The Physical Interaction of Cancer cells:Intravastion

Physical Interaction in Cancer

Definitions

Terminologies

Most definitions below will be based on the cancer or metastasis process.

  1. Integrin: Cell adhesion transmembrane receptors that serve as extracellular matrix (ECM)-cytoskeletal linkers and transduce biochemical and mechanical signals between cells and their environment.[1]
  2. Collagen Crosslinking: Occurs when enzymes like lysyl oxidase (LOX) create chemical bonds between collagen fibers, making the ECM more rigid and densely packed.
  3. Focal adhesions: Integrin clusters located at the basal surface of adherent cells that connect the extracellular matrix to the cytoskeleton through focal adhesion proteins.
  4. Lamellipodia: Large cytoplasmic projections found primarily at the leading edge of migrating cells, particularly on two-dimensional substrates.
  5. Filopodia: Thin finger-like protrusions composed of bundled actin filaments, often extended from the edges of the lamellipodium. They help find a direction to migrate.
  6. Pseudopodial protrusions: Temporary, irregular projections of cell membranes, less structured than lamellipodia and filopodia.
  7. Matrixtraction: Mechanical forces exerted by cells on surrounding ECM to deform it, allowing them to adhere, migrate, and remodel their environment.
  8. Rate-limiting step: A reaction step in biochemistry that controls the rate of a series of biochemical reactions.
  9. Interstitial flow: Refers to the movement of fluid within the interstitial space of tissue (small gaps between cells and extracellular matrix). It helps transport nutrients, waste products, and signaling molecules.
  10. Lamina: A thin layer or sheet of tissue (nuclear lamina) composed of lamin protein that provides structural support and regulates nuclear processes.
  11. Actin: A globular protein that polymerizes to form filaments and is a key component of the cytoskeleton. It provides structural support and enables cell shape changes, forming structures for lamellipodia and filopodia that allow cells to move.
  12. Haemodynamic force (HDFs): The forces that occur when blood and the surrounding tissues exchange forces.
  13. Cytokeratin: A family of intermediate filament proteins in epithelial cells that are part of the cytoskeleton, providing structural support and shape to the cell.

Physical Interaction in Invasion

Epithelial-to-Mesenchymal Transition

Mesenchymal cell: Refers to a cell derived from the mesenchyme (a type of embryonic connective tissue). They can differentiate into many cells (bone, cartilage, fat, muscle). Key characteristics include:

  • Motile, can migrate through ECM.
  • Interact loosely with other cells.

Epithelial-to-Mesenchymal Transition: A cellular process during which epithelial cells acquire mesenchymal phenotypes and behaviors following the down-regulation of epithelial characteristics. EMT is triggered in response to signals from the microenvironment. During EMT, changes in gene expression and post-translational regulation mechanisms lead to the repression of epithelial characteristics and the acquisition of mesenchymal traits.[2][3]

In particular, the detachment of carcinoma cells from the epithelium and subsequent invasion of the underlying stroma resemble well-characterized EMT in embryogenesis.[3] These cells also express matrix metalloproteinases (MMPs) on their surface, promoting the digestion of the laminin- and collagen IV-rich basement membrane.[4]

In the vicinity of a mammary tumor, the matrix is often stiffer than in normal tissue due to enhanced collagen deposition[5] and lysyl-oxidase-mediated cross-linking of collagen fibers by tumor-associated fibroblasts.[6]

Since tumor-associated fibroblasts improve ECM stiffness by depositing collagen and promoting cross-linking, stiffness increases integrin signaling, stimulating more fibroblasts and tumor cells to remodel the ECM further, creating a positive feedback loop. Such changes in the physicochemical properties of the matrix can enhance cell proliferation and invasion.

Whether stiffening of the stromal matrix occurs in other solid tumors beyond mammary tumors has yet to be determined. Despite recent technological advances, little is known about the molecular and physical mechanisms driving motile cancer cells away from the primary tumor and into the stromal space, especially at the subcellular level.

Motility in Three Dimensions

Similarly, several cellular features that are important in 3D like nuclear deformation, MMP production, and major reorganization of the ECM are of no importance in 2D cells. A recent study shows focal adhesion is altered when cells are embedded inside a 3D matrix.[9] A cell is much larger than the diameter of the fibers of the ECM, which are typically on the order of 100nm. So from a cell perspective, the collagen fibers in the ECM appear quasi-one- dimensional (1D) but in the 2D substrate, the size of collagen fiber is 1-10 μm in size, much larger than the fiber diameter of the ECM.[10, 11, 12]This limited size effect limits the size of focal adhesions and associated cluster of integrin that can be formed in cells embedded in a 3D matrix. Unlike in a 2D culture, where the cell has a continuous flat su rface to interact with, in a 3D environment, the cell’s contacts are smaller and more localized due to the structure of the matrix(confined local contact with quasi-1D fibers). Nevertheless, in 3D the collagen fiber still can support the formation of small and highly dynamic integrin clusters, with sizes on the order of tens of nanometers and a few seconds lifetime, which still may help in motility. Moreover, cells in vivo( inside the actual body not in vitro/lab dish) could promote the bundling of collagen fiber through the generation of contractile forces produced by cellular protrusions(extensions). They enhance the surface available and potentially promote the formation of larger adhesion.[13] Actomyosin stress fiber which contains bundled acting filament has an important role in 2D cell migration by providing the contractile forces required for the regulated detachment of the rear of the cell from the substratum and establishing actin flow at the leading edge of the cell.[10]But in 3d there are few stress fibers and these are either localized to cell cortex or radiate from nucleus towards the plasma membrane to form pseudopodial protrusions. [14]. In a 3D matrix, cancel cells and epithelial or endothelial cells have fewer focal adhesions and stress fibers and typically do not form the characteristics of wide lamellipodium and associated filopodia protrusions at the periphery. Instead, they display a limited number of pseudopodial protru- sions typically of 10-20 μm thickness, which is intermediate between both.[9] Traction microscopy suggests that in 2D substrate, the lamellipodium actively pulls the rest of the cell through nascent focal adhesions at the edge of the lamellipodium.[15]But in 3D traction microscopy it revealed that cells inside a 3D matrix never push the surrounding matrix and only pull on surrounding fibres.[14, 16]. Substantial matrix traction only occurs in the presence of productive pseudopodial protrusions which is typically between only one to five per cell at any time.[14].In a 3D matrix, pseudopodial protrusions pull with approximately equal force in the rear and trail edge. However, due to the asymmetric release of pseudopodia from the collagen fiber, it creates a defect in the matrix in the wake of the cell. The partial digestion of the ECM in the wake of the cell results in biased motion. This defect does not allow the cell to retrace the tunnel formed during migration and, therefore promotes highly persistent migration in a 3D matrix, compared to less persistent migration of the same cell in the 2D substrate. [17]Since pseudopodial protrusion correlates with 3D cell speed, protrusion dynamics is crucial in establishing 3D motility.

A recent study shows focal adhesion is altered when cells are embedded inside a 3D matrix.[9]

Protein and Dimensionality

  • Protein p130CAS: Mediates a high number and high growth rate of protrusions.
  • Mechanosensing protein zyxin: Represses protrusion activity and diminishes the rate of protrusion growth along collagen fibers.

A recent study showed that the number of protrusions per unit time and the growth rate of protrusions, as modulated by focal adhesion proteins, correlated strongly with tumor cell motility in 3D matrices. [9] p130CAS-depleted cells moved more slowly and zyxin-depleted cells moved more rapidly than control cells in 3D matrices, these depleted cells displayed opposite motility phenotypes on flat surfaces. However, no correlation exists between adhesion protein depletion and motility in 2D substrates. For example,vinculin-depleted cells move at a similar speed to control cells on flat substrates, whereas they move faster than control cells inside a 3D matrix.[9] Such results suggest that pharmacological screens for drugs that limit motility on 2D substrates could be misleading. Since, many features observed in vivo by intravital microscopy have been recapitulated in 3D matrix constructs, including highly persistent migration of single cells away from tumors, the role of actomyosin contractility in collective migration to the lymphatic vessels, and the crucial role of MMPs in cancer cell dispersion from primary tumor site.[3]. Much more is needed to validate 3D models for in vitro cancer studies.

The Role of Cell Mechanics in Intravasation

A cellular mechanism is a process that occurs at the cellular level, such as how a cell interacts with itself, other cells, or the environment. The cell contains cytoplasm, which is a complex composite system that behaves like an elastic material at high deformation rates but more like a viscous material that exhibits yield stress at low deformation rates.[18] As MMP-mediated digestion of matrix seems to be only partial, the rate-limiting step in the migration of cancer cells within a matrix or across an endothelium may be the deformation of the interphase nucleus(the largest organelle in cell[?] and is approximately ten times stiffer than the cytoplasm. [19, 20] The elasticity of the nucleus seems to be determined by nuclear lamina underlying nuclear envelope[20] and by both chromatin organization [21] and LINC. LINC (Linkers of nucleus and cytoskeleton ) are protein assemblies that span the nuclear envelope and mediate physi- cal connections between nuclear lamina and cytoskeleton.[22] This connection is mediated by (the SUN domain-containing protein and KASH). Depletion of LINC component causes nuclear shape defect.[23, 24, 25]. The nuclear lamina and LINC complex molecules have crucial roles in collec- tive 2D migration[26, 27]; however, their role in 3D migration is yet to be studied. The mutation that occurs in nesprins and Lamin A?C that have been found in breast cancer[28] could cause a change in LINC-mediated connections between the nucleus and cytoskeleton which affect cancer cell 3D motility and invasiveness. Biophysical measurements that compare the mechanical properties of normal and cancer cells have consistently shown that cancer cells are softer than normal cells and that this cellular com- pliance correlates with increased metastatic potential. [22, 29].However, the reason for cancer cells being softer than non-transformed is yet to be found. Migration through a 3D matrix and penetration through an endothelium is likely to require optimal mechanical properties. If they are too stiff or too soft, cells cannot deform the highly crosslinked collagen fiber of the matrix to migrate efficiently. However, single-cell measurements have consistently revealed that induced cells of particular cell types are usually heterogenous(cells are not uniformly soft or stiff).[3] and 6 display a wide range of mechanical properties. This suggests inheritance because the traits are not randomly distributed at every cell division. Instead, the persistence of a broad distribution implies that cells with optimal mechanical traits maintain and pass these properties over gener- ations. Random determination at cell division would lead to a more uniform distribution over time, which contradicts observations. An important question is whether the physical attributes of cancer cells, such as stiffness are passed on from generation to generation. If these physical properties are inherited we can stop them using pharmacological inhibition or activation of pro- teins affecting their cell mechanics to stop stromal invasion. Different optimal mechanical properties probably are required for each step in the metastatic cascade. To survive such a harsh changing environment carcinoma cells need to adapt to survive.

Shear Stress and the Circulatory System

Circulating tumor cells(CTCs) are subjected to hemodynamic forces, immunological stress, and collisions with host cells such as blood cells and endothelial cells lining the vessel wall. Only cells that can overcome or even exploit the effects of fluid shear and immunosurveillance will adhere to the vascular endothelium of distant organs, exist the circulation, and successfully infiltrate tissue. A tiny fraction of CTCs survive to generate metastases; most CTCs die or remain dormant.[?] Assuming the reader does know about viscosity and how it works. Something interesting about blood is its nature, at shear rates greater than 100s−1, blood is considered a Newtonian fluid, implying that the shear stress increases linearly with shear rate. The mean blood velocityvav in arteries for a vessel of diameter d=4 mm is 0.45ms−1, whereas vav=0.1s−1 in a 5 mm vein. Shear flow influences the translational and rotational motion of CTCs and the time constant associated with receptor-ligand interactions that lead to adhesion but the magnitude of these effects and their influence on occlusion and adhesion remain to be determined. [3]

References

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