Biology subjects

Girardo, S.

Publications and source records attributed to Girardo, S..

13 recordsLinked to original sources

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

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↗

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

Endothelial calcium firing mediates extravasation of metastatic tumor cells.

Metastatic dissemination is driven by genetical, biochemical and biophysical cues that favor the distant colonization of organs and the formation of life-threatening secondary tumors. We have previously demonstrated that endothelial cells (ECs) actively remodel during extravasation by enwrapping arrested tumor cells (TCs) and extrude them from the vascular lumen while maintaining perfusion. In this work, we dissect the cellular and molecular mechanisms driving endothelial remodeling. Using high-resolution intravital imaging in zebrafish embryos, we demonstrate that the actomyosin network of ECs controls tissue remodeling and subsequent TC extravasation. Furthermore, we uncovered that this cytoskeletal remodeling is driven by altered endothelial- calcium (Ca2+) signaling caused by arrested TCs. Accordingly, we demonstrated that inhibition of voltage-dependent calcium L-type channels impairs extravasation. Lastly, we identified P2X4, TRP, and Piezo1 mechano-gated Ca2+ channels as key mediators of the process. These results further highlight the central role of endothelial remodeling during extravasation of TCs and open avenues for successful therapeutic targeting. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/587188v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@16fdc30org.highwire.dtl.DTLVardef@e0ff22org.highwire.dtl.DTLVardef@18e2311org.highwire.dtl.DTLVardef@a06320_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

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↗

bioRxiv2022

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 {micro}m wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe1 that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

biophysics↗

bioRxiv2020

Combined fluorescence, optical diffraction tomography and Brillouin microscopy

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

biophysics↗

bioRxiv2019

Using real-time fluorescence and deformability cytometry and deep learning to transfer molecular specificity to label-free sorting

The identification and separation of specific cells from heterogeneous populations is an essential prerequisite for further analysis or use. Conventional passive and active separation approaches rely on fluorescent or magnetic tags introduced to the cells of interest through molecular markers. Such labeling is time- and cost-intensive, can alter cellular properties, and might be incompatible with subsequent use, for example, in transplantation. Alternative label-free approaches utilizing morphological or mechanical features are attractive, but lack molecular specificity. Here we combine image-based real-time fluorescence and deformability cytometry (RT-FDC) with downstream cell sorting using standing surface acoustic waves (SSAW). We demonstrate basic sorting capabilities of the device by separating cell mimics and blood cell types based on fluorescence as well as deformability and other image parameters. The identification of blood sub-populations is enhanced by flow alignment and deformation of cells in the microfluidic channel constriction. In addition, the classification of blood cells using established fluorescence-based markers provides hundreds of thousands of labeled cell images used to train a deep neural network. The trained algorithm, with latency optimized to below 1 ms, is then used to identify and sort unlabeled blood cells at rates of 100 cells/sec. This approach transfers molecular specificity into label-free sorting and opens up new possibilities for basic biological research and clinical therapeutic applications.

biophysics↗

bioRxiv2019

3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.

bioengineering↗

bioRxiv2018

Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

Mechanical stress exerted and experienced by cells during tissue morphogenesis and organ formation plays an important role in embryonic development. While techniques to quantify mechanical stresses in vitro are available, few methods exist for studying stresses in living organisms. Here, we describe and characterize cell-like polyacrylamide (PAAm) bead sensors with well-defined elastic properties and size for in vivo quantification of cell-scale stresses. The beads were injected into developing zebrafish embryos and their deformations were computationally analyzed to delineate spatio-temporal local acting stresses. With this computational analysis-based cell-scale stress sensing (COMPAX) we are able to detect pulsatile pressure propagation in the developing neural rod potentially originating from polarized midline cell divisions and continuous tissue flow. COMPAX is expected to provide novel spatiotemporal insight into developmental processes at the local tissue level and to facilitate quantitative investigation and a better understanding of morphogenetic processes.

biophysics↗

bioRxiv2018

Standardized microgel beads as elastic cell mechanical probes

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acylamide (PAAm) microgel beads mimicking biological cells. We produced mono-disperse beads at rates of 20 - 60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.\n\nSignificance StatementOften vastly different cell mechanical properties are reported even for the same cell type when employing different measurement techniques. This discrepancy shows the urgent need for standardized calibration particles to cross-compare and validate techniques. Microgel beads can serve this purpose, but they have to fulfil specific requirements such as homogeneity, sizes and elasticities in the range of the cells, and they have to provide comparable results independent of the method applied. Here we demonstrate the standardized production of polyacrylamide microgel beads with all the features an elastic cell-mimic should have. These can not only be used as method calibration particles, but can also serve as cell-scale sensors to quantify normal and shear stresses exerted by other cells and inside tissues, enabling many new applications.

biophysics↗

bioRxiv2017

Real-time fluorescence and deformability cytometry - flow cytometry goes mechanics

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument -- opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.

biophysics↗