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Small cell size preserves the function of hematopoietic stem cells (HSCs); HSC enlargement during aging causes their dysfunction.

AbstractExisting preclinical 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, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1−2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-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.

AbstractExisting preclinical 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, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1−2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-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.

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

© 2020 National Academy of Sciences. All rights reserved. Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Sizedependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency., National Cancer Institute (Grants P30-CA14051 and CA217377)

Rotational dynamics often challenge physical intuition while enabling unique realizations, from the rotor of a gyroscope that maintains its orientation regardless of the outer gimbals, to a tennis racket that rotates around its handle when tossed face-up in the air. In the context of inertial sensing, which can measure mass with atomic precision, rotational dynamics are normally considered a complication hindering measurement interpretation. Here, we exploit the rotational dynamics of a microfluidic device to develop a modality in inertial sensing. Combining theory with experiments, we show that this modality measures the volume of a rigid particle while normally being insensitive to its density. Paradoxically, particle density only emerges when fluid viscosity becomes dominant over inertia. We explain this paradox via a viscosity-driven, hydrodynamic coupling between the fluid and the particle that activates the rotational inertia of the particle, converting it into a ‘viscous flywheel’. This modality now enables the simultaneous measurement of particle volume and mass in fluid, using a single, high-throughput measurement.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection1–3. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell 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 B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3−/− naive B cells 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−/− B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold 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−/− B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells 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 do

HSP90 is critical for the maintenance of cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we describe the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane-bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in the absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors.

© 2020, The Author(s). The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal.

© 2020 National Academy of Sciences. All rights reserved. Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Sizedependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2. Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

As the use of nanoparticles is expanding in many industrial sectors, pharmaceuticals, cosmetics among others, flow-through characterization techniques are often required for in-line metrology. Among the parameters of interest, the concentration and mass of nanoparticles can be informative for yield, aggregates formation or even compliance with regulation. The Suspended Nanochannel Resonator (SNR) can offer mass resolution down to the attogram scale precision in a flow-through format. However, since the readout has been based on the optical lever, operating more than a single resonator at a time has been challenging. Here we present a new architecture of SNR devices with piezoresistive sensors that allows simultaneous readout from multiple resonators. To enable this architecture, we push the limits of nanofabrication to create implanted piezoresistors of nanoscale thickness (∼100 nm) and implement an algorithm for designing SNRs with dimensions optimized for maintaining attogram scale precision. Using 8-in. processing technology, we fabricate parallel array SNR devices which contain ten resonators. While maintaining a precision similar to that of the optical lever, we demonstrate a throughput of 40 »000 particles per hour - an order of magnitude improvement over a single device with an analogous flow rate. Finally, we show the capability of the SNR array device for measuring polydisperse solutions of gold particles ranging from 20 to 80 nm in diameter. We envision that SNR array devices will open up new possibilities for nanoscale metrology by measuring not only synthetic but also biological nanoparticles such as exosomes and viruses.

Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.

The Navier slip condition describes the motion of a liquid relative to a neighboring solid surface, with its characteristic Navier slip length being a constitutive property of the solid-liquid interface. Measurement of this slip length is complicated by its small magnitude, expected to be in the nanometer range based on molecular simulations. Here, we report an experimental technique that interrogates the Navier slip length on individual nanoparticles immersed in liquid with subnanometer precision. Proof-of-principle experiments on individual, citrate-stabilized, gold nanoparticles in water give a constant slip length of 2.7 ± 0.6 nm (95% C.I.), independent of particle size. Achieving this feature of size independence is central to any measurement of this constitutive property, which is facilitated through the use of individual particles of varying radii. This demonstration motivates studies that can now validate the wealth of existing molecular simulation data on slip.

The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1- driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies., National Cancer Institute (Grant CA217377), Koch Institute (Grant P30-CA14051)

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis., National Institutes of Health (Grant 1R01-CA184956, Grant 5U24AI118672, Grant 1U54CA217377, Grant 1R33CA202820, Grant 2U19AI089992, Grant 1R01HL134539, Grant 2RM1HG006193 and Grant 2P01AI039671), National Institutes of Health (Award 1DP2GM119419), National Cancer Institute (Grant P30-CA14051)

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging. Optimal cell function requires maintenance of a narrow range of DNA:cytoplasm ratios and when cell size exceeds this ratio cytoplasmic dilution contributes to senescence, National Institutes of Health (Grant HD085866), National Institutes of Health (Grant 1U54CA217377)

The monitoring of mechanics in a single cell throughout the cell cycle has been hampered by the invasiveness of mechanical measurements. Here we quantify mechanical properties via acoustic scattering of waves from a cell inside a fluid-filled vibrating cantilever with a temporal resolution of < 1 min. Through simulations, experiments with hydrogels and the use of chemically perturbed cells, we show that our readout, the size-normalized acoustic scattering (SNACS), measures stiffness. To demonstrate the noninvasiveness of SNACS over successive cell cycles, we used measurements that resulted in deformations of < 15 nm. The cells maintained constant SNACS throughout interphase but showed dynamic changes during mitosis. Our work provides a basis for understanding how growing cells maintain mechanical integrity, and demonstrates that acoustic scattering can be used to noninvasively probe subtle and transient dynamics.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon (POLE) proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations., National Institute of Environmental Health Sciences (Core Center Grant P30-ES002109), National Institute of Allergy and Infectious Diseases (U.S.) (Grant R21AI110787)

Measuring the size of micron-scale particles plays a central role in the biological sciences and in a wide range of industrial processes. A variety of size parameters, such as particle diameter, volume, and mass, can be measured using electrical and optical techniques. Suspended microchannel resonators (SMRs) are microfluidic devices that directly measure particle mass by detecting a shift in resonance frequency as particles flow through a resonating microcantilever beam. While these devices offer high precision for sizing particles by mass, throughput is fundamentally limited by the small dimensions of the resonator and the limited bandwidth with which changes in resonance frequency can be tracked. Here, we introduce two complementary technical advancements that vastly increase the throughput of SMRs. First, we describe a deconvolution-based approach for extracting mass measurements from resonance frequency data, which allows an SMR to accurately measure a particle's mass approximately 16-fold faster than previously possible, increasing throughput from 120 particles/min to 2000 particles/min for our devices. Second, we describe the design and operation of new devices containing up to 16 SMRs connected fluidically in parallel and operated simultaneously on the same chip, increasing throughput to approximately 6800 particles/min without significantly degrading precision. Finally, we estimate that future systems designed to combine both of these techniques could increase throughput by nearly 200-fold compared to previously described SMR devices, with throughput potentially as high as 24 000 particles/min. We envision that increasing the throughput of SMRs will broaden the range of applications for which mass-based particle sizing can be employed. Keywords: Particle size analysis; Signal processing; Microfluidic devices; Phase lock loop; Cantilever; Resonator device; MEMS devices; Mass measurement, Cancer Systems Biology Consortium (Grant R33 CA191143), National Cancer Institute (Grant U54 CA217377), National Cancer Institute. Koch Institute Support (Grant P30-CA14051)