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Interferon-induced transmembrane protein3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1-3. Here we studied clinical cohorts of patients with Bcell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting Bcells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the Bcell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In Bcell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3-/- naive Bcells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3-/- Bcells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant Bcells. Mechanistic experiments revealed that IFITM3 functions as a PIP3scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3-/- Bcells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after Bcells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of Bcells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.

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)

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)

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)

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell’s biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean., National Science Foundation (U.S.) (OCE-1129359), Simons Foundation (337262), United States. Army Research Office (W911NF-09-D-0001)

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)

We report on a fabrication and packaging process for a microsystem consisting of a mass-based protein detector and a fully integrated preconcentrator. Preconcentration of protein is achieved by means of a nanofluidic concentrator (NC), which takes advantage of fast nonlinear electroosmotic flow near a nanochannel-microchannel junction to concentrate charged molecules inside a volume of fluid on the order of 1 pL. Detection of preconcentrated protein samples is accomplished by passing them through a suspended microchannel resonator (SMR), which is a hollow resonant cantilever serially connected to the NC on the same device. The transit of a preconcentrated sample produces a transient shift in the cantilever's resonance frequency that is proportional to the density of the sample and, hence, the concentration of protein contained in it. A device containing both NC and SMR structures was produced using a novel fabrication process which simultaneously satisfies the separate packaging requirements of the two structures. The initial testing of this prototype device has demonstrated that the integrated SMR can accurately measure the concentration of a bovine serum albumin solution, that was preconcentrated using the integrated NC. Future improvements in the fabrication process will allow site-specific surface modification of the device and compatibility with separation methods, which will create opportunities for its application to immunoassays and universal detection., National Cancer Institute (U.S.) (Contract R01CA119402), National Institutes of Health (U.S.) (NIH Cell Decision Process Center P50-GM68762)

SUMMARY 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., Graphical Abstract, In Brief 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 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)

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.

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter., National Institutes of Health (U.S.) (EUREKA Exceptional, Unconventional Research Enabling Knowledge Acceleration (R01GM085457)), National Institutes of Health (U.S.) (contract R21CA137695), National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874)

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the “baby machine” was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing “baby machine” is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic “baby machine” in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic “baby machine” by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase., National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874), National Institute of General Medical Sciences (U.S.) (EUREKA R01GM085457)