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Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted. How trisomy 21 contributes to Down syndrome phenotypes, including increased leukemia risk, is not well understood. Mowery et al. use per-cell normalization approaches to reveal global transcriptional amplification in Down syndrome models. HMGN1 overexpression is sufficient to induce these alterations and promotes lineage-associated transcriptional programs, signaling, and B cell progenitor phenotypes.

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecularbond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up." Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8+ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2Kb:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies., National Institutes of Health (U.S.) (U54 CA143874), National Institutes of Health (U.S.) (R21 AI110787)

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites., Sir Henry Wellcome Fellowship (103972/Z/14/Z), European Research Council (research grant ERC- 2012-StG 309255-EndoTox), National Institutes of Health (U.S.) (NIH Exploratory R21 grant 1R21AI123746), United States. Army Research Office. Institute for Collaborative Biotechnologies (grant W911NF-09-0001)

We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology., National Institutes of Health (U.S.) (Contract R21AI110787), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Science Foundation (U.S.). Graduate Research Fellowship, National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award F32CA1800586), Kinship Foundation. Searle Scholars Program, Beckman Young Investigator Program, National Institutes of Health (U.S.) (New Innovator Award DP2 OD020839)

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Institutes of Health (U.S.) (Contract R01GM085457), National Cancer Institute (U.S.) (Fellowship F31CA167872), National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334)

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines., Janssen Pharmaceutical Ltd., National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), MIT-Harvard Center of Cancer Nanotechnology Excellence (Grant 26697290-47281-A), Stand Up To Cancer, National Institutes of Health (U.S.). P41 Biotechnology Resource Center, National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051)

Osmotic regulation of intracellular water during mitosis is poorly understood because methods for monitoring relevant cellular physical properties with sufficient precision have been limited. Here we use a suspended microchannel resonator to monitor the volume and density of single cells in suspension with a precision of 1% and 0.03%, respectively. We find that for transformed murine lymphocytic leukemia and mouse pro–B cell lymphoid cell lines, mitotic cells reversibly increase their volume by more than 10% and decrease their density by 0.4% over a 20-min period. This response is correlated with the mitotic cell cycle but is not coupled to nuclear osmolytes released by nuclear envelope breakdown, chromatin condensation, or cytokinesis and does not result from endocytosis of the surrounding fluid. Inhibiting Na-H exchange eliminates the response. Although mitotic rounding of adherent cells is necessary for proper cell division, our observations that suspended cells undergo reversible swelling during mitosis suggest that regulation of intracellular water may be a more general component of mitosis than previously appreciated., National Cancer Institute (U.S.). Physical Sciences-Oncology Center (MIT, (U54CA143874)), National Cancer Institute (U.S.) (Koch Institute Support (core) grant P30-CA14051), Samsung Scholarship Foundation

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis., United States. Dept. of Defense. Congressionally Directed Medical Research Programs (Postdoctoral Award W81XWH-12-1-0466), Smith Family Foundation, Burroughs Wellcome Fund, Damon Runyon Cancer Research Foundation, Stern Family, American Association for Cancer Research, National Cancer Institute (U.S.) (NIH 5P30CA1405141), National Cancer Institute (U.S.) (R01CA168653)

Therapeutics that block kinases, transcriptional modifiers, immune checkpoints and other biological vulnerabilities are transforming cancer treatment. As a result, many patients achieve dramatic responses, including complete radiographical or pathological remission, yet retain minimal residual disease (MRD), which results in relapse. New functional approaches can characterize clonal heterogeneity and predict therapeutic sensitivity of MRD at a single-cell level. Preliminary evidence suggests that iterative detection, profiling and targeting of MRD would meaningfully improve outcomes and may even lead to cure., Koch Institute-Dana-Farber/Harvard Cancer Center Bridge Project, National Cancer Institute (U.S.) (R33 CA191143), National Cancer Institute (U.S.). Cancer Systems Biology Consortium (U54 CA217377)

Cells must duplicate their mass in order to proliferate. Glucose and glutamine are the major nutrients consumed by proliferating mammalian cells, but the extent to which these and other nutrients contribute to cell mass is unknown. We quantified the fraction of cell mass derived from different nutrients and found that the majority of carbon mass in cells is derived from other amino acids, which are consumed at much lower rates than glucose and glutamine. While glucose carbon has diverse fates, glutamine contributes most to protein, suggesting that glutamine's ability to replenish tricarboxylic acid cycle intermediates (anaplerosis) is primarily used for amino acid biosynthesis. These findings demonstrate that rates of nutrient consumption are indirectly associated with mass accumulation and suggest that high rates of glucose and glutamine consumption support rapid cell proliferation beyond providing carbon for biosynthesis., National Institutes of Health (U.S.) (Grant U54CA143874)

Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell's biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor's record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell's deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories., National Cancer Institute (U.S.) (Grant P30-CA14051)

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics. Keywords: Single-cell RNA-Seq, Mass, Growth, Serial suspended microchannel resonator, Multi-omics, Single cell, T cell activation, Glioblastoma, GBM, Drug response, Microfluidics, Biophysical properties

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes., United States. Army Research Office (Grant W911NF-09-D-0001), National Science Foundation (U.S.) (Grant 1129359), National Cancer Institute (U.S.) (Grant U54CA143874), National Cancer Institute (U.S.) (Grant P30-CA14051), National Cancer Institute (U.S.) (Grant R33-CA191143), National Institutes of Health (U.S.) (Grant T32-GM008334), National Institute of General Medical Sciences (U.S.) (Grant T32-GM008334)

Background: Growth rate is determined not only by extracellular cues such as nutrient availability but also by intracellular processes. Changes in cell morphology in budding yeast, mediated by polarization of the actin cytoskeleton, have been shown to reduce cell growth. Results: Here we demonstrate that polarization of the actin cytoskeleton inhibits the highly conserved Target of Rapamycin Complex 1 (TORC1) pathway. This downregulation is suppressed by inactivation of the TORC1 pathway regulatory Iml1 complex, which also regulates TORC1 during nitrogen starvation. We further demonstrate that attenuation of growth is important for cell recovery after conditions of prolonged polarized growth. Conclusions: Our results indicate that extended periods of polarized growth inhibit protein synthesis, mass accumulation, and the increase in cell size at least in part through inhibiting the TORC1 pathway. We speculate that this mechanism serves to coordinate the ability of cells to increase in size with their biosynthetic capacity., American Cancer Society (Postdoctoral Fellowship), National Cancer Institute (U.S.). Physical Sciences Oncology Center (Contract U54CA143874)

Simultaneously measuring multiple eigenmode frequencies of nanomechanical resonators can determine the position and mass of surface-adsorbed proteins, and could ultimately reveal the mass tomography of nanoscale analytes. However, existing measurement techniques are slow (<1 Hz bandwidth), limiting throughput and preventing use with resonators generating fast transient signals. Here we develop a general platform for independently and simultaneously oscillating multiple modes of mechanical resonators, enabling frequency measurements that can precisely track fast transient signals within a user-defined bandwidth that exceeds 500 Hz. We use this enhanced bandwidth to resolve signals from multiple nanoparticles flowing simultaneously through a suspended nanochannel resonator and show that four resonant modes are sufficient for determining their individual position and mass with an accuracy near 150 nm and 40 attograms throughout their 150-ms transit. We envision that our method can be readily extended to other systems to increase bandwidth, number of modes, or number of resonators., United States. Army Research Office (Grant W911NF-09-0001), Center for Integration of Medicine and Innovative Technology (Contract 09-440), National Science Foundation (U.S.) (Grant 1129359)

Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Cancer Institute (U.S.). Cell Decision Process Center (P50GM68762), National Institutes of Health (U.S.) (Contract R01GM085457)

Human red blood cells (RBCs) lose ~30% of their volume and ~20% of their hemoglobin (Hb) content during their ~100-day lifespan in the bloodstream. These observations are well-documented, but the mechanisms for these volume and hemoglobin loss events are not clear. RBCs shed hemoglobin-containing vesicles during their life in the circulation, and this process is thought to dominate the changes in the RBC physical characteristics occurring during maturation. We combine theory with single-cell measurements to investigate the impact of vesiculation on the reduction in volume, Hb mass, and membrane. We show that vesicle shedding alone is sufficient to explain membrane losses but not volume or Hb losses. We use dry mass measurements of human RBCs to validate the models and to propose that additional unknown mechanisms control volume and Hb reduction and are responsible for ~90% of the observed reduction. RBC population characteristics are used in the clinic to monitor and diagnose a wide range of conditions including malnutrition, inflammation, and cancer. Quantitative characterization of cellular maturation processes may help in the early detection of clinical conditions where maturation patterns are altered., United States. Army Research Office (Institute for Collaborative Biotechnologies Contract W911NF-09-D-0001), National Institutes of Health (U.S.) (Director's New Innovator Award), National Institutes of Health (U.S.) (Grant 1DP2DK098087)

Nanomechanical resonators can quantify individual particles down to a single atom; however the applications are limited due to their degraded performance in solution. Suspended micro- and nanochannel resonators can achieve vacuum level performances for samples in solution since the target analyte flows through an integrated channel within the resonator. Here we report on a new generation suspended nanochannel resonator (SNR) that operates at approximately 2 MHz with quality factors between 10,000-20,000. The SNR is measured to have a mass sensitivity of 8.2 mHz/attogram. With an optimized oscillator system, we show that the resonator can be oscillated with a mass equivalent frequency stability of 0.85 attogram (4 parts-perbillion) at 1 kHz bandwidth, which is 1.8 times the calculated stability imposed by the thermal noise. We demonstrate the use of this mass resolution by quantifying the mass and concentration of nanoparticles down to 10 nm in solution.

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell’s passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces., National Cancer Institute (U.S.) (Contract CCNE-T (Grant 26697290-47281-A)), National Cancer Institute (U.S.) (Physical Sciences Oncology Center U54CA143874), Stand Up To Cancer (SU2C/AACR)

We introduce a microfluidic system for simultaneously measuring single-cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 h of growth data from mouse lymphoblast and pro–B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis., National Cancer Institute (U.S.) (Physical Sciences Oncology Center, U54CA143874), National Cancer Institute (U.S.) (Physical Sciences Oncology Center, R21 CA137695), National Institute of General Medical Sciences (U.S.) ((NIGMS) EUREKA R01GM085457), Kwanjeong Educational Foundation (Korea) (Graduate Fellowship), National Science Foundation (U.S.) (Graduate Research Fellowship), European Commission. Community Research and Development Information Service (Marie Curie International Reintegration Grant PIRG-GA-2010-277062), Israeli Centers of Research Excellence (I-CORE) (Program, Center no. 41/11)