title: Analysis of immune cell migration in three dimensional environments
title: Analyse des Migrationsverhaltens von Immunzellen in dreidimensionalen Umgebungen
Creators: name: Czerwinski, Tina Linda
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2024
Subjects: subject: Biophysik
subject: Migration
subject: Immunzellen
subject: 3D
subject: DDC Classification::5 Naturwissenschaften::50 Naturwissenschaften::500 Naturwissenschaften und Mathematik
subject: DDC Classification::5 Naturwissenschaften::53 Physik::530 Physik

description: The immune system plays a key role in recognising and combating pathogens and tumour cells in the body. To achieve their function, immune cells constantly patrol a connective tissue of densely packed mesenchymal cells and extracellular matrix fibres with pores smaller than the cells, at high migration speeds of 5 - 10 μm/min. Immune cell migration has been extensively studied over the past decades, both in vitro using flat, two dimensional (2D) substrates and three dimensional (3D) matrices, and more recently in living animals using intra-vital imaging techniques. The 3D migratory ability of immune cells is commonly quantified by the migration speed and the directional persistence of cell trajectories. In this thesis, I demonstrate that the motile cell fraction is a vastly more sensitive indicator for the motility of immune cells in health, under pathological conditions and under different treatments, whereas migration speed and directional persistence remain surprisingly stable. The motile fraction indicates the fraction of cells in a population that are moving freely in a 3D matrix. Moreover, the remaining non-motile fraction is still viable and may only be temporarily unable to move. Hence, the motile fraction increases with measurement time. Taken together, this implies in a rather fundamental way that immune cells appear to either migrate at full speed, or they stop moving. In this thesis, I present new methods to accurately quantify the motile fraction of immune cell populations in different 3D matrices. Since the standard error of average values for the measured single cell parameters such as cell speed, persistence and motile fraction scales with the square root of the number of observed cells, an accurate estimate of the motile fraction requires live tracking of a large number of individual cells. Therefore, I developed a high-throughput 3D migration assay to track 1000 to 3000 individual cells per experiment, facilitating the detection of even very small changes in cell behaviour. With this tool in hand, I investigated and uncovered some of the fundamental laws that govern the ability of immune cells to infiltrate dense tissue. To mimic the process of immune cell migration through the connective tissue of different organs, I used biopolymer fibre networks as in vitro models, composed of collagen with tunable mechanical and structural properties, with pore sizes ranging from 3.5 μm to 5.7 μm and stiffness values (shear modulus) ranging from 100 Pa to 1200 Pa. To investigate the underlying cause of the sensitivity of the motile fraction in response to small changes in culture conditions or the microenvironment, I combined the 3D migration assay with high-speed 3D traction force microscopy (TFM). Current understanding suggests that immune cells infiltrate tissue using a so-called amoeboid migration mode, characterised by weak matrix adhesions and low traction forces. While my migration and traction force data confirm that immune cells (B cells, T cells, NK cells, neutrophils and monocytes) predominantly migrate in an amoeboid fashion, these data also demonstrate that the amoeboid migration mode is only used when the cells can move freely through matrix pores. However, when the cells encounter a narrow pore, they avoid getting stuck by switching to a highly contractile, mesenchymal-like mode of migration, which helps to pull the cell body through the narrow pore. Surprisingly, the magnitude of traction forces that immune cells are able to generate can reach values seen previously only for mesenchymal cells such as fibroblasts and cancer cells after endothelial-to-mesenchymal transition (EMT). Accordingly, the motile fraction appears to reflect the ability of an immune cell population to overcome the steric hindrance in a mechanically challenging environment. In support of this hypothesis, I demonstrate that NK92 cells, an NK cell line, as well as ex vivo expanded NK cells and primary NK cells, neutrophils, monocytes, T cells and B cells, all exert short bursts of contractile forces during 3D migration. These short contractile bursts occur predominantly after a cell slowed down on its way through the matrix and is in danger of getting stuck. After increasing their traction forces, cells tend to speed up. Decreasing cellular force generation by inhibiting actomyosin contraction decreases the motile fraction, while increasing cellular force generation by inhibiting microtubule polymerisation increases the motile fraction. I also show that the transmission of force to the extracellular matrix by immune cells is integrin-dependent, further highlighting the similarity of this mode of migration to mesenchymal cell migration. The motile fraction during 3D migration thus provides an indirect but surprisingly accurate and specific readout for the ability of immune cells to switch to a contractile mode of migration to overcome steric hindrance. With these novel insights and methods in hand, I next turned my attention to the development of improved expansion protocols that not only activate immune cells, but also enhance immune cell motility. Regulatory T cells are a prime target for ex vivo expansion because of the low number of these cells circulating in the peripheral blood. My data show that a clinical expansion protocol strongly increases the motility of expanded regulatory T cells by increasing the motile fraction, while cell speed and directional persistence remain nearly unchanged before versus after expansion. This finding strongly supports my earlier findings that a universal property of immune cells is that they are able to achieve the same cell speed and directional persistence under different conditions, but the number of cells that get stuck in the matrix varies significantly. Hence, the motile fraction is a clinically highly relevant and crucial parameter for optimising expansion protocols. Finally, I addressed the question of why current immune cell therapies fail in patients with solid tumours. I measured the motile fraction of NK cells derived from peripheral blood monocytes using a Good Manufacturing Practice (GMP) compliant NK cell expansion protocol. These protocols are used in a clinical context to generate a large, highly activated NK cell population from a patient’s blood sample, with the aim of reinjecting these expanded cells into the patient to enhance the immune response against blood-borne cancers or solid tumours. At the end of the expansion period, the cell population is cryopreserved to bridge the time required for biosafety testing. I found that after cryopreservation, the cytotoxicity (i.e. the killing ability of these cells) as well as the cell speed and directional persistence of motile NK cells is almost unchanged, while the motile fraction is decreased 6-fold. This finding may help explain the persistent failure of NK cell therapy in patients with solid tumours. In summary, the key finding of my thesis is that the default amoeboid migration mode of immune cells can be interrupted by a switch to a highly contractile, mesenchymal-like migration mode when the cell encounters a local microenvironment with a high degree of steric hindrance. This switch helps the cell to avoid getting stuck in the matrix. The ability to overcome the steric hindrance of the matrix can be readily estimated by the motile fraction in 3D migration assays. Together, these insights may be helpful for clinical applications including expansion protocols and novel treatments that target immune cell function.