Mechanobiology

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

Abstract

The NIBIB’s Section on Mechanobiology focuses on developing advanced atomic force microscopy (AFM) techniques to assess the micro- and nano-scale mechanical properties of cells and tissues. AFM involves probing samples using a micron-sized cantilever, with or without an indenter, to non-invasively measure material properties in complex biological systems —from molecules to tissues— under physiologically relevant conditions. A key advantage of AFM is that it avoids chemical manipulation, making it the primary biophysical approach in our lab. This is the main reason why AFM is the main biophysical approach used in our lab. Project #1: Pancreatic cancer cells and solid tumor mechanics: role of cellular actomyosin cytoskeleton, glycocalyx, and microenvironment architectures promoting mechanical heterogeneity, anisotropy, and regulation. Pancreatic cancer remains one of the most lethal malignancies due in part to issues with early detection and high levels of drug resistance. With a 5-year survival rate of about 10%, pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Partly to blame for these issues is the glycocalyx, an extracellular structure found on most cells that is aberrantly glycosylated and has overexpressed biopolymers in cancerous cells. In pancreatic cancer, this includes higher levels of mucin expression, increased sialylation, and more hyaluronic acid production. Of note regarding sialic acid, numerous studies suggest that aberrant sialylation can affect various pathways in cancer, including immune attenuation, enhanced metastasis, and altered apoptosis. Although there is considerable work detailing the biochemical role of these glycocalyx modifications, we sought to understand their role in the architecture and mechanical properties of the cell. The mechanical profiling of cells is an emerging field, yet the role of glycocalyx from a mechanical standpoint remains less understood. Thus, understanding the ways in which the solid TME and cancer cell cytoskeletal and glycocalyx aberrations are related to changes in mechanical properties at timescales of biological relevance may reveal new therapeutic strategies to prevent or at least slow down cancer progression and metastasis. (Aim1) Actomyosin and glycocalyx remodeling differentially regulates viscoelastic behavior of pancreatic cancer cells. We hypothesized that a systematic analysis of the actomyosin and glycocalyx structures and mechanics of pancreatic cancer cells at different malignancy stages would provide insight into the role of the glycocalyx in metastasis and in serving as a physical barrier to chemo- and immuno-therapies. Postdoctoral fellow Dr. Andrew Massey performed a wide survey of the mechanical properties (actomyosin cortical tension, Young's modulus, Storage Modulus and Loss Modulus, hydrostatic intracellular pressure) of pancreatic cancer cell lines at different stages of malignancy. To elucidate these properties, we enzymatically degraded different components of the glycocalyx commonly found to be aberrantly expressed in pancreatic cancer (e.g., N-glycans, sialic acids, mucins, and hyaluronic acid) and visualized changes in the structure of the membrane via atomic force microscopy (AFM), confocal fluorescence microscopy, and scanning electron microscopy. First, we observed a profound reduction in microvilli density and thickness that was the most consistent with sialic acid removal across all three cell lines investigated. Using AFM-based nanomechanical mapping, we investigated changes in cell surface mechanics and observed a significant reduction in the viscoelastic properties (elastic storage and viscous loss moduli) when removing sialic acid. This observation suggests that the cell surface softens and fluidizes in response to desialylation. Similarly, removal of mucins also led to reduced microvilli density, softening, and fluidization in pancreatic cells - to a degree that was matched by removal of sialic acids. We are currently performing glycomics studies, thus far preliminary results revealed unique changes in the structure of N- and O-glycans, with significantly more heterogeneity in the structure of N-glycans on pancreatic cancer cells, and O-glycans showing a particularly higher degree of sialic acid deposition. Future studies will attempt to translate in vitro observations of de-glycosylation with patient tissue viscoelastic data to highlight the role of glycocalyx modulation at an intratumoral level to better understand chemo and immune therapy resistance, with the goal of determining the therapeutic potential of aberrant sialylation as a target for novel therapies. (Aim 2) Upregulated expression of bulky glycoproteins modulate integrin-based adhesions in pancreatic cancer cells. Compared to other carcinomas, PDAC is highly desmoplastic, therefore leading us to hypothesize upregulated expression of bulky glycoprotein leads to aberrant integrin adhesion complexes (IACs) and altered integrin-based signaling, contributing to PDAC’s aggressive nature. To investigate this, Postdoctoral fellow Dr. Elijah Marquez enzymatically degraded several aberrantly expressed glycocalyx components in PDAC: mucins, sialic acids, hyaluronic acid, and N-glycans. Then we used confocal microscopy to visualize differences in IAC structure across various representative pancreatic cancer cell lines. We observed cell line-dependent alterations in IAC distribution, size, and length. Our results suggest that IAC distribution and size change as PDAC progresses and encounters different physiological cues, thus possibly impacting integrin-based signaling. Following glycocalyx-targeting treatments, cleaving mucins appears to significantly decrease individual IAC size and total IAC area. Cleaving hyaluronic acid, sialic acids, and N-glycans did not significantly affect individual IAC size or total IAC area. These results suggest that a more complex glycocalyx-IAC regulatory axis exists, rather than solely a kinetic trap imposed by the glycocalyx’s thickness and density. Future studies will proceed in two main directions. First, we will investigate how PDAC stages and glycocalyx treatments affect cellular mechanics such as cell traction forces and cell-ECM (extracellular matrix) adhesion strength. Second, we will elucidate the role of intratumoral mechanics and ECM on controlling IACs throughout PDAC malignancy progression. Project #2: Understanding T lymphocytes mechanobiology during the formation of the immunological synapse. The immunological synapse (IS), which is the specialized cell-cell junction between a T lymphocyte and an antigen-presenting cell, plays a crucial role in T cell activation. This process begins when the T-cell receptor (TCR) binds to its specific antigenic peptide. Several studies have highlighted that the mechanical properties of T cells during activation significantly impact various cellular functions, including proliferation, migration, and cytotoxic activity. The IS formed between T lymphocytes and their targets is a dynamic cellular structure capable of exerting mechanical force. The formation and maintenance of the IS are facilitated by force generation through the dynamic interaction of the actomyosin and microtubule cytoskeletal networks. However, the modulation of mechanical properties by T lymphocytes within the immune synapse remains poorly understood. Therefore, we hypothesize that T lymphocytes modify their viscoelastic properties to enhance immunological synapse formation, maintenance, force generation, and killing capacity. To investigate this, Postdoctoral fellow Dr. Mazen Mezher utilized high spatiotemporal resolution AFM to measure the viscoelastic response of T cells through common mechanical parameters (storage and loss moduli) across different timescales at the nanometer scale. Additionally, Traction Force Microscopy was used to quantify the traction stresses generated by T cells on soft silicone hydrogels during IS formation. Our findings revealed that T cells exhibit structurally diverse viscoelastic properties at the nanoscale during IS formation, triggered by CD3/CD28/LFA-1 co-stimulation. Specifically, we observed significantly higher elastic and viscous properties at the edge and center of the IS, while the peripheral transition region appeared softer and more fluid. These observations correspond to changes in the actomyosin cytoskeleton structure in these areas. Furthermore, our results demonstrated that perturbations in cytoskeletal proteins regulating filamentous actin caused substantial changes in T cell elasticity and fluidity, as well as alterations in the traction stresses generated during IS formation. Notably, we observed significant softening, fluidization, and a decrease in traction stresses when detaching the actin cortex from the plasma membrane by inhibiting Ezrin activity. In summary, understanding the relationship between key cytoskeletal structures at the IS and the mechanical property of the cell is essential for maintaining and forming the IS, offering valuable insights into potential strategies to engineer T cells with enhanced activation and cytotoxic abilities. We are also currently exploring the role of key filamentous actin cytoskeletal regulatory proteins known to play an important role in immune cell activation and in the formation and maintenance of the immune synapse, including non-muscle myosin II, ERM proteins, formin, and septins.