Microscopical Imaging of the Cell, Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
David H Koch Center for Applied Research of Genitourinary Cancers, Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
Microscopical Imaging of the Cell, Department of Cell Biology (283), Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9191, 6500 HB Nijmegen, The Netherlands.
Cancer cell invasion results from actomyosindependent movement of the cellular body through or along tissue structures. After metastasis to secondary organs, local invasion by cancer cells may lead to secondary organ infiltration, thereby engaging with a tissue environment that differs from the primary organ 1. At any invasion stage, cancer cells are confronted with non-neoplastic tissue composed of cells and cell-derived extracellular matrix (ECM). Cancer cells constitutively express adhesion receptors that bind to ECM as well as to resident stromal cells which not only activate the actomyosin machinery to generate traction forces needed to migrate, but also deliver important signals for their growth and survival 2. Initially cancer invasion is largely non-destructive, but over time leads to substantial tissue remodelling, local destruction with ulceration, vessel disruption, and, ultimately, loss of function of invaded organs 3.
Two principal types of interstitial tissues are transmigrated by tumour cells: (i) collagen-rich interstitial connective tissue present in most parenchymatous organs of the body and (ii) the interstitium of the brain. Both share significant similarities but also show important anatomic and molecular differences that impact upon the invasion process. The understanding of how physical and molecular guidance mechanisms converge, and how these principles apply to different tissues and organ systems, is important for developing experimental in vitro models of cancer invasion; it is further important for addressing and interpreting cell invasion patterns and routes in vivo by histopathological analysis. Using brain parenchyma and breast stroma as prototypic models for nervous and peripheral connective tissue invasion, respectively, we here review key tissue structures and their molecular properties that enable and guide cancer invasion and discuss the implications for designing experimental models and molecular anti-invasion therapy.
Cell–cell and cell–matrix receptors
During cell–tissue interaction and migration in most tumours, integrin and non-integrin cell surface receptors simultaneously engage with locally available ECM and cell surface ligands (Table 1).
Table 1. Cell–matrix and cell–cell adhesion molecules expressed in glioma and carcinoma cells compared with normal brain and breast tissue
Expression in histopathological samples compared with normal human tissue: , not altered; —, not expressed; ↑, up-regulation; ↓, down-regulation. D = ductal breast carcinoma; L = lobular breast carcinoma. References often show trends, with divergent expression regulation in patient subgroups.
Integrins are heterodimeric transmembrane glycoproteins consisting of non-covalently linked α and β chains, which both determine ligand binding strength and specificity. Eight distinct α and 18 β chains combine to form 24 different heterodimers with distinct ligand specificity for ECM proteins (eg collagens, laminins, fibronectin, vitronectin, tenascins, thrombospondin, and fibrin) and cell surface receptors (eg ICAM-1, VCAM-1, and L1-CAM) 4, 5. By these means, each cell type maintains a selective and activation-dependent integrin repertoire and thus ligand preference. The cytoplasmic integrin domains connect, via cytosolic adaptor and signalling proteins, to the actin cytoskeleton and mediate intracellular mechanocoupling and signal transduction 6. Consequently, integrins are important mediators for cell adhesion and migration. They further contribute to cell–cell contacts via direct interactions with counterpart cell receptors or indirectly, by bridging intercellular ECM molecules, including fibronectin or vitronectin 7.
Syndecans are a family of transmembrane heparan sulphate proteoglycans with four members, syndecans 1 to 4. Syndecans function mainly as co-receptors by binding to their ECM ligands in conjunction with other receptors, notably integrins 8. Through their heparan sulphate side chains, syndecans may further engage directly in ligand binding 9.
Dystroglycans are heterodimeric complexes consisting of non-covalently associated α and β subunits with extracellular ligand-binding and transmembrane functions, respectively 10. Dystroglycans are a part of the larger dystrophin-associated protein (DAP) complex that connects basement membranes to the cytoskeleton, particularly via α2 laminins and perlecan 10.
Ig superfamily members consist of immunoglobulin-like and fibronectin type III domains involved in homophilic and heterophilic cell–cell adhesion 11. The superfamily includes a variety of cell adhesion molecules (CAMs) with distinct ligand-binding specificities, including ICAM (intercellular), NCAM (neural), Ep-CAM (epithelial), L1-CAM, VCAM (vascular), ALCAM (activated leukocyte), and JAM (junctional adhesion molecule), among others 11.
Cadherins are transmembrane proteins consisting of several tandemly repeated cadherin domains that mediate calcium-dependent homophilic cell–cell contacts 12, 13. The cadherin superfamily comprises a total of more than 100 different members 14, with E- (epithelial) and N-cadherin (neural) most widely expressed in epithelial and neural tissues, respectively.
Link module superfamily of HA-binding proteins (hyaladherins)
CD44 is the main HA receptor expressed by all nucleated cells in vertebrates 15. Besides the standard form (CD44s), multiple splice variants encoded by variable exons v1–10 (CD44v1–10) can be expressed depending on the cell differentiation and activation state 16. Interactions of CD44 with multiple other molecules, including collagens, laminins, and fibronectin, have been shown in vitro; however, the relevance of these interactions to processes in vivo is not clear.
These receptors mediate tissue recognition and mechanical coupling and, with their expression levels and tissue context, whether cells remain immobile, migrate slowly or rapidly; and they further determine whether migration occurs individually with cell–cell junctions absent or collectively, as multicellular groups with cell–cell junctions present 2, 17.
Guidance structures of brain tissue
Brain tissue harbours both primary tumours of neural origin and secondary metastases originating from other tissues. The structures that guide invasion into brain tissue are cell processes of neuronal and astrocytic origin, blood vessels, and tissue gaps present along vessels and brain surfaces.
Brain cells and vessels
The grey matter of the brain cortex consists of a dense network of neurons, including their dendrites and myelinated axons, together with astrocytes (Figure 1A). The top layer of the cortex, termed glia limitans superficialis, is formed by astrocyte processes which interact tightly with the pia mater, consisting of a basement membrane and an outward monolayer of mesothelial cells (Figure 2A). Besides contributing to the outer meninges, both pia mater and glia limitans also extend into the brain parenchyma, where they cover and ensheath arteries, arterioles, and larger veins 18. Glia limitans with its basement membrane, but without pia mater, further surrounds small venules and capillaries 18. Thus, all larger blood vessels in the brain differ from vessels in other organs by their dual-layer microanatomy with a continuous perivascular space bordered by pia mater and glia limitans (Figure 1D) 19. Through this connection, the perivascular space conducts interstitial fluid and passenger leukocytes towards the subarachnoid space and ventricular cavities, thus fulfilling a draining function, which is similar to that of lymphatic vessels that are absent in the brain (Figure 2A) 20. As an exception, in capillaries, the basement membranes of glia limitans and endothelial cells are fused to a singular layer and obliterate the perivascular space 20.
Adjacent to the cortex, the white matter consists of myelinated axon tracks with adjacent oligodendrocytes providing axonal myelin sheaths and astrocytes providing an interstitial stroma by their interconnected processes, but lacks neuronal cell bodies (Figures 1B and 1C). Myelinated fibres thus form elongated tracks with interspersed astrocyte processes and a gap-like interstitium filled with ECM (Figures 1B and 1C). The brain anatomy thus consists of cell-fibre and perivascular space-track systems, which both provide constitutive trails of least resistance to moving cells.
Molecular composition of brain ECM and basement membranes
The brain ECM is mainly deposited by astrocytes and oligodendrocytes and comprises an estimated 20% of the brain volume in adults 21. The main ECM components are hyaluronic acid (HA), tenascin R, and lecticans, which interconnect with each other non-covalently and form molecular networks filling the intercellular space 22.
HA is a non-sulphated, linear, high-molecular-weight glycosaminoglycan which, due to its water-binding capacity, controls the high water content of the brain interstitium 23. Besides tenascins and lecticans, HA binds to cell surface receptors including CD44 and ICAM-1, which together contribute to both ECM organization and cell–matrix interaction. Tenascin R, a brain-specific member of the tenascin family comprising also tenascins C, X, and W, is a homotrimer with both lectican and integrin binding sites forming an adhesion bridge between the ECM and cells 24. Lecticans comprise a family of chondroitin sulphate proteoglycans with four members (brevican, versican, neurocan, and aggrecan), whereby brevican and neurocan are brain-specific 25. Lecticans contain HA and tenascin R binding sites and thus act as link molecules in protein–proteoglycan–glycosaminoglycan networks 26. Compared with peripheral interstitial tissues, a distinctive feature of the brain ECM is the absence of fibrillar collagen networks, which results in a low stiffness of the brain parenchyma 18. In a restricted expression pattern, fibrillar collagens I and III are, however, deposited by leptomeningeal cells, pericytes, and smooth muscle cells in blood vessels and the brain meninges, including the pia mater 18, 27, 28 (Figure 1).
Basement membranes are dense sheet-like meshworks of 50–100 nm thickness, which are particularly permissive for guiding cell migration 29. Basement membranes consist of interconnected collagen IV, laminins, heparan sulphate proteoglycans (perlecan), and nidogen/entactin 29. Collagen IV is a heterotrimer composed of three α-polypeptide chains 30, which interconnect with laminins, also heterotrimeric molecules, consisting of α, β, and γ chains 31.
Guidance of glioma cells
Gliomas are the most common primary brain tumours in adults. Gliomas presumably originate from transformed glial progenitors and, depending on their differentiation state, several subtypes exist, including astrocytomas, oligodendrogliomas, and ependymomas 32, 33. Glioma cells can form tumours in any brain region and infiltrate adjacent parenchyma diffusely, thereby, arguably, recapitulating the migration of glial progenitor cells during brain development 32, 34, 35. Glioma cell invasion into brain tissue occurs along pre-existing brain structures, with notable preference for myelinated fibres and blood vessels (Figure 2) 36, 37. Ultimately, through the perivascular space, glioma cells can reach and populate the subarachnoid space 37, 38. As a consequence of such extended tissue invasion, surgical resection of gliomas is usually non-curative and followed by cell survival and regrowth from invasion zones beyond the resection margins 35. For as yet unknown reasons, glioma cells usually do not invade vessel lumens; thus, unlike most other malignancies, systemic dissemination and metastasis in non-neuronal organs are a rarity in brain neoplasia 39.
Guidance by blood vessels
Glioma cells migrate along blood vessels using two biomechanically distinct routes: (i) the glia limitans along the outward vessel–parenchyma interface (Figure 2B) and (ii) inside the lumen of the perivascular space (Figures 2C). For the interstitial-type migration along the glia limitans, glioma cells may displace astrocytes bordering the blood vessels and employ the basement membrane as a substrate 40, 41, whereas the role of simultaneous glioma cell–astrocyte interactions is unclear. Glioma cells further penetrate through the glia limitans and its basement membrane to reach the barrier-free lumen of the perivascular space and migrate along the inner basement membranes of vessels 40, 41 (Figure 2C).
In vivo, glioma cells express and/or up-regulate a range of receptors binding to laminins and collagen IV, including integrins (α2β1, α3β1, α6β1, α6β4, , ), dystroglycans, and syndecans 42–45 (Table 1). Consequently, laminins and collagen IV are the main ligands supporting glioma cell invasion along basement membranes, with α3β1 integrin as the major laminin receptor and laminins 332 and 511 as the matching ligands 43, 46. Candidate cell–cell adhesions for the invasion route along the glia limitans comprise integrins connecting to cell surface-associated fibronectin and vitronectin, together with homophilic engagement of N-cadherin, L1-CAM, and NCAM (Figure 2B). Thus, whereas molecular guidance by the basement membrane appears to predominate in perivascular invasion, the role of glioma–astrocyte interactions remains to be clarified.
Guidance by nerve tracks
The mechanisms of glioma cell invasion along myelinated fibres are likely multifactorial, whereby each relative contribution remains to be elucidated (Figure 2D). Myelin sheaths formed by oligodendrocyte processes are considered as non-permissive substrates for cell migration, because they lack commonly known pro-migratory ECM proteins (eg laminins, collagens) and cell surface receptors that can bind these molecules 47. In addition, myelin sheaths express repelling receptors, including Nogo-A, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, which together inhibit axonal growth and cell migration 48, 49. Glioma cells, due to their altered protease expression profile, including increased MMP-14/MT1-MMP and MMP-2 levels 50, are, however, able to proteolytically cleave inhibitory molecules from the myelin surface using MMP-14 and thereby remove otherwise dominant anti-migration signals 51. Thus, an anti-migratory brain scaffold is conditioned by ECM remodelling and processing of cell surface receptors towards a migration-enhancing tissue environment.
Due to their high water content and non-covalent intermolecular linkages, the ECM networks between myelinated fibres formed by HA, tenascin R, and lecticans are likely soft and deformable, compared with collagen-rich peripheral tissues. It is not known whether and how glioma cells use this HA-based matrix as a migration substrate; however, the consistent up-regulation of the HA receptors CD44 and ICAM-1, and of the tenascin R receptor α9β1 integrin in glioma cells, is suggestive of a pro-invasive cell–ECM interaction 42, 52, 53. Glioma cells further up-regulate ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs), which is secreted, cleaves brevican, and thereby likely disrupts ECM networks and lowers the physical interfibre resistance 26, 54, 55. The distance between myelinated fibres in white matter tracks is less than 1 µm; thus, glioma cells have to expand the inter-fibre space and deform their nuclei, which are the largest and most rigid organelle in cells 56. Consequently, glioma cell invasion along myelinated fibres in a rat brain model also depends on cell squeezing in a myosin II-dependent manner 57.
Simultaneously with myelinated fibres themselves, astrocyte processes localizing ubiquitously in all brain regions may contribute to glioma cell guidance (Figure 1). The border of gliomas shows an increased number of reactive astrocytes, which together with glioma cells secrete various pro-migratory molecules, including laminins, vitronectin, fibronectin, and thrombospondin 46, 58–61. Because of their polyvalency (ie one molecule comprises more than one receptor-binding site), they adsorb to and decorate cell surfaces and provide a multimeric ECM meshwork available for counterpart cell surface receptor engagement (Figures 2B and 2D) 7. Thus, glioma cells employing myelinated fibres and astrocyte processes as a substrate for migration engage multiple receptor–ligand systems for polarization and generating actomyosin-mediated traction forces, together with pericellular proteolysis.
Guidance of metastatic tumour cells in brain tissue
Besides glioma cells, circulating tumour cells of other organs (mainly carcinoma and melanoma cells) may penetrate through blood vessels, establish metastatic brain lesions, and invade the brain. Similar to glioma cell invasion, metastatic brain invasion occurs preferentially along the blood vessels but spares white matter tracks and thus commonly lacks diffuse infiltration 62, 63. The vascular basement membranes are considered the most important structures for initial growth and migration of carcinoma and melanoma cells metastasizing in the brain 64, 65. It is likely that the lack of glioma cell-specific capabilities to remove anti-migratory activity of white matter tracks confines metastatic cell movement to the perivascular space, reminiscent of retained structural and molecular cues present in the connective tissue environment that they originate from.
Cell and tissue structures of the mammary gland
The mammary gland is a prototypic peripheral organ consisting of the epithelium with an underlying basement membrane that forms the milk-producing alveoli and connecting ducts, and surrounding collagen-rich connective tissue including blood and lymph vessels and nerve tracks, as well as an adjacent layer of fat tissue (Figure 3A). In contrast to most other peripheral tissues, the postnatal mammary gland is structurally dynamic and undergoes glandular growth and sprouting during puberty and pregnancy, a process that is recapitulated in a pathological manner during breast cancer.
The basic structural units of the mammary gland are alveoli joined together to form a lobule. Each lobule has its own lactiferous duct converging with other ducts to form a tree-like branching network towards the nipple. Both alveoli and ducts consist of a bilayered epithelium, inner luminal cells surrounding the cavities, and outer myoepithelial cells engaging with the surrounding epithelial basement membrane 66. Mammary ducts remain quiescent until puberty, when hormone-induced elongation and secondary branching occur by sprouting-type movement of the terminal end bud (TEB), a specialized region at the end of each primary duct. During pregnancy, through a similar sprouting mechanism, additional lateral branching occurs and luminal epithelial cells differentiate in the secretory alveoli 67.
ECM of the mammary gland
The interstitial tissue of the mammary gland contains fibrillar type I collagen as a main structural component and interconnected glycoproteins 67, 68. After extracellular deposition by fibroblasts, the propeptides of the triple-helical type I procollagen molecule are proteolytically cleaved by the endopeptidases ADAMTS-2, 3, and BMP-1 (bone morphogenetic protein-1), resulting in monomeric tropocollagen, which spontaneously polymerizes to multimeric fibrils 69–72. The fibrous connective tissue of the normal breast stroma is heterogeneous in physical organization and density, with collagen fibrils measuring approximately 70 nm in diameter organized laterally into wavy fibre bundles (Figure 3) 73. Thereby, regions of dense collagen bundles intersperse with loosely organized networks and inter-fibre clefts of variable widths, ranging from 3 to 30 µm 74 (O Ilina, unpublished data). This spatial structure of interstitial collagen, including physical and molecular signalling properties, are strongly modulated by collagen cross-linking and the presence of regulatory proteins. Collagen fibrils are cross-linked covalently by lysyl oxidases released by fibroblasts, stably bridging neighbouring collagen monomers and increasing fibril stiffness 75, 76. The kinetics of fibril formation, their physical properties, and function towards cells are further modulated by type I collagen-binding ECM constituents, including fibrillar type III 77 and V collagens 78, fibronectin 79, decorin 80, and biglycan 81. Fibronectin is an abundant fibrillar constituent of the ECM, with multi-adaptor functions, coupling cells and matrix via α5β1 integrin and providing binding sites for collagen type I and heparan sulphate proteoglycans 82. Collagen-interacting molecules that modify its function further include proteoglycans (heparan sulphate, chondroitin sulphate) and hyaluronan, which together form a viscous, gel-like filling of inter-fibrillar spaces and provide binding sites for growth factors and cytokines. Heparan-sulphate proteoglycans bind growth factors, such as FGF1, FGF2, and HGF; chemokines incuding SDF-1α; and collagen-associated glycoproteins, including periostin 83. HA non-covalently binds to proteoglycans and ECM proteins, including osteopontin, tenascin C, and versican 84, 85.
The adipose tissue of the mammary gland comprises groups of adipocytes with a cell surface-associated basement membrane composed of collagen type IV, laminins, and perlecan 86. The interstitial ECM supporting adipocytes is composed of type I collagen fibres 87, interconnected with collagen types III, V, and VI, fibronectin, and proteoglycans 88, which form a loose reticular network with interstitial clefts 89 and interspersed cell- and vessel-rich fibrous septa (Figure 3B).
In human quiescent breast epithelium, α1β1, α2β1, α3β1, and α6β4 integrins are expressed by myoepithelial cells and are involved in the stable adhesion to ECM and thus cell anchoring to the matrix 90. The quiescent state of normal breast epithelium is further maintained by desmocollin-2 (Dsc-2) and desmoglein-2 (Dsg-2), which form hemidesmosomal junctions between luminal epithelial and myoepithelial cells and E-cadherin connecting luminal epithelial cells laterally 91. Thus, in the quiescent mammary gland, the structure of the ECM is mainly anti-migratory, stabilized by integrin- and cadherin-based cell–matrix and cell–cell adhesion.
Guidance of breast carcinoma invasion
The majority of breast carcinomas originate from dedifferentiated luminal epithelial cells, histopathologically classified as ductal or lobular lesions depending on their original location 92. Thereby, invading breast cancer cells appear to recapitulate the process of duct elongation occurring during development, although in a spatially and timely deregulated form. During gland formation, the terminal end buds invade the mammary stroma collectively as multicellular ducts with cell–cell junctions intact 93. Similarly, ductal carcinomas form multicellular invasion strands consisting of cancer cells with cell–cell junctions retained, which with decreasing differentiation lose apicobasal polarity and the lumen 92. During collective invasion, ductal carcinoma cells remain physically and functionally connected, with expressed cadherins (E-cadherin and P-cadherin), tight (JAM-A, claudin-3, and -4), and immunoglobulin-based junctions (ALCAM and L1-CAM) 2, 94–96. Unlike ductal carcinoma, most of these cell–cell junction proteins are down-regulated in invading lobular carcinoma cells, with the loss of E-cadherin as a parameter for the diagnostic differentiation between ductal and lobular carcinoma 92. Nonetheless, most invasive lobular carcinomas invade collectively as thin multicellular sheets and strands (Indian file pattern) parallel to collagen bundles, histologically with notable cell–cell contact retained or as detached individual cells with cell–cell junctions lost 97. Candidate adhesion mechanisms for multicellular cohesion within Indian files that lack E-cadherin include N-cadherin and other cadherins, yet their mechanical and signalling contribution remains to be defined. Irrespective of the type of invasion, and in contrast to TEB sprouting which occurs with a basement membrane largely intact, the transition to invasive carcinoma is initiated by the disappearance of the basement membrane which allows for initial tumour cell contact to and migration along fibrillar collagen 98.
In response to the direct tumour cell–stroma interaction, both tumour cell- and stromal cell-derived changes in intracellular signalling and reactive cytokine and growth factor release induce substantial molecular and physical reorganization of the ECM molecular and physical reorganization, enhancing pro-migratory cancer cell activation and guidance 99. The main structural and molecular guiding principles within the mammary gland are the ducts themselves, the surrounding bundled type I collagen fibres, their secondary decoration with pro-migratory ECM molecules, and intercellular spaces between the basement membranes of adipocytes.
As a not yet invasive precursor, carcinoma in situ is an intraluminal ductal or lobular accumulation of epithelial cells with disordered, multilayered epithelial organization, partially or completely obliterated lumen, but intact cell–cell junctions and basement membrane (Figures 4A and 4B). As a first in situ invasion route with basement membranes and stromal tissues intact, neoplastic epithelial cells proliferate within and likely move along the lumen of the duct, which leads to the expansive growth of tumour foci and outward pushing of the surrounding ECM but not interstitial invasion or metastatic dissemination 100.
The ECM surrounding invading breast tumours undergoes substantial changes in density, composition, and structural organization, termed a desmoplastic reaction 101, 102, including the accumulation of fibrillar collagen and other matrix components, which enhance neoplastic invasion and disease progression. With increased collagen I deposition, the fibres become aligned and bundled, and cross-linking is increased, resulting in elevated stiffness of peritumoural ECM 103. As mechanisms, elevated activity of lysyl oxidases and tissue transglutaminases in stromal cells mediate covalent collagen cross-linking 104 and thereby enhance carcinoma cell invasion 105. Adhesion-promoting additional ECM constituents, including laminin-332 106, non-basement membrane collagen IV 107, fibronectin 108, and elastin 102, are jointly synthesized by carcinoma cells themselves and stroma cells, and reinforce migration-promoting activities, as suggested by their chemotaxis- and haptotaxis-enhancing effects in vitro109. Likewise, the composition of core proteins and glycosaminoglycan chains is altered in the ECM surrounding breast carcinoma. Versican, the major chondroitin sulphate proteoglycan present in the peritumoural stroma 110, forms non-covalent cross-links to different matrix molecules, including HA, tenascins, and fibronectin, and is recognized by the cell surface molecules β1 integrin and CD44 111. Similarly, the expression of HA is strongly up-regulated in the peritumoural stroma and carcinoma cells themselves 112, 113, together with its receptor CD44 114, thus creating additional options for cell–ECM interactions.
Besides structural ECM components, numerous matricellular proteins are de novo deposited nearby the front of invading tumours, including thrombospondin-1 115, tenascin C 116, SPARC (osteonectin) 117, osteopontin 118, and periostin 119, all of which exert structural, growth factor binding and signalling functions towards tumour and stromal cells. Matricellular proteins, including osteopontin and thrombospondin-1, favour cell invasion in vitro in haptokinetic and 3D migration assays 120–122, and further support the growth and spontaneous metastasis of breast carcinoma cells in mouse models in vivo123.
As mechanisms, matricellular proteins likely support motility and metastasis by complexing with and interconnecting between ECM structures, including fibrillar collagen type I, fibronectin, and heparin, thus modulating tissue porosity and stiffness, and providing additional functional ligands for cell surface receptors. As examples, periostin and osteopontin display binding sites towards cell surface , , and integrins and CD44 120, 123, 124. Tenascins C and X bind to collagen type I and fibronectin fibres via periostin 125 and/or the leucine-rich proteoglycan decorin 126, which provides bridging and possibly bundling of ECM, and additional ligand sites for cell guidance. Thus, interactions of various de novo deposited ECM proteins and proteoglycans with the collagen-based scaffold lead to its decoration and ‘functionalization’, which adds a migration-promoting layer, often together with signals for growth and survival.
Physical guidance by collagen scaffolds
The molecular modifications of the peritumoural ECM microanatomy further lead to marked physical alterations, resulting in lateral association and bundling of collagen fibrils, and increased overall ECM stiffness. The curly normal structure of collagen fibres oriented parallel to ducts becomes straightened and realigned perpendicular to the tumour boundary, and fibre bundles are thickened with marked inter-bundle gaps and spaces 127. Despite the elevated collagen density, inter-fibre clefts present in desmoplastic regions of invading tumours likely represent a network of microchannels suited to provide contact guidance to invading cells (O Ilina, unpublished data).
As part of the underlying process of anatomic restructuring, carcinoma-associated fibroblasts mechanically remodel the collagen network by adhesion, compaction and contraction 128, and/or proteolytic cleavage and re-alignment in parallel order 129. Aligned collagen bundles are a strong pro-migratory stimulus for cell invasion and further correlated with enhanced metastasis in vivo128, arguably by providing unhindered tracks of least resistance 130 and contact guidance 131. Similar guidance is recapitulated by experimental microtracks in 3D collagen lattices enhancing breast cancer cell invasion in vitro132. As a consequence of entering narrow tracks, carcinoma cells adjust their calibre by first deforming themselves followed by lateral pushing, thereby accommodating the ECM geometry to the space required for invasion by a physical mechanism 132.
Guidance by adipose tissue
Following penetration of the stromal layer, breast carcinoma cells reach the adipose tissue, as an indication of particularly aggressive forms of invasive carcinomas 133. Ductal breast cancer cells invade as solid strands between adipocytes, followed by adipocyte inclusion and destruction by the tumour mass 133. Lobular carcinoma cells invade adipose tissue preferentially along collagen fibres between adipocytes as multicellular Indian files or as individual cells 134. These different routes likely depend on adhesion or chemokine receptors differentially expressed by breast cancer cells and available counterpart ligands in the adipose tissue. Molecular guidance mechanisms of differential adipose tissue invasion are unknown, yet candidate pathways include integrins interacting with basement membrane components (ie α3β1, α6β4, and ), fibronectin (α5β1), and collagen type I (α2β1) 134, 135 (Figure 4D). Carcinoma cells may further utilize barrier-free clefts formed by loosely organized ECM between adipocytes for unhindered invasion and expansive growth, thus adjusting the space by outward pushing of the fibrillar ECM network. Thus, peritumoural ECM provides a complex environment with distinct invasion-promoting molecular zones together with spaces of least resistance.
Adhesion receptors in breast carcinoma invasion
Together with migration-promoting ECM modifications, expression modulation of the adhesion receptor repertoire in breast carcinoma cells coincides with the acquisition of a mobile state. Numerous in vitro models implicate β1, β3, and β4 integrins as the major receptors for cell–matrix coupling and mechanotransduction in breast cancer cells, the blocking of which attenuates migration and invasion in 2D and 3D models 136, 137 (O Ilina, unpublished data). Histopathological data from human breast cancer lesions show marked down-regulation of most integrin subunits in tumour cells at the invasion front (Table 1 and references cited therein). Conversely, integrin, which is not expressed by normal breast epithelium, becomes up-regulated, which coincides with the increased stromal deposition of ECM ligands for integrin, including periostin, osteopontin, and tenascin C 138, 139. Because of their pro-adhesive functions, the down-regulation of many integrins at the tumour invasive edge might allow cancer cells to turn over stable contacts to basement membrane or other ECM components, and transit to a mobile state through alternative integrins engaging with neo-ligands of the reactive stroma. In vitro, tenascin C, thrombospondin-1, and osteonectin modulate otherwise stable integrin-dependent attachment, which favours the turn-over of adhesion sites and enhances cell migration 140; thus, lowering strong adhesion to ECM ligands may support invasiveness in a context-dependent manner. Consistently, β3 integrin supports the haptotactic migration of breast carcinoma cells towards osteopontin in vitro and enhances bone-specific spontaneous metastasis in vivo141. Likewise, integrins α6β4 and α3β1 mediate migration along or invasion into model basement membrane in vitro and furthermore are associated with enhanced systemic metastasis 135, 142, suggesting a role in promoting both cell migration and metastatic dissemination. In summary, the integrin profile in invading breast cancer cells likely matches and engages with an altered ligand repertoire in the reactive tumour stroma, which results in dynamic interactions with ECM and enhanced invasion.
In morphogenesis, the multicellular sprouting of breast epithelium during mammary gland development is not affected by the knockout of individual integrin subunits, including α2, α3, α4, and β4143–145, with reduced epithelial branching caused by α2 integrin deficiency 144 and mild defects of gland polarity and anchoring to the basement membrane after the loss of β1 integrin 145. This mild phenotype suggests the function of as yet unknown compensatory adhesion systems to mediate normal duct elongation and sprouting morphogenesis, with unclear implications for the redundancy of pro-invasive adhesion systems in breast cancer.
Besides their contribution to cell migration and metastasis, integrins mediate stroma-derived growth and survival signals in tumour cells 137. Consequently, the cellular mechanisms by which certain integrin subsets control poor outcome in breast carcinoma in vivo, ie their role in promoting invasion versus metastasis versus growth and survival, remain unclear.
Intravasation and metastasis of carcinoma cells
Whereas in other tumour types blood and lymph vessels serve as pro-invasive structures 37, 130 perivascular invasion is not a prominent feature of invading breast carcinoma in human lesions (O Ilina, unpublished data). However, similar to most metastasizing tumour types, breast cancer cells eventually reach the lumen of blood and lymph vessels. Tumour-associated blood vessels are less organized, with incomplete basement membrane, only loosely associated pericytes, and leaky cell–cell junctions 146, and thereby likely facilitate the vessel wall penetration and intravasation by tumour cells. In lymph vessels, carcinoma cells were shown to form tissue-like multicellular cords, ‘emboli’, and intravascular growing tumour foci, which eventually spread further and reach distant organs 147, 148. A range of invasion-promoting factors locally up-regulated in the tumour stroma, including periostin 123, osteopontin 149, and tenascin C 116, strongly support distant breast cancer metastasis in mouse models in vivo, whereby their local and systemic effects 150 likely converge to enhance both local invasion and metastatic seeding at a distant site (eg by generating a ‘pre-metastatic niche’).
Tumour cell invasion in brain and mammary gland—similarities and differences
Despite their distinct invasion routes and molecular mechanisms, the invasion of glioma and carcinoma cells into the brain and mammary gland, respectively, shares the principle of guidance by the tissue scaffold, likely by a combination of molecular and physical mechanisms guiding cells along interfaces through pre-existing trails of least resistance. In the brain interstitium, which lacks fibrillar collagen, major invasion routes are basement membranes and intercellular tracks provided by myelinated axons and astrocyte processes. Basement membranes guide via laminin- and collagen-IV-mediated integrin engagement, whereas white matter tracks guide by cell–cell contacts and as yet unknown adhesion and force generation mechanisms. In the mammary gland, guided invasion follows gaps between bundled collagen fibres and interstitial spaces bordered by adipocyte-associated basement membrane, and likely depends on restricted integrin-mediated mechanocoupling. However, in both cases, it remains unclear how physical and molecular guidance principles are coordinated, how both principles synergize, and where they negatively impact each other.
Mechanically, migration through a porous interstitial space, along a viscoelastic protein polymer such as collagen bundles or basement membranes or along cell surfaces such as myelinated axons, represents a near barrier-free migration type with little or no enzymatic tissue modification required. Interface-mediated guidance is recapitulated in vitro by 2D culture in which cells move along a functionalized surface or by engineered 3D ECM-based models guiding cell invasion along tissue gaps and trails 17, 132. In diffuse brain infiltration, the inter-scaffold substrate is soft ECM dominated by HA non-covalently linked to scaffold proteins; thus, the mechanical resistance is likely low and allows glioma cells to squeeze through the gaps and push the gel-like interstitial matrix aside. In the reactive stroma of breast tumours, the ECM content between collagen bundles is poorly contrasted by immunohistochemistry or non-linear (multi-photon) microscopy; thus, their composition and mechanical properties in native state are unknown. Whereas the dimensions and function of the inter-fibre gaps in vivo likely provide space for invasion by cell squeezing, pulling, and pushing in collagen-based in vitro models 132, their guidance function in desmoplastic stroma remains to be shown. Histologically, invading cancer cells seem to ‘respect’ cell and ECM boundaries, resulting in proteolytic tissue destruction as a late consequence rather than as a prerequisite of invasion.
As a further shared event between brain and connective tissue invasion, tumour cells themselves, or the reactive tumour stroma, provide pro-migratory tissue conditioning. In breast carcinoma, the invasion-promoting properties of fibrillar collagen are increased by pro-migratory proteins deposited into the existing scaffold. In glioma, the proteolytic removal of otherwise anti-migratory activity is required for white matter infiltration, and it is not known whether similar mechanisms are involved in the invasion of peripheral connective tissue.
Despite extensive in vitro and in vivo investigations, for most invasion routes the combined molecular and physical mechanisms are not clear, and likely several complementary mechanisms converge to influence invasion outcome. Whereas molecular pathways have been studied extensively, the physical guidance mechanisms, including mandatory surface receptors and proteases required for contact guidance, remain poorly addressed. Thus, a mechanistic understanding of guidance, including its central signalling or mechanocoupling nodes that could be targeted, is required to design effective inhibition approaches, besides the targeting of integrins and other adhesion systems. It is likely that, even with integrins inhibited, major invasion pathways are still active in cancer cells, similar to leukocytes 151; therefore, models are needed to identify additive effects and compensation in order to establish rational targeting of tissue invasion.
Structural and molecular insight into tissue invasion by cancer cells is further mandatory for the development of experimental in vitro and in vivo models exploring the mechanisms of cancer invasion. Whereas for breast cancer invasion, protein-scaffold-based collagen, basement membrane, and interconnecting accessory protein-containing ECM models appear sufficient as they reflect the natural main routes and ligands, glioma invasion is likely more complex and requires the simultaneous presence of cell and ECM scaffolds in an interconnected manner. The design of an appropriate cell environment comprising the main tissue components critical for each cancer cell invasion route in vivo will improve the clinically relevant identification of key molecules that guide cancer cell invasion, and dissect the relative contribution of physical versus molecular guidance.
We thank Pieter Wesseling, Peter Bult, and Han van Krieken for providing human brain and mammary gland samples and for helpful discussion. This work was supported by Pieken in the Delta Oest Nederlad (PID082022) and the European Union, FP7 European Tissue Transmigration Training Network (T3Net 237946).
Author contribution statement
PG and OI performed the experiments. All the authors wrote the manuscript.