201511809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787208PMC
February 2016

Biocompatible fluorinated polyglycerols for droplet microfluidics as an alternative to PEG-based copolymer surfactants.

Lab Chip 2016 Jan 2;16(1):65-9. Epub 2015 Dec 2.

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany.

In droplet-based microfluidics, non-ionic, high-molecular weight surfactants are required to stabilize droplet interfaces. One of the most common structures that imparts stability as well as biocompatibility to water-in-oil droplets is a triblock copolymer surfactant composed of perfluoropolyether (PFPE) and polyethylene glycol (PEG) blocks. However, the fast growing applications of microdroplets in biology would benefit from a larger choice of specialized surfactants. PEG as a hydrophilic moiety, however, is a very limited tool in surfactant modification as one can only vary the molecular weight and chain-end functionalization. In contrast, linear polyglycerol offers further side-chain functionalization to create custom-tailored, biocompatible droplet interfaces. Herein, we describe the synthesis and characterization of polyglycerol-based triblock surfactants with tailored side-chain composition, and exemplify their application in cell encapsulation and in vitro gene expression studies in droplet-based microfluidics.
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http://dx.doi.org/10.1039/c5lc00823aDOI Listing
January 2016

Microfluidic Production of Alginate Hydrogel Particles for Antibody Encapsulation and Release.

Macromol Biosci 2015 Dec 21;15(12):1641-6. Epub 2015 Jul 21.

Harvard University, School of Engineering and Applied, Cambridge MA 02138, USA.

Owing to their biocompatibility and reduced side effects, natural polymers represent an attractive choice for producing drug delivery systems. Despite few successful examples, however, the production of monodisperse biopolymer-based particles is often hindered by high viscosity of polymer fluids. In this work, we present a microfluidic approach for production of alginate-based particles carrying encapsulated antibodies. We use a triple-flow micro-device to induce hydrogel formation inside droplets before their collection off-chip. The fast mixing and gelation process produced alginate particles with a unique biconcave shape and dimensions of the mammalian cells. We show slow and fast dissolution of particles in different buffers and evaluate antibody release over time.
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http://dx.doi.org/10.1002/mabi.201500226DOI Listing
December 2015

Simple Microfluidic Approach to Fabricate Monodisperse Hollow Microparticles for Multidrug Delivery.

ACS Appl Mater Interfaces 2015 Jul 2;7(27):14822-32. Epub 2015 Jul 2.

‡Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki FI-00014, Finland.

Herein, we report the production of monodisperse hollow microparticles from three different polymers, namely, pH-responsive acetylated dextran and hypromellose acetate succinate and biodegradable poly(lactic-co-glycolic acid), at varying polymer concentrations using a poly(dimethylsiloxane)-based microfluidic device. Hollow microparticles formed during solvent diffusion into the continuous phase when the polymer close to the interface solidified, forming the shell. In the inner part of the particle, phase separation induced solvent droplet formation, which dissolved the shell, forming a hole and a hollow-core particle. Computational simulations showed that, despite the presence of convective recirculation around the droplet, the mass-transfer rate of the solvent dissolution from the droplet to the surrounding phase was dominated by diffusion. To illustrate the potential use of hollow microparticles, we simultaneously encapsulated two anticancer drugs and investigated their loading and release profiles. In addition, by utilizing different polymer shells and polymer concentrations, the release profiles of the model drugs could be tailored according to specific demands and applications. The high encapsulation efficiency, controlled drug release, unique hollow microparticle structure, small particle size (<7 μm), and flexibility of the polymer choice could make these microparticles advanced platforms for pulmonary drug delivery.
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http://dx.doi.org/10.1021/acsami.5b04824DOI Listing
July 2015

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

Cell 2015 May;161(5):1187-1201

Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2015.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441768PMC
May 2015

Platelet bioreactor-on-a-chip.

Blood 2014 Sep;124(12):1857-67

Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition,micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168343PMC
http://dx.doi.org/10.1182/blood-2014-05-574913DOI Listing
September 2014

Microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein.

Blood 2015 Jan 19;125(5):860-8. Epub 2014 Nov 19.

Division of Translational Medicine and.

Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged β1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia.
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http://dx.doi.org/10.1182/blood-2014-09-600858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311231PMC
January 2015