Molecular and Cellular Pathophysiology 20211 Migration and invasiveness in cancer, cell movement, Epithelial-mesenchymal transition Jaromír Gumulec j.gumulec@med.muni.cz @jarogumulec Define footer – presentation title / department2 Cytoskeleton ̶ protein fibers that are involved in ̶ cell shape and cell mechanic properties (no cell wall in animal cell) ̶ providing mechanical strength ̶ cell movement ̶ chromosome separation ̶ intracellular transport of organelles ̶ enable cell communication ̶ cytoskeletal fibers + motor proteins ̶ dynamic instability, ̶ self-assembly Define footer – presentation title / department3 Define footer – presentation title / department4 Define footer – presentation title / department5 Cytoskeleton in eukaryotic cells 6 Microfilaments Intermediary filaments Microtubules build of G-actin/F-actin various a-tubulin/ b-tubulin diameter 7 nm 10-12 nm 25 nm molecular motors myosins none kinesin / dynein polymeration fuel ATP none GTP function structure stabilisation, muscle contraction, cytokinesis, cell movement mechanical stability, cell-specific intracel. transport, mitotic spindle Define footer – presentation title / department7 8 end Actin filament formation end F-Actin networking G-actin polymer depolymeration polymeration Imaging the time course of the polymerization of ATP-actin. Length scale bar (L) is 10 μm and timescale bar (T) is 1000 s. (C) Lengths of 13 filaments as a function of time https://www.sciencedirect.com/science/article/pii/S000 6349505732057 Formation and depolymeration of actin filaments. Like microtubules, actin filaments are dynamically instable 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z- bunecne-fyziologie/ Define footer – presentation title / department9 Actin cytoskeleton ̶ higher order structures – connecting proteins (eg plastin 3) ̶ polarized, grow on + end (by ATP hydrolysis) ̶ associated with other protein complexes ̶ specialized functions in various parts of cells Define footer – presentation title / department10 Myosin motor protein families ̶ The head is both actin-binding and ATP binding; the purple light chain has a regulatory role. Myosin II is muscle myosin. ̶ 18 different myosin families have been identified (I –XVIII) Myosin head energized, with bound ADP and Pi,, not attached to actin Myosin head attaches to actin Head rotates - Powerstroke transmits force to thin filament – head is deenergized ATP replaces ADP and Pi on myosin head Head detaches from thin filament ATP hydrolyzed HEAD DETACHED HEAD ATTACHED 12 Muscle contraction molecular mechanism. Actin Myosin Tropomyosin Troponin Calcium ions https://twitter.com/drewb erryIV/status/12644313 44689414146 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/13 microvilli adhesion belt cell skeleton contractille ring stress fibers myosin filaments actin filaments contraction actin fillaments Actin filaments location in cells. actin shown green muscle contraction: motor molecule of myosin interacts with actin, resulting in contraction. by hydrolysis of ATP and resulting morphology changes 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803- vybrane-kapitoly-z-bunecne-fyziologie/ Define footer – presentation title / department14 Fillopodia Lamellipodia Stress fibers morphology thin protrusions at leading edge veil-like cytoplasm extension actin bundles in cells regulation ↓ RhoA + ROCK ↑ RhoA + ROCK function environment probing modulation of adhesion migration in 2D and 3D cellular contractility, force for adhesion, migration Define footer – presentation title / department15 Leading edge in migrating cell ̶ Formation of polarised cells: ̶ change its morphology + intracellular organization. ̶ and integrins forming focal adhesions, localise to the leading edge. Ngalim 2010 10.1155/2010/363106 16 PFA fixation test: the effects on live cell imaging In this sense, while chromatin, nuclear membrane and mitochondria were distinguishable before fixation, their shape and density were affected after PFA was added to the medium. This same phenomenon was observed on the cellular membrane, with cells shrinking. Several blebs appeared throughout the sample. https://www.nanolive.ch/pfa-fixation-test-the-effects-on-live-cell-imaging/ 17 Cytoskeleton is a highly dynamic structure Am I the only one who likes to watch the #LeadingEdge all day long? The orange bundles make #RetrogradeFlow easy to follow. #Actin assembly @VUCellImaging @VUBasicSciences #VandyCytoskeleton https://twitter.com/i/status/1251982 170421428224 Define footer – presentation title / department18 F-actin associated structures Adherens junctions focal adhesions associated protein cadherins (α- / β-catenin) + others integrins + 200 others function cell-cell adhesion cell-ECM adhesion, mechanotransduction, migration Define footer – presentation title / department19 Focal adhesions ̶ connection between a cell's cytoskeleton and ECM. ̶ sub-cellular structures that mediate the regulatory effects of a cell in response to ECM adhesion ̶ in a state of constant flux: proteins associate and disassociate with it continually as signals are transmitted to other parts of the cell, relating to anything from cell motility to cell cycle ̶ contact with ECM via integrins: integrins bind to extracellular proteins via short amino acid sequences ̶ white blood cells migrate along the connective endothelium Actin Filament Induced Movement II (Cell Adhesion) https://www.youtube.com/watch?v=sHFN 48il9YY 2017 Collins http://dx.doi.org/10.1016/B978-0-12-803394-4.00001-X20 Four layers of Focal adhesions ̶ integrin extracellular layer, • responsible for binding to the ECM. • extracellular domain “outside-in” signaling: integrin-ECM binding changes in intracellular signaling. • cytoplasmic domain “outside-in” signalling: intracellular signaling affect extracell integrin binding. ̶ integrin signaling layer ̶ force transduction layer FA stabilization and mechanosensitive signaling via focal adhesion kinase (FAK), paxillin, talin, vinculin. ̶ actin regulatory layer: the regulation of actin assembly, disassembly, and actomyosin contractility Define footer – presentation title / department21 integrin extracellular layer, • responsible for binding to the ECM. • extracellular domain “outside-in” signaling: integrin-ECM binding changes in intracellular signaling. • cytoplasmic domain “outside-in” signalling: intracellular signaling affect extracell integrin binding. integrin signaling layer force transduction layer FA stabilization and mechanosensitive signaling via focal adhesion kinase (FAK), paxillin, talin, vinculin. actin regulatory layer: the regulation of actin assembly, disassembly, and actomyosin contractility http://media.cellsignal.com/www/html/science/landscapes/adhesion 22 Filopodia during cell migration as "mechanical antennae“ • probing ECM stiffness • Fillopodia function as signaling platforms: • probing ECM topography • probing ECM stiffness • at the leading edge regulates Arp2/3-mediated actin remodeling and polymerization to drive lamellipodia membrane protrusions and forward cellular movement. migration Filopodia probe the ECM by assembling specialized adhesion complexes at specific sub-filopodial locations Fillopodia A spinning disk is a device that enables high signal:noise fluorescence microscopy by eliminating out of focus light. https://twitter.com/TyskaLabActual/status/1277 592591882768385 Define footer – presentation title / department23 Filopodia during cell migration actin polymeration = strenght generated for fillopodia and lamellipodia growth fillopodia formation preceeds lamellipodia formation formation facilitated by • insulin-receptor substrate p53 (IRSp53) and others: deform and/or tubulate the plasma membrane, and • by motor activity of myosin-X - actin fiber convergence at the cell periphery. Fillopodia 24 Fillopodia GFP-stained transmembrane protein, colors code for depth, spining disk miscoscopy https://twitter.com/TyskaLabActual/ status/1428127612497317892 Define footer – presentation title / department25 Filopodia, invadopodia and filopodia-like structures. In 2D, cells form well-defined finger-like, actin-rich structures including filopodia and invadopodia. Filopodia transient and extend out from the advancing lamellipodium associated with myosin-X and fascin, Invadopodia actin-rich, more stable, localize beneath the cell body possess substrate degradation properties. assoc: cortactin and the ECM-degrading protease MT1- MMP spaces used for consequent migration 26 Structure, components and secreted enzymes of an invadopodium Mierke, 2020, https://doi.org/10.3389/fcell.2020.583226 27 Tubulin, actin and mitochondria anchoring Schematic of mitochondria (red), microtubules (blue), and f-actin (green) distribution in an undifferentiated cell. (b) Mitochondria associate with microtubules (blue, bottom) and with actin (green, top) via motor/adaptor complexes. Dynein/dynactin associate with mitochondria via TRAK and Miro to drive retrograde mitochondrial motility. In contrast, Kinesin-1 coordinates anterograde motility toward the cell periphery. Myo19 can associate with the mitochondria outer membrane either directly or through Miro. Syntaphilin anchors mitochondria to microtubules. https://doi.org/10.1016/j.cophys.2018.03.003 Petrie 2012 10.1242/jcs.09373229 Extracellular matrix, 2D vs 3D ̶ in 2D use actin polymerisation to extend leading edge ̶ in 3D this is just one of the movement strategies. ̶ migration modes (in 3D) dependent on physical properties of ECM ̶ ECM properties distinguished by leading edge cells ̶ single cell can switch between leading edge structures Define footer – presentation title / department30 Filopodia, invadopodia and filopodia-like structures. In 3D and in vivo, cells form filopodia-like protrusions, (lack of clear classification criteria) filopodia, invadopodia, filopodium-like protrusions and actin spikes. molecular machinery associated is poorly understood metastatic prostate cancer cell line PC3 Paul, 201732 Migration ̶ Single cell migration ̶ ameboid ̶ mesenchymal ̶ lobopodial ̶ pseudopodial ̶ Collective migration ̶ Cell migration in which groups of cells migrate while in physical contact and in the same net direction. This is in contrast to single cell migration in which cells move individually and are not in physical contact with other Define footer – presentation title / department33 Types of movements ̶ mesenchymal x pseudopodial migraiton? 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/34 collective strategy individual strategy mesenchymal ↓ cell-cell adhesion ameboid ↓ cell-cell adhesion ↓ cell-extracellular matrix adhesion movement through ECM due cleavage of ECM by matrix metalloproteases loss of cell polarity membrane blebbing push through spaces in ECM extracellular matrix (ECM) Main types of migration in cancer cells cancer cells employ variety of invasivenes modes migration strategies can be switched Key switches: Rho, Rac moleculles Rac1 = Ras-related C3 botulinum toxin substrate RhoA = Transforming protein RhoA). 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803- vybrane-kapitoly-z-bunecne-fyziologie/ 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803-vybrane-kapitoly-z-bunecne-fyziologie/35 Location of movement structuresin the mesenchymal type of movement. Formation of structures enabling cell movementis significantly regulated by the activity of small GTPase from the Rho family • Rac1 = Ras-related C3 botulinum toxin substrate 1, • Cdc42 = Celldivision control protein 42 homolog • RhoA= Transforming protein RhoA 2018 Raudenská https://www.lekarskeknihy.cz/produkt/109803- vybrane-kapitoly-z-bunecne-fyziologie/ stress fibers actin filaments movement growth factors + various signals 4 steps of migration: protrusion, adhesion, contraction, retraction. F-actin • short, branched F-actin at the leading edge • long, unbranched F-actin stress fibres at the rear Microtubules with ends emanating from the MTOC Strong and weak focal adhesions The gradients of active Rho and Rac 37 Cytoskeleton is a highly dynamic structure visualization of static (arteficially stabilised) and dynamic actin filaments by co-imaging SiR-actin (green) and LifeAct (magenta). https://twitter.com/joachimgoedhart /status/1402234543646679042 Define footer – presentation title / department38 Rho GTPases in the cell motility cycle. 1. A migratory cell enters the cell motility cycle in response to a chemoattractant signal. 2. Cdc42 determines the direction of motion. 3. Rac induces the formation of actin-rich lamellopodial protrusion at the leading edge. 4. New protrusion is stabilized by the formation of new adhesions to the underlying substratum, a process controlled mainly by Rac and RhoA . 5. Rho acts at the rear end leading to the formation of stress fibers and actin–myosin contractility providing tension for the cell to retract its tail and move forward. 2013 Hanna http://dx.doi.org/10.1016/j.cellsig.2013.04.009 Define footer – presentation title / department39 A migration of mesenchymal cell within collagen matrix. Cells were embedded within bovine collagen gel (1mg/ml) and observed using CCHM. Tolde 2018 https://doi.org/10.1038/s41598-018-30408-7 Mesenchymal characteristics • elongated morphology • pseudopodial protrusions • adhesion to substrate generates coordinated action of MMPs and actomyosin machinery „paving the way“ Pseudopodial migration • filopodial, lamellipodial or other migration relying on protrusions driven by actin polymerisation follower cells are MMP-independent Paul 2017 https://www.nature.com/articles/nrc.2016.123 Paul 2017 Define footer – presentation title / department40 How much ATP is required for actin-driven motility? • actin filaments in moving goldfish epithelial keratocytes polymerize at the same rate that the cell moves—about 0.2 μm/s • each filament must grow by about 100 monomers/s to support motility, which costs ≈100 ATP per polymerizing filament per second • Lamellipodium is about 20 μm long and contains roughly 200 actin filaments per micron • this value turns out to be a very minor ATP requirement (cells produce 109 ATP/second) Milo et al., Cell biology by Numbers, 2016, p.202 Define footer – presentation title / department42 Translocation of an amoeboid cell through a narrow pore. An amoeboid cell embedded in rat-tail collagen (1 mg/ml) was observed using CCHM. This video demonstrates the dynamic cell body deformation during invasion through a narrow pore Tolde 2018 https://doi.org/10.1038/s41598-018-30408-7 Disorders associated with impaired cytoskeleton • Tumor diseases – Metastasis is responsible for the greatest number of cancer deaths Define footer – presentation title / department44 ̶ Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. ̶ For cancer cells to metastasize, they must successfully complete all of the steps of the metastatic cascade. primarytumordisseminationsecondarytumor cell division morphology abnormal differentiated tumor cell cancer stem cell basal membrane tumor cell nontumor cell loss of polarity and adhesion normal tissue benign hyperplasia dysplasia carcinoma in situ local invasion migration motility capacity anoikis resistance Epithelial mesenchymal transition blood stream, lymphatic dissemination intravasationextravasation micrometastasis formation adaptation to new microenvironment differentiated tumor cell cancer stem cell immune cells cancer associated fibroblasts growth of secondary tumor stromal cell growth factor synthesis cleavage of extracellular matrix Define footer – presentation title / department45 ̶ Cancer cells in the primary tumour acquire the ability to detach from the primary tumour and migrate through the surrounding ECM and stroma. ̶ Degradation of the vascular basement membrane and travel across the endothelium, termed intravasation. ̶ Tumour cells transport through the vasculature, arrest in a capillary bed and cross the vasculature (termed extravasation). ̶ Disseminated cells grow and interact with the extracellular environment to form metastatic tumours. primarytumordisseminationsecondarytumor cell division morphology abnormal differentiated tumor cell cancer stem cell basal membrane tumor cell nontumor cell loss of polarity and adhesion normal tissue benign hyperplasia dysplasia carcinoma in situ local invasion migration motility capacity anoikis resistance Epithelial mesenchymal transition blood stream, lymphatic dissemination intravasationextravasation micrometastasis formation adaptation to new microenvironment differentiated tumor cell cancer stem cell immune cells cancer associated fibroblasts growth of secondary tumor stromal cell growth factor synthesis cleavage of extracellular matrix Define footer – presentation title / department46 2D vs 3D ̶ Migration in unconfined 2D surfaces X in confined spaces ̶ migration in confining microenvironments not predicted by 2D assays Mierke, 2020, https://doi.org/10.3389/fcell.2020.58322647 Migration and invasion of cells ̶ Metastasis regulated by biochemical and mechanical cues of microenvironment ̶ Decreased cell adhesion → enhanced invasive capacity ̶ Migration subject to fluctiations due alteration of ̶ cytoskeleton, ̶ matrix mechanics, ̶ organelle mechanics Typical protrusive structures in invasive cancer cells ̶ Cancer formation of structures: ̶ plasma membrane blebs, invadopodia or pseudopodia ̶ actin-dependent ̶ Nonapoptotic blebs are highly dynamic protrusions in which the plasma membrane bulks out owing to increased hydrostatic pressure on regions of weak cortical actin. ̶ :10.1038/nrc3003 Define footer – presentation title / department49 Migration through confining tracks ̶ endogeneous: features of tissues ̶ made by tumors/tumour associated cells ̶ types of migration ̶ 2dx3d 50 Cancer cell migration occur in pre-defined paths. ECM alignment provides migration cues collagen alignment and bundling at tumour periphery provide cues for directed migration. unbundled ECM (fibrillar collagen), which present pore-like migration spaces Microtracks intravascularly and perivascularly between epithelial or endothelial surfaces (eg between muscle and nerve fibres). Paul 2017, https://www.nature.com/articles/nrc.2016.123 Paul, 2017, https://www.nature.com/articles/nrc.2016.12351 Physical limits for migration ̶ Nuclear size and stiffness control confined migration ̶ as confinment increases, deformation and squeezing is challenging ̶ knockdown of lamin A, (component of the nuclear lamina) decreases nuclear stiffness and enhances the transmigration ̶ progerin (a mutant form of lamin A) increases nuclear stiffness and suppresses confined cell migration 52 Nucleus stiffness. lamin A/C as limitin factor in migration We use combined AFM and side-view SPIM to study how forces correlate with nuclear shape change under compression in live cells. https://twitter.com/C_M_Hobson/status/1227278696798539777 Hobson 2020 https://doi.org/10.1091/mbc.E20-01-0073 Define footer – presentation title / department53 Homing of Endothelial Progenitor Cells to tumor Cells migrate to tumor in a chemotaxis response to tumor-secreted cytokines (VEGF, IL-8, CCL5, and others) which interact with their respektive receptors de la Puente 2013 10.1158/1078-0432.CCR-13- 0462 2015 Jansen, 2021 Raudenska, 2021 Espina54 Durotaxis: mechanosensing and transduction in action directed cell motility in response to gradiens in substrate rigidity ̶ Fillopodia - affinity for stiffer ECM ̶ ECM stiffness favor migration and attract movement to stiffer parts of ECM) ̶ explain migration to vasculature: tumor-associated vessels stiffer than nontumor Gradient cause asymetry in cells ̶ front: higher rigidity → ↑ FA asembly → larger FA ̶ rear: the softer substrate → FA disassembly. ̶ net flow of FA proteins to the leading edge → mature FAs in front promote protrusion extension and establish the direction of migration to the stiffer regions of the substrate. 2015 Jansen, 1945 Weiss55 Contact guidance ̶ orientation of cells and stress fibers influenced by geometrical patterns of stroma ̶ directed cell migration/orientation based on microenvironment alignment 1945 Weiss https://doi.org/10.1002/jez.1401000305 Define footer – presentation title / department56 intrinsic and extrinsic cues influence the migration migrate proteolytically through the secretion of matrix metalloproteinases (MMPs), which create microtracks for migration. Follower cells moving through these tracks and cells moving through microenvironments with pre-existing migration tracks use diverse migration mechanisms that depend on the levels of adhesion and cell contractility, and are thus dependent on both the cell and the microenvironment. • when cell adhesions to the substrate are present, tumour cells migrate using a pseudopodial-based mechanism that is dependent on protrusions. • when high contractility - fibroblasts move using a lobopodial migration mode. • low cellular adhesion migrate using a bleb-based mode of amoeboidal migration dependent on high cortical contractility. • contractility is inhibited, tumour cells may use a protrusion-based amoeboidal migration mode (A1 bleb-based migration) dependent on actin at the leading edge. • absence of actin polymerization, cell movement is achievable through frontto-rear flow of water through the cell (which is termed osmotic engine migration). Paul 2017 https://www.nature.com/articles/nrc.2016.123 van Helvert https://doi.org/10.1038/s41556-017-0012-057 Cell migration modes in 3D environments, including single-cell and collective migration. Define footer – presentation title / department58 ̶ ability of cancer cells to invade via ̶ MMP-independent amoeboidal mode versus ̶ an MMP-dependent mesenchymal mode ̶ may not solely be attributed to cell-intrinsic properties ̶ but also to the 3D architecture of the local microenvironment. ̶ mouse mammary gland:significantly less fibrous tissue than the corresponding human Comparison of human and mouse mammary glands. (A) Hematoxylin & eosin (H&E) stained section of human breast tissue showing a terminal ductal lobular unit comprised of ducts and acini embedded in a fibrous connective tissue stroma. (B) Schematic representation of a human terminal ductal lobular unit, emphasizing the intimate association of epithelial structures with interstitial fibrous connective tissue stroma and the more distant adipose tissue. (C) H&E stained section of the mouse mammary gland, showing ducts imbedded in a stroma composed of adipose tissue. (D) Schematic representation of the mouse mammary gland, displaying ducts in intimate contact with fibroblasts and adipocyte Parmar et al 2004 10.1677/erc.1.00659 Define footer – presentation title / department59 Collective migration ̶ movements of group of cells and the emergence of collective behavior from cell-environment interactions and cell-cell communication. ̶ essential process for embryonic development, wound healing and cancer spreading Define footer – presentation title / department60 FaDu head and neck cancer cells, collective migration and division, QPI, 10X Define footer – presentation title / department61 Mesenchymal and hybrid epithelial–mesenchymal (EM), basal, cancer-associated fibroblast (CAF) and tumour-associated macrophage (TAM) represent four major categories of leader cell that drive collective cancer invasion. Multiple leader cell types may arise in a tumour, though not necessarily all together. Key functions of leader cells include generating a migration path, coordinating with nearby cells to enable collective movement and enhancing the survival and metastatic capabilities of the tumour. Leader cells perform these functions using several molecular programmes such as • matrix remodelling, • cell mechanics and cell signalling, • cell reprogramming. ECM, extracellular matrix. Mercedes 2021 https://www.nature.com/articles/s41568-021-00376- 8 Define footer – presentation title / department62 Leader cells communicate with other cells and their environments mechanically. This illustration highlights the key components of the cell mechanics cascade for cell–cell and cell– environment coordination. Leader and follower cells modulate RHO signalling according to their cell type and matrix density; this can in turn activate other pathways such as mitogenactivated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and transforming growth factor-β (TGFβ) pathways in leader cells. Mercedes 2021 https://www.nature.com/articles/s41568-021-00376-8 63 no EMT needed Raudenska 10.1039/c9mt00049f Define footer – presentation title / department64 Cytoskeleton therapeutic target ̶ Actin targetable by mycotoxins: ̶ block polymeration: cytochalasins ̶ block depolymeration: phalloidin Death cap (Amanita phalloides) amantadin + phalloidin phallotoxins are highly toxic to liver cells, they have since been found to add little to the death cap's toxicity, as they are not absorbed through the gut 65 Stress fiber recovery after Cytochalasin D washout is enhanced by activated FHOD1 U2OS cells expressing mCherry-Actin together with EGFP-FHOD1 constructs (not shown) were subjected to Cytochalasin D washout to stimulate stress fiber formation. Left: FHOD1 WT; right: FHOD1 V228E. Images were acquired by time-lapse confocal microscopy The original research can be accessed at http://dx.doi.org/10.1242/jcs.134627 j.gumulec@med.muni.cz | @jarogumulec | www.med.muni.cz/masariklab