Biology subjects

Guck, J.

Publications and source records attributed to Guck, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Impact of chemotherapy on leukocyte stiffness -A longitudinal RT-DC study in a breast cancer patient

BackgroundThe physical properties of leukocytes, such as cell size and stiffness, are critical for their circulation in microcapillary networks where rapid shape changes are required to squeeze through vascular constrictions. Alterations in the cells physical phenotype can promote venous thromboembolism (VTE), a common cause of death in cancer patients receiving chemotherapy. While biochemical VTE predictors are well studied, physical properties of blood cells receive less attention. MethodsUsing real-time deformability cytometry (RT-DC), we monitored for the first time the physical phenotype of leukocytes in a longitudinal study of a breast cancer patient treated with epirubicin/cyclophosphamide (EC) and paclitaxel (Pax). ResultsThe leukocyte counts extracted from RT-DC were in good agreement with standard clinical leukograms and EC had no immediate effect on leukocyte properties. However, Pax caused a significant softening of granulo/monocytes and a stiffening of lymphocytes immediately after administration. Leukocyte size was constant throughout the therapy, but we observed an overall increase in leukocyte stiffness, which was restored to normal values 45 weeks post treatment. ConclusionTaken together, our data reveal chemotherapy-induced specific alterations of leukocyte stiffness potentially critical for microcirculation. Thus, RT-DC measurements can add important, yet currently not available information to VTE prediction in cancer patients.

biophysics↗

bioRxiv2026

Quantitative assessment of mesoscale cellular order and organization in the mouse hippocampus

The hippocampus is characterized by a stereotypical macroscopic structure, where the nuclei are densely and heterogeneously packed among different subregions of the hippocampus. Despite the fact that tissue-specific cellular organization has been implicated in neural function, it has been technically challenging to quantitatively analyze mesoscopic cellular organization in the hippocampus due to its high cellular density. To overcome this technical hurdle, we developed Computational Biophysical Histomorphometry Software (CBHS), an automated image-analysis pipeline, aimed at quantifying nuclear shape and the order of the cellular ensemble in high-density areas. When applied to the subfields of hippocampus, we found that denser regions, most notably the dentate gyrus, were the most positionally, but least orientationally ordered. Nuclear shape exhibited a dependence on the local environment in a packing-dependent manner. This association was cell-type specific, with neurons, but not astrocytes displaying nuclear shape that varied with neighbour proximity, although astrocytes demonstrated greater intrinsic shape variance. The results reveal the presence of reproducible mesoscale cell packing order in hippocampal tissue, and are consistent with a nucleus-driven mechanical coupling between neighbouring cells. The present study provides a quantitative framework with which to understand mesoscopic tissue organization, thus enabling the formulation of testable hypotheses for future investigation.

neuroscience↗

bioRxiv2025

Quantitative phase deformability cytometry (QP-DC) for precise and clinically relevant multiparametric immune cell profiling

Imaging flow cytometry enables the detailed analysis of cell morphology and internal structures through high-throughput cell imaging, and quantitative phase imaging (QPI)-based microfluidic approaches have extended this by providing label-free measures such as dry mass and refractive index (RI). Building on these developments, we present quantitative phase deformability cytometry (QP-DC), which integrates QPI with deformability cytometry to simultaneously measure morphology, mechanics, and intrinsic biophysical parameters such as mass density and dry mass. Numerical refocusing ensures in-focus images independent of axial position, improving precision in contour detection and feature extraction. Using microspheres and whole blood, we validated QP-DC and then applied it to neutrophils under lipopolysaccharide (LPS) stimulation and from patients with systemic lupus erythematosus (SLE). QP-DC revealed LPS-induced reductions in neutrophil mass density and identified heterogeneous subpopulations in SLE. These results demonstrate the capability of QP-DC for precise biophysical and mechanical characterization, offering significant potential for research and clinical diagnostics.

biophysics↗

bioRxiv2025

Adipose-mimetic granular hydrogels uncover biophysical cues driving breast cancer invasion

1.Breast cancer cells invade mammary adipose tissue during initial stages of metastasis but how the physical properties of adipose tissue regulate this process remains unclear. Here, we combined single cell mechanical characterization of primary adipocytes with microfluidic hydrogel fabrication, quantitative multiparametric imaging, Discrete Element Method (DEM) simulations, and in vivo experiments to elucidate these connections. First, we quantified the heterogeneous size and stiffness of primary adipocytes, and replicated these properties by fabricating adipocyte-sized polyacrylamide (PAAm) beads with tunable elasticity. Subsequently, we embedded these beads into type I collagen, the primary fibrillar extracellular matrix (ECM) component of breast adipose tissue, to form 3D granular hydrogels mimicking aspects of native adipose tissue architecture. Granular hydrogels embedded with beads demonstrated increased breast cancer cell invasion relative to bead-free controls, an effect that was more pronounced with soft versus stiff beads and correlated with increased collagen fiber alignment and hierarchical organization. In addition, live cell imaging and DEM simulations revealed that soft beads promoted invasion relative to stiff beads by deforming in response to confined cancer cell migration. Fiber alignment and adipocyte deformation trends were validated in vivo via intravital imaging of cancer cell migration in mammary fat pads of mice, and suggest that adipocyte mechanics regulate breast cancer invasion by coordinating both ECM architecture and cellular confinement. Ultimately, this work highlights the utility of tunable PAAm bead-collagen composites as micromechanical models to study the effect of adipose tissue structure on cancer cell invasion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/684224v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@d3ce0org.highwire.dtl.DTLVardef@117abb1org.highwire.dtl.DTLVardef@10917faorg.highwire.dtl.DTLVardef@55f3e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Polyacrylamide bead granular hydrogels recapitulate adipose tissue structure and alter cancer cell invasion in a stiffness-dependent manner. The invasion of primary breast cancer cells through mammary adipose is influenced by the heterogeneous mechanical properties of native stromal components, namely adipocytes and extracellular matrix (left). Customizable granular hydrogels provide insight into the physical regulation of breast cancer progression by mimicking adipose tissue extracellular matrix structure (collagen I, i.) and stromal cell mechanics (polyacrylamide bead, ii.), whereby the inclusion of soft beads and/or dilute matrix promotes cancer cell migration compared to stiffer hydrogels (right). C_FIG

bioengineering↗

bioRxiv2025

Self-organization of tumor heterogeneity and plasticity

Phenotypic heterogeneity and plasticity drive tumor growth, metastasis, therapy resistance, and relapse. This heterogeneity is mainly interpreted as a response to external signals from the microenvironment. However, here we show that cancer cells also follow intrinsic self-organized programs that are sufficient to coordinate the spatiotemporal patterning of tumor cell states. By combining quantitative measurements in tumors and organoids with theoretical modeling, we reveal emergent mechanical gradients that orchestrate cell state transitions during colorectal tumor growth. Compression at the tumor center induces a transition from a fetal-like state into a cancer stem cell (CSC) state. The CSC compartment exhibits a characteristic size determined by tumor rheological properties. Once this size is surpassed, a translationally arrested apoptotic core emerges, triggering a shift from homogeneous proliferation to a hierarchical cell turnover. These findings uncover stereotyped programs of self-organization that likely cooperate with the microenvironment to shape tumor heterogeneity and plasticity.

cancer biology↗

bioRxiv2025

Direct, high-throughput linking of single cell imaging and gene expression

Understanding sequence-phenotype relationships in biology requires large-scale datasets that provide one-to-one mapping of sequence to phenotype. Here, we address this challenge by introducing multimodal droplet barcoding, enabling individual microfluidic droplets to be tracked across both optical and sequencing measurements. We demonstrate the power of this approach through multimodal single-cell analysis. We pair imaging with gene expression profiling of individual cells, identifying genes that correlate with phenotypic features.

genomics↗

bioRxiv2025

Fine-tuning cell-mimicking polyacrylamide microgels: Sensitivity to microscale reaction conditions in droplet microfluidics

The ability to shape polyacrylamide (PAAm) hydrogels using droplet microfluidics enables the production of microgel particles that mimic the cellular physical properties, opening new avenues in mechanobiology. Precisely controlling microgel size and elasticity is crucial yet complex, as various factors influence polymerization and the resulting network structure. While it is well established that chemical reactions in microdroplets are typically faster and more homogeneous than in bulk systems, an often-overlooked aspect is the increased sensitivity of these microreactors: minor variations in chemical or physical parameters can lead to significant changes in the resulting microgel properties. Our study identifies flow conditions as a key factor influencing both microgel elasticity and size, by modulating interfacial transport during gelation. Using a flow-focusing microfluidic chip, we generated pre-gel droplets with identical composition dispersed in an oil phase, systematically varying the PAAm-to-oil flow rate ratio while keeping the total flow rate constant. This approach yields droplets with minimal diameter variation (<1 {micro}m), yet produces beads with distinct Youngs moduli, despite identical total monomer concentrations. Further analysis revealed that the catalyst transport across the oil-water interface significantly affects the polymerization efficiency and polymer network. Our findings highlight that despite the advantages of droplet-based polymerization, achieving reproducible microgel properties requires careful control of flow parameters. This underscores the importance of precise microfluidic control in advancing PAAm microgel applications in biophysics.

biophysics↗

bioRxiv2025

Hierarchical Heterogeneities in Spatio-Temporal Dynamics of the Cytoplasm

The physical properties of the cytoplasm fundamentally constrain the dynamics and fidelity of cellular processes. Endogenous fluctuations carry signatures of these properties, yet how their collective dynamics across mesoscopic spatiotemporal scales relate to cytoplasmic heterogeneity and organization remains poorly understood. Here, we use differential dynamic microscopy (DDM) to quantify these fluctuations in eukaryotic cytoplasms. In Xenopus laevis egg extracts, we identify signatures of subdiffusive fractional Brownian motion (fBm) and non-Gaussian displacement distributions, consistent with a crowded, heterogeneous environment. We introduce an empirical model combining fBm with an inverse Gaussian diffusivity distribution, enabling robust estimation of fractional diffusivities and scaling exponents across conditions. Denser cytoplasm exhibits lower fractional diffusivity and scaling exponent, while energy addition induces non-stationary dynamics. Stabilization of microtubule networks introduces a secondary timescale, rationalized by a two-state fBm model separating cytosolic and network-associated contributions. In HeLa cells, the framework reveals comparable scaling exponents but lower diffusivities than in egg extract. Upon microtubule depolymerization, the dynamics become slower yet more Brownian-like. This highlights dual roles of the cytoskeleton as confinement but also as fluidizer of the cytoplasm. These results establish label-free DDM as an interpretable, collective, multiscale readout of how composition, activity, and cytoskeletal organization shape hierarchical cytoplasmic dynamics.

biophysics↗

bioRxiv2025

Viscoelastic characterization of cells in microfluidic channels with 3D hydrodynamic focusing

The viscoelastic nature of biological cells has emerged as an increasingly important research subject due to its relevance for cellular functions under physiological and pathological conditions. Advancements in microfluidics have made this technology a promising tool to study the viscoelasticity of cells. However, significant challenges remain, including the complex distribution of stresses acting on cells depending on the channel geometry, and the difficulty of keeping cells in the focal plane for imaging. Here, we report a new approach using hyperbolic channels for measuring cell viscoelasticity. A channel height much larger than the typical cell size minimized shear stresses so that normal stresses in the hyperbolic region dominated the stress distribution. Reducing the complexity of the stress-strain relationship allowed us to use polyacrylamide microgel beads to calibrate the stress curve. Additionally, we introduced 3D hydrodynamic focusing which enabled us to focus cells and microgel beads in the center of the channel. Finally, Kelvin-Voigt and power-law rheology models were employed to extract the mechanical properties of microgel beads and human leukemia HL60 cells. The measurement technique described here will help establish the viscoelastic properties of cells as an important readout in biophysical research in health and disease.

biophysics↗

bioRxiv2025

High-Throughput Mechanomic Screening Reveals Novel Regulators of Single-Cell Mechanics

The mechanical properties of cells are dynamic, allowing them to adjust to different needs in different biological contexts. In recent years, advanced biophysical techniques have enabled the rapid, high-throughput assessment of single-cell mechanics, providing new insights into the regulation of the mechanical cell phenotype. However, the molecular mechanisms by which cells maintain and regulate their mechanical properties remain poorly understood. Here, we present a genome-scale RNA interference (RNAi) screen investigating the roles of kinase and phosphatase genes in regulating single-cell mechanics using Real-Time Fluorescence and Deformability Cytometry (RT-FDC). Our screen identified 80 known and novel mechanical regulators across diverse cellular functions from 214 targeted genes, leveraging RT-FDCs unique capabilities for comprehensive, high-throughput mechanical phenotyping with single-cell and cell cycle resolution. These findings refine our understanding of how signaling pathways coordinate structural determinants of cell mechanical phenotypes and provide a starting point for uncovering new molecular targets involved in biomechanical regulation across diverse biological systems. SIGNIFICANCECell mechanical properties are tightly regulated and play pivotal roles in processes ranging from tissue morphogenesis to disease progression. Despite their importance, the genetic regulation of single-cell mechanics remains largely unexplored. This study represents one of only a few large-scale mechanomic investigations conducted to date. It is the first study to leverage RT-FDCs unique capability for high-throughput mechanical phenotyping with single-cell and cell cycle resolution to detect gene impacts that may be overlooked in lower-throughput or population-level studies. The mechanical genes identified here provide valuable data points for understanding how cells control their mechanical state and serve as a foundation for future studies exploring the molecular basis of biomechanical regulation.

biophysics↗

bioRxiv2025

Biphasic inflammation control by dedifferentiated fibroblasts enables axon regeneration after spinal cord injury in zebrafish

Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS is not understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and subsequently resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a+ fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a+ fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics inhibits axon regeneration and alters the mechano-structural properties of the lesion environment. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration. ONE SENTENCE SUMMARYDedifferentiated fibroblasts sequentially induce and resolve neutrophil-driven inflammation through cytokine release to facilitate axon regeneration after spinal cord injury in zebrafish. HIGHLIGHTSO_LITime-resolved single-cell transcriptomics of zebrafish spinal cord regeneration. C_LIO_LISpinal cord injury induces fibroblast dedifferentiation. C_LIO_LIDedifferentiated fibroblasts sequentially induce and resolve inflammation. C_LIO_LIDysregulation of inflammation dynamics alters mechano-structural tissue properties. C_LI

neuroscience↗

bioRxiv2024

Cell viscosity influences hematogenous dissemination and metastatic extravasation of tumor cells

Metastases arise from a multi-step process during which tumor cells change their mechanics in response to microenvironmental cues. While such mechanical adaptability could influence metastatic success, how tumor cell mechanics directly impacts intravascular behavior of circulating tumor cells (CTCs) remains poorly understood. In the present study, we demonstrate how the deformability of CTCs affects hematogenous dissemination and identify the mechanical profiles that favor metastatic extravasation. Combining intravital microscopy with CTC-mimicking elastic beads and mechanically-tuned tumor cells, we demonstrate that the inherent properties of circulating objects dictate their ability to enter constraining vessels. We identify cellular viscosity as the key property that governs CTC circulation and arrest patterns. We further demonstrate that cellular viscosity is required for efficient extravasation and find that properties that favor extravasation and subsequent metastatic outgrowth can be opposite. Altogether, we identify CTC viscosity as a key biomechanical parameter that shapes several steps of metastasis.

cancer biology↗

bioRxiv2024

Optical quantification of molecular interaction strength in protein condensates

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

biophysics↗

bioRxiv2024

Beyond comparison: Brillouin microscopy and AFM-based indentation reveal divergent insights into the mechanical profile of the murine retina

Mechanical tissue properties increasingly serve as pivotal phenotypic characteristics that are subject to change during development or pathological progression. The quantification of such material properties often relies on physical contact between a load-applying probe and an exposed sample surface. For most tissues, these requirements necessitate animal sacrifice, tissue dissection and sectioning. These invasive procedures bear the risk of yielding mechanical properties that do not portray the physiological mechanical state of a tissue within a functioning organism. Brillouin microscopy has emerged as a non-invasive, optical technique that allows to assess mechanical cell and tissue properties with high spatio-temporal resolution. In optically transparent specimens, this technique does not require animal sacrifice, tissue dissection or sectioning. However, the extent to which results obtained from Brillouin microscopy allow to infer conclusions about potential results obtained with a contact-based technique, and vice versa, is unclear. Potential sources for discrepancies include the varying characteristic temporal and spatial scales, the directionality of measurement, environmental factors, and mechanical moduli probed. In this work, we addressed those aspects by quantifying the mechanical properties of acutely dissected murine retinal tissues using Brillouin microscopy and atomic force microscopy (AFM)-based indentation measurements. Our results show a distinct mechanical profile of the retinal layers with respect to the Brillouin frequency shift, the Brillouin linewidth and the apparent Youngs modulus. Contrary to previous reports, our findings do not support a simple correlative relationship between Brillouin frequency shift and apparent Youngs modulus. Additionally, the divergent sensitivity of Brillouin microscopy and AFM-indentation measurements to cross-linking or changes post mortem underscores the dangers of assuming both methods can be generally used interchangeably. In conclusion, our study advocates for viewing Brillouin microscopy and AFM-based indentation measurements as complementary tools, discouraging direct comparisons a priori and suggesting their combined use for a more comprehensive understanding of tissue mechanical properties.

biophysics↗

bioRxiv2023

High-throughput viscoelastic characterization of cells in hyperbolic microchannels

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress-strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress-strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, has a constant extensional viscosity of 0.5 Pa s up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation time of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid-liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Youngs moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with Latrunculin B and Nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.

biophysics↗

bioRxiv2023

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Colorectal cancer tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is unknown. Using CRC patient derived organoids (PDOs), we found that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing scRNA-seq expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

cancer biology↗

bioRxiv2023

Estimation of the mass density of biological matter from refractive index measurements

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms by utilizing models that explicitly depend on physical observables. One of the basic biophysical properties is the mass density (MD), which determines the degree of crowdedness. It impacts the dynamics in sub-cellular compartments and further plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge as it requires detailed knowledge of the biochemical composition of the sample. Here we propose a methodology on how to account for a priori and a posteriori assumptions about the biochemical composition of the sample as well as respective RI measurements. To that aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations as well as Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well characterized complex mixtures given by bovine milk and intralipid emulsion. Further, we employ it to estimate the mass density of trunk tissue of living zebrafish (Danio rerio) larvae. Our results enable quantifying changes of mass density estimates based on variations in the a priori assumptions. This illustrates the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

biophysics↗

bioRxiv2023

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes

The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

biophysics↗