Friday, September, 30th, 2022: 09:00 am
Chairs: Marta Urbanska and Oliver Otto
Integrating biophysical perspective into the description of cellular behaviors — typically studied from a biochemical angle — fosters comprehensive understanding of health and disease. In its broad understanding, mechanocytometry encompasses all methods that measure mechanical properties of the cells, such as stiffness or deformability. Classically, methods such as micropipette aspiration or atomic force microscopy-based indentation are used to characterize such properties. More recent developments aim at addressing challenges such as measuring of cell mechanical properties in situ in developing tissues (for example using Brillouin microscopy or intratissue bead sensors), or characterizing the mechanical properties of singles cells at high throughput in a flow cytometry-like manner (using a class of methods called deformability cytometry). The goal of measuring the mechanical properties of cells is to unravel their role in physiological and pathological processes such as tissue development and cancer metastasis, or to harness their potential as a diagnostic marker in various diseases. Our session will showcase new developments in the field of mechanocytometry, and present how cell mechanics can play a role in mammalian development and in breast cancer progression as ßstudied in 3D model systems with adaptable environment stiffness.
Mechanical control of mammalian ovarian folliculogenesis
Principal Investigator, Mechanobiology Institute, National University of Singapore, Singapore
Assistant Professor, Department of Biological Sciences, National University of Singapore, Singapore
The formation of ovarian follicles that house the functional eggs (oocytes) is a critical process in mammalian development as it ensures successful reproduction and species propagation. While past molecular genetics studies have revealed genes that are critical for regulating the bidirectional communication between the oocyte and the somatic cells, the underlying mechanisms driving follicular growth remain enigmatic. Recent work suggests that follicle growth is sensitive to surrounding matrix stiffness, which calls for a need to understand cell-cell interactions within the follicle. Here, we investigate the mechanical functions of theca cells (TCs), which are the outer cells encapsulating the follicles. Using both in vivo staining and in vitro reconstitution assay, we demonstrate that the TCs are highly contractile and generate significant compressive stress to modulate follicle growth. TCs are also mechanosensitive and can modulate their proliferation rate and YAP signalling in response to cell stretch and substrate stiffness, suggesting that follicle growth or basement membrane stiffness may regulate TC functions in vivo. Finally, we found that the follicles are relatively compressible, and perturbing mechanical stress can influence somatic cell proliferation, oocyte signalling and growth. Overall, our data suggest that the interplay between tissue pressure and cell mechanics may control robust follicle morphogenesis and oocyte maturation through mechanotransduction pathways. These findings will have profound implications for future understanding and treatment of ovarian diseases and infertility.
Chii Jou (Joe) Chan studied theoretical physics at the University of Cambridge (B.A., M.Phil.), and received his doctorate from Cambridge on biophysical studies of cells and nuclei under the supervison of Prof. Jochen Guck. Inspired by how physical forces shape early development of living organisms, he joined the group of Dr. Takashi Hiiragi at EMBL Heidelberg, where he made a major discovery in hydraulic regulation of mouse embryo size and cell fate specification. Since 2021, Chan was a Principal Investigator at the Mechanobiology Institute at the National University of Singapore, where his lab investigates the biophysics and mechanobiology underlying ovarian follicular development and tissue hydraulics in mammalian ovaries, and their relevance in ovarian ageing and diseases. Chan has been awarded the Singaporean Teaching and Academic Research Talent Inauguration Grant in 2021. His interdisciplinary productivity is reflected in the diversity of his collaborators (cell and developmental biologists, experimental biophysicists, theorists) across the world.
Studying the mechanical and morphological phenotype of Cancer-associated fibroblasts of the prostate
Biotechnology Center, Technische Universität Dresden, Germany
Reciprocal interactions between prostate epithelial cells and their adjacent stromal
microenvironment not only are essential for tissue homeostasis but also play a key role in tumor development and progression. Malignant transformation is associated with the formation of a reactive stroma where cancer-associated fibroblasts (CAFs) induce matrix remodeling and thereby provide atypical biochemical and biomechanical signals to epithelial cells. Previous work has been focused on the cellular and molecular phenotype as well as on matrix stiffness and remodeling, providing potential targets for cancer therapeutics. So far, biomechanical changes in CAFs and adjacent epithelial cells of the prostate have not been explored. Here, we compared the mechanical properties of primary prostatic CAFs and patient-matched non-malignant prostate tissue fibroblasts (NPFs) using atomic force microscopy (AFM) and real-time deformability cytometry (RT-FDC). We found that CAFs exhibit an increased apparent Young’s modulus, coinciding with an altered architecture of the cytoskeleton compared with NPFs. In contrast, co-cultures of benign prostate epithelial (BPH-1) cells with CAFs resulted in a decreased stiffness of the epithelial cells, as well as an elongated morphological phenotype, when compared with co-cultures with NPFs. Moreover, the presence of CAFs increased proliferation and invasion of epithelial cells, features typically associated with tumor progression. Altogether, this study provides novel insights into the mechanical interactions between epithelial cells with the malignant prostate microenvironment, which could potentially be explored for new diagnostic approaches.
Since 2020 I have been a group leader at the Biotechnology Center of the TU Dresden. I have a background in bioengineering with more than 10 years research experience in the biophysics field. Over the past years I could contribute to a better understanding of cell and tissue mechanics and changes associated with disease. My group, supported by the Mildred Scheel Early Career Center Dresden, currently focuses on mechanical tumor cell- microenvironment interactions. To study the role of cancer cell and microenvironment mechanics on cancer cell growth, invasion and metastasis, we employ different tools in the lab, e.g. atomic force microscopy (AFM), realtime deformability cytometry (RT-DC), and Brillouin microscopy. Besides mechanical characterization of patient tumour samples, bioengineered 3D in vitro models are developed to systematically study the influence of microenvironment stiffness on cancer cell behaviour. In particular, mechanisms by which cells sense
and adapt to their mechanical environment in 3D are explored and how this can be potentially used for new diagnostic approaches. I have (co-)authored more than 50 publications in peer-reviewed (h-index = 32) with over 3,493 citations.
Bob Fregin (Short Talk)
ZIK HIKE, Institute of Physics, University of Greifswald, Greifswald, Germany
Dynamic real-time deformability cytometry to decipher the response of bats to heterothermy
Dynamic real-time deformability cytometry (dRT-DC) is a label-free method to capture the full viscoelastic properties of suspended cells at a throughput of up to 100 per second. Cellular shape-changes along the entire length of the microfluidic channel are tracked in real-time and are subsequently analyzed by a Fourier decomposition. We demonstrate that this approach allows disentangling the cell response to the complex hydrodynamic environment at the inlet from the steady-state stress distribution inside the channel. A superposition of both effects is present in almost all microfluidic systems and potentially biases label-free cytometric measurements relying on steady-state flow conditions. We show that dRT-DC allows for cell mechanical assays at the millisecond time scale fully independent of cell shape.
Using dRT-DC we address for the first time the role of red blood cell viscoelasticity in temperature control in hibernating animals. We measured blood from the bat species Nyctalus Noctula over 3 years to understand how hibernating animals can maintain red blood cell function at low body temperatures during torpor. For that purpose, we analyzed the mechanical properties at three different temperature levels, ranging from approximately 10 °C to 37 °C. We also acquired data from a non-hibernating bat species, Rousettus Aegyptiacus, and humans to compare red blood cell mechanics among these 3 different species and corresponding regulatory effects. While between the bat species, only minor variations exist, we observe a clear difference in comparison to humans.
Benedikt Hartmann (Short Talk)
Max Planck Institute for the Science of Light & Max Planck Zentrum für Physik und Medizin, Erlangen, Germany
Linking mechanical properties of cells with their ability to circulate using real-time deformability cytometry
Blood cells are sequentially squeezed in many constrictions during their round trip through the body. Especially in the capillary bed in organs, such as the lung, their deformability determines how quickly they pass through these constrictions. Yet the deformability is not just a matter of a cell’s elasticity, but also their viscosity. Real-time deformability cytometry (RT-DC) is an established technique to quantify mechanical properties of cells and offers a way to measure viscoelastic properties of cells as they are exposed to shear stress in a microfluidic channel. From the steady state deformation at the end of the channel, the elasticity can be calculated, while from the time course of the deformation over the length of the channel one can estimate the viscosity. Using custom channel designs, including constrictions of various width and length, the influence of viscoelastic properties as well as other parameters, like cell size, can be linked with passage time through a constriction in order to provide inside into the question “What determines how good a cell can squeeze through a constriction?”.
Martin Kräter (Short Talk)
Max Planck Institute for the Science of Light and Max-Planck-Zentrum für Physik und Medizin, 91058 Erlangen, Germany
Clinical application of physical characterization of major blood cell types during COVID-19 and beyond
Research over the last decades revealed that single-cell physical properties, such as mechanical features, can serve as label-free markers of cell state and function. Thus, mechanical changes are a sign of alterations in the cell’s molecular composition. With the advent of microfluidic techniques that assess physical properties of single cells in high-throughput, the transition of research knowledge to clinical application became possible. Here we present medical applicability of real-time deformability cytometry (RT-DC) on the example of blood measurements from severely ill COVID-19 patients and compare them to recovered individuals and healthy controls. We found changes in the physical properties of all major blood cell types in these patients, indicative for an overall activated immune status as well as changes in the deformation of red blood cells, critical for circulation. Some of these changes also persisted months after the infection which led to the idea of a possible causal link of altered blood mechanics and the persistent symptoms in Long Covid. As an outlook we´ll highlight the steps that we are doing to transfer RT-DC from a technology in basic research towards a tool in daily clinical routine.