Tissues are dynamically shaped by bidirectional communication between resident cells and the extracellular matrix (ECM) through cell-matrix interactions and ECM remodelling. Tumours leverage ECM remodelling to create a microenvironment that promotes tumourigenesis and metastasis. In this review, we focus on how tumour and tumour-associated stromal cells deposit, biochemically and biophysically modify, and degrade tumour-associated ECM. These tumour-driven changes support tumour growth, increase migration of tumour cells, and remodel the ECM in distant organs to allow for metastatic progression. A better understanding of the underlying mechanisms of tumourigenic ECM remodelling is crucial for developing therapeutic treatments for patients. Tumors are more than cancer cells — the extracellular matrix is a protein structure that organizes all tissues and is altered in cancer. Here, the authors review recent progress in understanding how the cancer cells and tumor-associated stroma cells remodel the extracellular matrix to drive tumor growth and metastasis. These ECM components are modified post-translationally by an array of secreted remodelling enzymes, such as oxidases and proteases. In addition, the ECM binds soluble factors, such as growth factors and other ECM-associated proteins. Cell surface receptors interact with ECM components and ECM-bound factors to mediate cell adhesion and cell signalling thereby regulating processes as diverse as proliferation, differentiation, migration and apoptosis2. ECM can also demonstrate very different mechanical and topographical properties, which, importantly, can influence cell fate and function via different mechanosignalling routes3.Cellular phenotypes and molecular functions are fundamentally dependent on signals from outside the cell such as the interactions with the extracellular matrix (ECM). The core matrisome is composed of ~300 unique matrix macromolecules and can be classified into collagens, proteoglycans  and glycoproteins 4. In cancer, remodelling of the interstitial ECM induces a broad range of biophysical and biochemical changes affecting cell signalling, ECM stiffness, cell migration and tumour progression5. In contrast, the basement membrane is a more stable, sheet-like, dense structure that lines the basal surface of, for example, epithelial and endothelial cells, surrounds muscle cells and adipocytes6, and separates tissues into different, well-organised compartments. The basement membrane consists mainly of collagen IV and laminins, which are interconnected through different network-bridging proteins such as nidogen and heparan sulphate proteoglycans (HSPGs)7. Binding of cells to the basement membrane is essential for establishing epithelial cell polarity and is crucial for many developmental processes and maintenance of tissue homoeostasis8. Remodelling of the basement membrane is required for cancer cells to invade stromal tissue and become a malignant tumour9.The ECM has two main forms, which differ in function, composition and location. The interstitial matrix forms porous three-dimensional networks around cells that interconnect cells in the stroma and can connect to the basement membrane, which is the other form of ECM structure. The interstitial matrix guarantees the structural integrity of tissues and organs but also modulates processes such as cell differentiation and migration. The protein composition of the interstitial matrix mainly includes collagens I, III, V, etc., fibronectin and elastin. Abundance and composition of the interstitial matrix vary between tissue types, between microenvironments within the same tissue and can be remodelled in response to force stress or trauma such as wound repair or tissue regeneration1, change overall abundance, concentration, structure and organisation of individual ECM components, thereby affecting the three-dimensional spatial topology of the matrix around cells, its biochemical and biophysical properties and consequently its effect on cell fate. ECM remodelling is an essential and tightly regulated physiological process in development and in restoring tissue homoeostasis during wound repair10. However, it is not surprising that cells dysregulate this process in pathologic conditions such as inflammatory diseases, tissue fibrosis, and cancer11. Recent research highlights the importance of the tumour-mediated systemic aberrations of the ECM for the establishment of metastasis. In this review, we discuss remodelling mechanisms of extracellular matrices and the implications of these mechanisms during cancer development, and describe recent concepts of ECM remodelling shaping tissues for tumour cells to metastasise. Increasing understanding of these processes opens up the possibilities of therapeutic approaches to target the aberrant ECM and/or the underlying pathologic mechanisms of its remodelling and prevent malignancy.Complex ECM remodelling processes, involving over 700 proteinsChanges in the ECM are a result of different remodelling mechanisms that can be divided into four main processes: (1) ECM deposition, which changes the abundance and composition of ECM components, thereby affecting biochemical and mechanical ECM properties; (2) chemical modification at the post-translational level, which alters the biochemical properties and structural characteristics of the ECM Fig. ; (3) pro12.Tissue homoeostasis is dependent on the tight regulation between ECM deposition, modification, degradation and organisation that give rise to the biochemical and biophysical ECM properties. Changing single elements can turn over the delicate balance of ECM remodelling events. ECM alterations have implications on complex cellular signalling networks since ECM components serve as ligands for various cell surface receptors such as integrins, syndecans and receptor tyrosine kinases. Thus, it is not surprising that cancer cells and tumour-associated stromal cells modify all four ECM remodelling mechanisms, creating a cancer-supporting matrix that actively contributes to the pathology of the tumour Fig. 12.Fig. 215. However, both in healthy tissue and tumours, the major producers of ECM in the interstitial matrix are fibroblasts, especially when activated and transformed to myofibroblasts; chondrocytes and osteoblasts are also significant, depending on tissue and context16. Myofibroblasts have a combined phenotypic characteristic of fibroblasts and smooth muscle cells, whereby they secrete ECM and exert contractile functions enabling mechanical alterations of the topology of the ECM, respectively. They can originate from different cell types and are mostly characterised by de novo expression of α-smooth muscle actin (α-SMA), although there are other markers of myofibroblasts in different conditions (reviewed in refs. 17). Activation of myofibroblasts is triggered by various pro-inflammatory factors18, most importantly transforming growth factor β (TGF-β)19. ECM remodelling by myofibroblasts is an essential physiologic process, for example in tissue regeneration and wound repair, which includes their inactivation after restoring homoeostasis. However, sustained inflammatory stimuli and subsequent TGF-β secretion by immune cells such as macrophages and tumour cells can generate deregulated, hyperproliferative and overactive myofibroblasts that are the major culprits in pathologic conditions such as fibrosis and cancer20.In principle, ECM components can be produced and deposited by any cell. Tumour cells themselves show altered expression of ECM components, such as collagens I and III and ECM-modifying enzymes, such as lysyl oxidases (LOX) and LOX-like proteins (LOXL) Fig. 12–15. Ho21. Recent evidence indicates that several of these stromal activation signals can be secreted in exosomes, which may impact the activation and differentiation to a myofibroblast phenotype, as exosomal TGF-β elevates FGF-2 production in fibroblasts compared to non-exosomal TGF-β22.Tumour cells primarily orchestrate the recruitment and activation of stromal cells that are the major depositors of ECM components in the tumour microenvironment Fig.  by the s24. These activated stromal cells are of multiple origins, prominently including tissue-resident or bone marrow-derived fibroblasts25, but also, for example, adipocytes26. Interestingly, stress-induced adrenergic signalling in ovarian cancer cells can also activate CAFs to deposit elevated amounts of collagen that can be inhibited by β-blockers27. Furthermore, in aged tissue, stromal cells show alterations in ECM remodelling properties such as changes in the expression of matrix metalloproteinases (MMPs) and chemokines that support tumour development and metastasis28. In melanoma, aged fibroblasts express lower amounts of the ECM-modifying protein hyaluronan and proteoglycan link protein 1 (HAPLN1) that leads to reduced integrity of lymphatic vasculature and increased metastasis29. Changes in the tumour ECM can also further activate stromal cells in a feed-forward mechanism31.The tumour-derived activation factors induce the differentiation of stromal cells towards so-called cancer-associated fibroblasts (CAFs), which function as myofibroblasts and remodel the ECM to support tumour growth34; this fosters the recruitment and activation of immune cells, which, as discussed below, contribute to the establishment of the cancer niche.ECM deposition by CAFs may depend on their spatial arrangement in the tumour. In pancreatic ductal adenocarcinoma (PDAC), CAFs in direct interaction with adjacent tumour cells show active TGF-β signalling and deposit collagen, whereas CAFs at a greater distance are also activated by the tumour cells, but are unresponsive to TGF-β, deposit HA and establish a tumourigenic, pro-inflammatory environment through the expression of cytokines such as IL-6, or antigen presentation36. CAFs also may have tumour-suppressive functions such as restraining tumour vasculature or supporting immune surveillance, since the ablation of CAFs in murine models of PDAC led to a more aggressive tumour phenotype, which also correlates with patient data38. Hence, the heterogeneity, spatial distribution and potential plastic nature of CAFs need to be considered carefully in the context of targeting the stromal compartment of tumours.It is important to note that subpopulations of CAFs additionally support tumour growth through mechanisms independent of ECM remodelling, such as promoting cancer stemness or preventing cancer cell recognition by T-cells39). The tumour-resistance of the longest-lived rodent, the naked mole-rat, involves the expression of a unique HA with high molecular mass (HMM-HA) as a major ECM component. HMM-HA signalling through the CD44 receptor activates the expression of key tumour suppressor genes40, leading to a hypersensitive cell-cycle arrest, a common mechanism of tumour suppression41. In contrast, high levels of HA, and in particular small HA oligosaccharides (LMM-HA), are associated with poor prognosis in several tumours such as colorectal, breast and prostate cancer46. Here, dysregulated HA synthetase and HA-degrading hyaluronidase lead to the accumulation of LMM-HA47. LMM-HA directly interacts with cell surface receptors regulating pro-tumourigenic signalling cascades48, including glycolysis, the main source of energy in tumours, and promotes migration49. LMM-HA signalling through CD44 also increases the resistance to cellular stress and thereby may promote tumour development50.ECM components possess both tumour-suppressing and tumour-promoting properties. For example, depending on its molecular weight hyaluronan (HA) functions as a tumour suppressor or a tumour promoter , to modify FGF-2 signalling leading to increased cell proliferation57.The most common tumourigenic alteration of ECM homoeostasis is an increased deposition of fibrillar collagen58. Unique ECM signatures that are characteristic of some tumours change during tumour progression and are predictive of clinical outcomes61. The combined pathologic alterations of the ECM provide a protein fingerprint by the release of cancer-specific ECM fragments into the circulation that may have diagnostic implications as it was shown for lung, ovarian, breast and colorectal cancer64. Moreover, even before tumour development, increased deposition of collagen I and proteoglycans lead to increased breast density, which is the greatest independent risk factor for the development of breast cancer  deposition and further cross-linking and alignment events which all together also result in stiffer breast tissue. A complex interplay of hormonal, inflammatory and environmental factors may all influence breast density277. So far, the detailed underlying mechanisms that trigger these ECM changes and how breast density increases breast cancer risk278 are unclear.In the breast, mammographic density is indicative of a higher risk for breast cancer development, which is used for preventive screening279. Whether or not pregnancy protects or promotes breast cancer is still controversial and largely depends on the age of the woman during her first birth280. During pregnancy, the breast epithelium and tissue matrix are intensively remodelled to prepare for lactation. After nursing, the lactating glands and surrounding breast stroma are remodelled to its pre-pregnancy-like state in a process called involution. These remodelling processes are exceptionally rapid and comprehensive. Each state is characterised by a massive change in ECM abundance and composition. Following pregnancy, the breast retains a more developed lobular architecture281, which may also affect the proportion of epithelial cell types in the breast282. Furthermore, even the fully regressed mammary gland shows a higher abundance of ECM components, in particular collagen I, compared to the pre-pregnancy (nulliparous) state283. During involution, massive ECM proteolysis mediated by various upregulated MMPs leads to the disruption and degradation of the basement membrane, thereby detaching epithelial cells, which triggers cell death of unnecessary secretory mammary epithelium. High MMP activity also leads to the release of bioactive matrix proteins, matrikines, and growth factors and the recruitment of ECM-clearing immune cells284. Interestingly, ECM remodelling during involution shows high similarities to processes during inflammation and wound healing, which was also shown to provide a bonafide environment for cancer development through pro-inflammatory TGF-β285. Together, these inflammatory conditions and the loss of the barrier function of the basement membrane temporally create a pro-tumourigenic environment that may increase the risk for more aggressive breast cancer280.Breast cancer risk is also influenced by the fundamental changes that the mammary gland undergoes during different reproductive states, which are characterised by profound ECM remodelling processes68). Post-translational modifications of ECM components affect matrix interactions with other molecules and cellular receptors, localisation within the tissue and ECM degradation69. We elaborate below how several key post-translational modifications are altered in a cancerous environment.Once synthesised, the ECM proteins undergo post-translational modifications (PTMs) inside and outside the cell, which enhances their complexity and three-dimensional organisation . Tissue transglutaminase 2 (TG2) cross-links ECM molecules such as fibronectin, HSPG, fibrinogen and collagen IV68 by the transamidation of glutamine residues to the amino group of a lysine residue of another protein chain. This transamidation process results in the formation of covalent N-γ-glutaminyl-ɛ-lysyl-isopeptide bonds, which are resistant to proteolytic degradation69.For example, collagen chains are synthesised as long precursors (procollagens) that are further modified by various post-translational modifications, for example, glycosylation of their lysine and hydroxylysine residues by addition of galactose and glucose; and hydroxylation of lysine residues through lysyl hydroxylases (encoded by procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD) genes)ess Fig. 73–75 , binding of growth factors, and mechanoresponses84. Interestingly, glycosylation patterns of surface proteins frequently change in cancer and may foster pathological cell behaviours86. Tumour cells upregulate bulky glycoproteins on the cell surface. The resulting bulky glycocalyx can apply tension to ECM-bound integrins, which are important receptors for ECM components, causing integrin clustering and thereby increasing pro-tumourigenic integrin signalling84.The majority of extracellular and transmembrane proteins are glycosylated. Transmembrane glycoproteins function as receptors for ECM molecules; for example, CD44 is heavily glycosylated and its ECM ligand HA is a glycosaminoglycan. Other glycosylated ECM molecules include HSPGs that bind growth factors and modify receptor tyrosine kinase signalling89. Sulf1 is downregulated in ovarian cancer and a subset of hepatocellular carcinoma, which prevents Sulf1-mediated inactivation of receptor tyrosine kinase signalling91. However, Sulf2 supports Wnt and PDGFRα signalling in vitro93. Since Sulfs act extracellularly, it is likely that stromal cells contribute to Sulf levels in the tumour ECM. Although both Sulf isotypes show similar substrate specificity, conflicting studies, in particular for Sulf1, suggest that the functional consequences of HSPG modification may depend on experimental conditions (in vitro vs. in vivo), context and tumour or tumour subtype.Modifications of ECM components also affect their capacity of binding to growth factors. Extracellular HSPGs regulate the activity of various receptor tyrosine kinase signalling pathways by their ability to store various growth factors, the release of which is regulated by extracellular modification enzymes such as endosulphatases (Sulfs). Sulfs, which are dysregulated in many cancers, modify the sulphation pattern of HSPGs and affect the binding of growth factors. Sulf1 can act as a tumour suppressor, whereas Sulf2 activates pro-tumourigenic signalling pathways94). These proteases are secreted primarily by stromal cells, including recruited bone marrow-derived cells, as well as by cancer cells 95. The substrates of the different proteases are still not completely understood. Recent advances in the sensitivity of proteomics techniques allow targeted degradomics studies to identify precise proteolytic cleavage targets from protease-substrate interactions97. These studies revealed that proteases cleave different substrates when they are bound to the cell membrane compared to their soluble form. ADAM10 and 17 are membrane-anchored and their ectodomains can be released by ADAM8. The soluble ectodomains show different target specificity98.The ECM is cleaved and degraded by target-specific proteases such as MMPs, disintegrin and metalloproteinases (ADAMs), disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS), and proteases that specifically cleave at serine, cysteine and threonine residues . Third, ECM-binding of soluble signalling molecules such as growth factors makes them insoluble and inactive and action of proteases liberate them. Fibronectin, for example, binds insulin-like growth factor binding-protein-3, FGF-2 and VEGF-A with high affinity105. The enhanced protease activity in tumours causes subsequent ECM degradation that releases the ECM-bound growth factors and thereby increases their bioavailability . Matrikines play an important role in angiogenesis and balance the angiogenic switch through promoting and inhibiting properties (reviewed in ref. 108). Anti-angiogenic functions are described for several NC1 domains of basement membrane collagens110, such as the matrikine endostatin111. A specific domain of the α3 chain of type IV collagen has anti-tumour effects through the inhibition of the expression of membrane-type 1-matrix metalloproteinase (MT1-MMP or MMP14) and integrin β3, which leads to impaired migratory abilities of tumour cells112. On the other hand, various elastin-derived matrikines, such as Val-Gly-Val-Ala-Pro-Gly (VGVAPG) or Ala-Gly-Val-Pro-Gly-Leu-Gly-Val-Gly (AGVPGLGVG) promote tumour progression113. These ECM fragments are products of the degradation of elastin through different proteolytic enzymes (elastases)114 and MMPs115. These matrikines can, in turn, also induce MMP expression and activation, including MT1-MMP and MMP-2, which would explain their tumour-promoting properties116.Last, the cleavage of long ECM components produces bioactive, shorter fragments with distinct functions that can be pro- or anti-tumourigenic compared to the full-length ECM component. These fragments often contain structures similar to chemokines or cytokines and are therefore termed matrikines . Cleavage patterns of FRET-based polypeptides thereby reflect specific enzyme activities when compared to purified proteases as standard. This method allows the detection of enhanced protease activity in patient samples such as cerebrospinal fluid, which is indicative of neoplastic meningitis and metastasis to the brain9, and MT1-MMP promotes basal extrusion during epithelial-to-mesenchymal transition (EMT) and basal mitosis noncatalytically118.It is important to note that, in addition to proteolysis of ECM, MMPs have significant non-proteolytic functions that can alter tumour progression. For example, MMP-3 binds and inhibits noncanonical Wnt5b, resulting in increased canonical Wnt signallingRemodelling of the tumour ECM impacts every stage of tumourigenesis. In this section we describe these effects on the development of a primary tumour, including how a tumourigenic ECM induces pro-tumour signalling in cancer cells, helps to create an immunosuppressive environment and further supports the progression of the tumour to grow, invade stromal tissue and promote vascularisation.120. Syndecans are cell surface-bound HSPGs that interact with integrins to support focal adhesion formation and migration80. Integrin-mediated signalling includes pro-survival and pro-apoptotic pathways and their crosstalk with growth factor receptors mediates cellular signalling (reviewed in refs. 123).ECM components bind to integrins which are heterodimeric transmembrane receptors with bidirectional signalling that couple them to the actin cytoskeleton within the cell. Force-mediated ECM-integrin interaction induces various downstream signalling events, known as mechanosignalling Fig. . Repeate125. The stiff matrix of the tumour leads to an altered mechanosignalling. High ECM deposition and increased stiffness combined with integrin overexpression in various cancers126 trigger tumour promotion (reviewed in Desgrosellier and Cheresh121).As a result of the alterations in the ECM, tumours often are stiffer than normal tissue, which can be applied in clinical diagnosis127. However, in PDAC, TGF-β loss in tumour cells is associated with increased desmoplasia and enhanced matrix tension. The pronounced desmoplasia triggers β1-integrin mechanosignalling that promotes tumour progression through STAT3 activation128. These observations show the complexity of TGF-β signalling in tumour and stromal cells, which can have pro- and anti-tumourigenic functions depending on the context.As mentioned above, tumour-secreted TGF-β activates the differentiation of stromal cells into ECM-remodelling CAFs. Additionally, TGF-β secreted by CAFs can induce STAT3 signalling in tumour cells129. The high abundance of collagen in the tumour microenvironment triggers the activation of the receptor tyrosine kinase DDR receptors. DDR1 expressed by gastric cancer cells supports invasion and tumour metastasis130. In breast cancer, the interaction of collagen I with DDR1 expressed by tumour cells leads to the induction of stemness-like signalling through the activation of STAT3, which is crucial to mediate the metastatic outgrowth131. Surprisingly, DDR1 deletion in a breast cancer mouse model increases ECM deposition and promotes tumour aggressiveness and metastasis132. DDR2 deficiency in hepatic stellate cells triggers differentiation of CAFs and fosters the establishment of a metastatic niche133. These studies suggest that DDR1 and DDR2 are expressed by tumour and stromal cells and depending on receptor isotype, cell type and context can either support or suppress tumour development132.Collagens also bind to unique transmembrane receptor tyrosine kinases, known as Discoidin Domain Receptors (DDR1 and DDR2). They induce cellular signalling in conjunction with other receptors such as integrins and Notch134. Components of the ECM can function directly as inflammatory stimuli, known as danger-associated molecular patterns (DAMPs)135 that induce immune responses through the interactions with pattern recognition receptors (PRRs) expressed by immune cells136. ECM-degrading proteases liberate these pro-inflammatory ECM components such as LMM-HA and biglycan, as well as matrix-bound growth factors and cytokines137. Biglycan, for example, activates PRRs including Toll-like receptor 4 (TLR4) and TLR2 expressed by macrophages that drive the expression of TNF-α and macrophage inflammatory protein–2 (MIP-2/CXCL2)138. Matrikines may also serve as DAMPs in tumours. Versican is degraded by ADAMTS proteases to reveal a bioactive fragment called versikine139. Increased intratumoural levels of versikine leads to increased tumour infiltration of CD8+ T-cells in colorectal cancer140 and myeloma141. Increased CD8+ T-cell infiltration results from versikine-induced expression of the transcription factor Interferon Regulatory Factor 8 (IRF8) in macrophages and enhances generation of CD103+ CD11chiMHCIIhi conventional dendritic cells (cDC) in a mouse model140.The high ECM remodelling activity mediated by tumour cells and CAFs contributes to an inflammatory tumour environment142 and constitute the major source of angiogenesis-inducing MMP-9  and tumour-associated neutrophils (TANs), are important sources for ECM remodelling proteases in the primary tumour environment Fig.  and metaP-9 Fig. 143. This12). LOX-mediated collagen cross-linking stiffens the ECM in a murine breast cancer model, which triggers an integrin-dependent invasive phenotype . Although under debate, these ECM migration barriers largely impair immune surveillance of tumours and have been associated with poor clinical outcome166.Proteolysis of ECM components is crucial for the active movement of cells through the interstitial matrix as it opens migratory tracks and reduces mechanical stress on the migrating cellsoma Fig. 162,163. 169. MT1-MMP is usually localised on invasive actin-rich cell structures called invadopodia170. There is now substantial evidence that these processes are supported by exosomes. Exosomes have been shown to promote maturation of invadopodia and ECM degradation via the extracellular delivery of MT1-MMP and other proteinases171. Additionally, integrins on the surface of secreted exosomes bind to fibronectin which thereby act as bridging molecules connecting integrins expressed on tumour cells. The local secretion of exosomes may, therefore, induce cellular integrin clustering that triggers adhesion assembly and result in more effective and directed cell migration172.In regions of dense collagen fibres in the tumour environment, pericellular proteolysis of the interstitial matrix is important for active cell migration and controlled by MMPs, particularly MT1-MMP on tumour cells and CAFs, which degrade the collagen migration barriers20. They are positioned in front of invading cancer cells and clear the interstitial matrix through proteolytic- and force-mediated remodelling processes. CAFs interact with collagen-rich ECM through the integrins α3 and α5, and Rho-mediated regulation of myosin light chain activity. These interactions apply force to the ECM thereby aligning the collagen fibres174. Additionally, fibronectin binding to integrins, such as integrins αvβ3 and α5β1, on CAFs allows receptor clustering, which applies force on fibronectin dimers changing their conformation and exposing binding sites to support self-assembly into fibrils4. The force-mediated fibronectin assembly creates anisotropic fibre orientation and can open gaps in the matrix that allow cell migration175. Collagen linearization can also be mediated by tumour-secreted factors such as the WNT1 inducible signalling pathway gene (WISP1)176, which is triggered by TGF-β.Owing to their high ECM-remodelling capacity, CAFs may serve as leader cells for tumour cell migration Fig. 20. They 177. The specific architecture of these collagen tracks guides tumour cells to travel longer distances along the aligned collagen fibres by forming fewer but longer protrusions or to use amoeboid blebbing for optimal migration. The aligned matrix thereby supports, in particular, the directed migration of small, plastic tumour cells with stem-like characteristics180. This may explain why the signature of highly aligned collagen fibres is associated with poor prognosis79.These processes create stable migratory tracks that are used for the efficient, proteolysis-independent collective invasion of the tumour cells that follow. Tumour cell migration is also actively guided by CAFs through force triggered E-cadherin/N-cadherin adhesionOverall, the physical properties of the tumour ECM together with its composition, density and the specific architecture of aligned fibres provide optimal migratory conditions for effective tumour cell dissemination and promote aggressive tumour phenotypes.181). Hypoxia triggers cellular signalling mediated by the hypoxia-inducible transcription factor (HIF‐1). HIF-1 also promotes ECM remodelling through the regulation of various MMPs, for example, an upregulation of MMP-2, MMP-9 and MT1-MMP183, and activates collagen fibre alignment via the induction of various collagen-modifying enzymes, like prolyl-4-hydroxylases (P4HA1 and P4HA2), PLOD1 and PLOD2, LOX and LOX-like proteins 186 in CAFs , and additionally upregulates integrins, thereby contributing to enhanced pro-tumourigenic mechanosignalling187. Deposition of collagen I by mesothelial cells is also enriched by a hypoxic tumour-mesothelial niche, which promotes the metastasis of ovarian cancer188.During tumour progression, rapid growth leads to a tissue architecture with tumour cells located distant from blood vessels with diffusion-limited oxygen and nutrient supply . For example, periostin has pro-angiogenic functions194; tenascin-C and fibronectin have both pro- and anti-angiogenic functions depending on context195 (reviewed in Obberghen-Schilling196), and thrombospondin-1 inhibits MMP-9 activation and angiogenesis and thereby suppresses spontaneous tumour development197 (reviewed in Lawler and Lawler198).The state of hypoxia is a key driver for angiogenesis. HIF-1 expression in hypoxic tumour cells induces the angiogenic switch through the upregulation and activation of pro-angiogenic proteins such as VEGF to recruit endothelial cells and build new vesselsECM Fig. 142. The 199. Vasculogenic mimicry of melanoma cells requires matrix remodelling through the expression of MMP-2, MT1-MMP and the deposition and cleavage of a specific laminin chain (γ2 chain of laminin 332)200. Interestingly, tumour-associated macrophages can also form vascular-like networks201. Besides providing an adequate blood supply for the tumour, vasculogenic mimicry is associated with an aggressive tumour phenotype and promotes metastasis199. Further research is needed to elucidate the underlying mechanisms of how the biochemical and biophysical properties of the ECM induce phenotypical changes in tumour cells and lead to formation of vascular-like structures.The architecture of dense collagen in tumours also triggers the formation of interconnected networks of cancer cells. These tumour cells form ECM-rich vasculature-like structures, in a process called vasculogenic mimicry, that supply the tumour with blood without inducing angiogenesis Fig. . A speciTaken together, the characteristics of a tumour microenvironment maintains and promotes itself through complex interdependent pathways that lead to enhanced angiogenesis and invasiveness with the ultimate goal of metastasising to a secondary organ.Crucial for cancer therapy is the prevention and elimination of cancer metastasis, which accounts for the majority of cancer-related deaths. Extensive research during the last decade has unravelled the significance of ECM remodelling at each stage of metastasis development, from surviving in circulation, to forming the pre-metastatic and metastatic niches Fig. .Fig. 4EC, TIMP-2, the proteoglycan decorin (Dcn), the glycoprotein osteonectin (Sparc), and fibronectin, as revealed by single-cell RNA-sequencing of pancreatic CTCs  through interaction with integrin-β1, expressed by both CTCs and NETs  in the blood upregulate expression of common stroma-derived ECM proteins, such as collagens TCs Fig. 202,203. ETs Fig. 206. The 207. Of note, at the metastatic site, ECM composition and remodelling processes are distinct from both the primary tumour and the healthy tissue210 (reviewed in Haye and Erler211). The most common alteration of the ECM in the primary tumour environment is increased collagen deposition. In contrast, mainly fibronectin is involved in the formation of the pre-metastatic niche along with glycoproteins and proteoglycans such as tenascin C, osteopontin and versican211.Potential sites of metastasis possess organ-specific microenvironments, which are very different from that of the primary tumour. The primary tumour triggers the formation of a favourable microenvironment at the distant tissue, which includes ECM remodelling, even before the presence of metastatic tumour cells, creating a so-called pre-metastatic niche214 and potentially also during pre-metastatic niche establishment. However, PAD4 secreted by colorectal cancer cells induces citrullination of collagen I in the liver215, which promotes the adhesion of disseminated tumour cells to the liver tissue. In the bone, tumour-derived factors create either an osteolytic (bone-degrading) or osteoplastic (bone-forming) pre-metastatic environment. The more common osteolytic metastasis occurs often in breast cancers as well as other cancers types, where bone homoeostasis is modulated by promoting the activity of bone-resorbing osteoclasts by factors such as IL-6, EGF-like growth factors, TGF-α, NF-κB ligand (RANKL) but also through tumour-derived LOX219. Moreover, ECM degradation is essential for releasing these soluble factors, such as the liberation of EGF-like growth factors and TGF-α in the primary tumour is mediated by MMP-1 and ADAMTS1219. Osteolytic bone metastasis includes the degradation of collagen I, the most common ECM component in the bone. Collagens can also undergo spontaneous non-enzymatic isomerization. Newly synthesised collagen is non-isomerised and is increased in bone metastasis to compensate for the high ECM turnover. Thus, the non-isomerised C-telopeptide of collagen I (α-CTX-I) can be used as a biomarker for bone metastasis from breast and prostate cancers instead of the isomerised form (β-CTX-I)220. In contrast, growth factors produced by prostate cancer cells trigger osteoblast differentiation, creating an osteoblastic lesion in the bone for tumour colonisation222. However, osteolytic events may also occur in osteoblastic bone metastasis since bone matrix-bound growth factors, which are released and activated upon ECM degradation, may be important for metastatic cell proliferation.Primary tumour-derived factors activate stromal cells in the pre-metastatic site to secrete new ECM molecules or remodel and modify the ECM directly Fig. . Citrull224. For example, hypoxia-induced LOX expressed by the primary tumour acts at the metastatic site, where it cross-links collagen IV, which provides the adhesion for bone marrow-derived cells, as shown in breast cancer225. With their potent ECM remodelling capabilities, bone marrow-derived cells, in turn, promote colonisation of the metastatic tumour cells227. In another example, colorectal cancer metastasises preferentially to the liver, which correlates with high systemic levels of the MMP inhibitor, TIMP-1. Although TIMP-1 inhibits ECM turnover, it has been implicated in the recruitment of neutrophils to the liver, which then remodel the tissue establishing a tumour-permissive niche228. CTCs may contribute to upregulated TIMP-1 levels229.Signals from the primary tumour also stimulate the recruitment and adhesion of specific bone marrow-derived cells  to the pre-metastatic niche Fig. 223,224. 230. Endothelial cells also deposit ECM components at the metastatic tissue. Mature endothelial cells can suppress breast cancer growth and mediate their quiescence through the secretion of thrombospondin-1. However, an MMP-mediated disrupted vasculature at the metastatic site230 results in the induction of newly sprouting endothelial tip cells that produce TGF-β1 and ECM molecules involved in pre-metastatic niche development, such as periostin, tenascin, versican and fibronectin231.Cancer cell-derived MMPs, such as MMP-3 and MMP-10, induce vascular disruption at the pre-metastatic site supporting the extravasation of CTCs and enhance ECM turnover, which opens up the way for infiltrating bone marrow-derived cells and tumour cells233.Likely, many of the tumour-derived factors acting in distant organs to prime the metastatic site for tumour cell colonisation may be transported in the form of exosomes. In their lumens, exosomes transport various proteins, such as proteases, and nucleic acids, mainly miRNAs, which are effective at the target site. The composition of exosomes is heterogeneous depending on cell type, and functional or developmental state of the cell and subject of intense research efforts234, suggesting that organ-specific tissue environments including their particular ECM may influence the growth of metastasis235. Surprisingly, tumour-derived exosomes may mediate the non-random pattern of metastasis and promote pre-metastatic niche development. Specific groups of integrins expressed on tumour-derived exosomes dictate their adhesion in a particular organ and thereby may determine the site of the future metastasis. Integrins α6β4 and α6β1 guide exosomes to the lung to target fibroblasts and epithelial cells, while exosomal integrin αvβ5 determines their transport to the liver for the uptake by Kupffer cells236. To date, it is unclear to what extent this role depends on ECM remodelling in the target tissue and which specific alterations in the ECM cause the organotropic exosome distribution. However, these tumour-derived exosomes create pro-inflammatory and fibrotic environments that recruit bone marrow-derived cells, further promoting ECM remodelling to host metastatic cells. Pancreatic cancer-derived exosomes contain macrophage migration inhibitory factor (MIF) that induces TGF-β secretion by Kupffer cells in the liver, which stimulates an enhanced fibronectin deposition by neighbouring hepatic stellate cells. The resulting fibrotic environment recruits bone marrow-derived macrophages237 and ultimately fosters liver metastasis. These studies link the well-known preferred organotropic metastasis pattern of different tumour entities with the novel concept of pre-metastatic niche establishment through organ-specific, exosome-mediated intercellular communication cascades.There is now mounting evidence that metastases of certain cancers show a tissue specificity238.To form metastases, CTCs need to extravasate from the circulation into the new tissue, a process that is dependent on ECM remodelling. Endothelial cells in the liver of colorectal cancer patients deposit and assemble fibrillar fibronectin that allows the attachment of CTCs to the vascular lumen, induces integrin-dependent focal adhesion and eventually the extravasation into the liver tissue Fig. 238.239. Glycoproteins, such as osteopontin, periostin and tenascin C are deposited either by infiltrating tumour cells or by stromal cells at the metastatic site and are important for the colonisation of tumour cells by inducing stemness-like signalling pathways242. Breast cancer cells in the lung produce tenascin C, which triggers Notch and Wnt signalling240. Periostin deposition by activated lung fibroblasts also increases Wnt signalling in metastatic breast cancer cells. The expression of osteopontin and tenascin C depends on the c-Jun transcription factor. Interestingly, chemotherapeutic treatment can activate the c-Jun N-terminal kinase (JNK) pathway and the deposition of these glycoproteins, fuelling metastatic progression243. CTCs exhibit mesenchymal characteristics, whereas primary and established metastatic tumour cells show more epithelial phenotypes242. Versican deposition by bone marrow-derived cells promotes metastasis by inducing the transition from mesenchymal to epithelial phenotype in metastatic cells244, suggesting that this proteoglycan may promote the establishment and progression of metastasis242.After colonisation, stromal and metastatic cells both contribute to create a metastatic niche and actively change the ECM to promote metastastic growth. Interestingly, ECM remodelling of the metastatic niche is different for the same tumour at different sites, indicating tissue tropism. Unique combinations of matrisomal and ECM-associated proteins are deposited by stromal and cancer cells, respectively, at brain, lung, liver and bone metastases in a breast cancer mouse model245. In an inflammatory environment, neutrophil extracellular traps bind to extracellular laminin and, owing to the presence of laminin-degrading proteases like neutrophil elastase and MMP-9, induce laminin degradation. This proteolytic processing results in the exposure of a specific laminin epitope. This matrikine triggers an integrin-mediated signalling cascade, which leads to the awakening of dormant cancer cells at sites of metastasis and their subsequent proliferation  can provide a quantitative map of the proteomic landscape of single cells in primary tumours and metastases253. With these advanced technologies, biomedical research can take the next step in uncovering the comprehensive interactions of individual cells within their environment, which can lead to the development of more effective ECM-targeted therapies.Progress in investigating cells in their complex intervening three-dimensional networks has resulted in the development of various organoid models that may provide better in vitro tools to investigate the effects of specific matrix perturbations on complex cell–cell and cell–matrix interactionsBox 2 Therapies targeting ECM remodelling in cancer and metastasis286. Tumourigenic ECM modifications and stiffness can also influence responses to anticancer agents in vitro287. Therapies that resolve such stiffness may allow for improved drug penetration to the tumour cells. On the other hand, ECM degradation has been linked to cancer cell migration, invasion and induction of angiogenesis288. As such, a regulated approach for the administration of therapies for either ECM stiffness or ECM degradation is critical for effectively controlling the effects of either without inducing the other.ECM stiffness increases cancer cell proliferation and survival, and induces immunosuppression, even during therapy289.To date, several promising therapies targeting the ECM Table  have not121. Another therapeutic approach being explored for targeting ECM remodelling with promising but so far limited effect is targeting the TGF-β pathway, known for its role in inducing increased collagen I deposition and decreased degradation when activated. This pathway is being targeted at the ligand, receptor-ligand and intracellular levels with trabedersen, an antisense oligonucleotide that targets TGF-β2; fresolimumab, an anti-TGF-β monoclonal antibody that prevents ligand-receptor interaction; and galunisertib, a small molecule inhibitor of TGF-βRI that prevents signal transduction290. MMPIs such as marimastat, incyclinide, etc., that were designed to target multiple MMPs and control ECM degradation, did not improve patient survival due to lack of specificity and severe side effects291. Studies suggest that targeting individual MMPs at an advanced stage of the disease with monoclonal antibodies such as anti-TIMP-1 monoclonal antibody might improve specificity and patient outcomes as MMPs are significantly increased at advanced stages of cancers292. Because the existing therapies seem to be complicated and ineffective, there is a need for more research to improve these therapies and find effective therapies for cancer treatment.Inhibiting LOX-mediated collagen cross-linking, which is necessary for the development of ECM stiffness by targeting the β1-containing integrins that bind to collagen I fibres has been proposed for the prevention of cancer pre-metastatic niche formation and for the treatment of cancer metastasis. However, because β1-integrins interact with multiple ECM components in addition to collagen I, β1-integrin antagonists may have unintended effects


Oxygen-enhanced magnetic resonance imaging (MRI) and T1-mapping was used to explore its effectiveness as a prognostic imaging biomarker for chemoradiotherapy outcome in anal squamous cell carcinoma.t-test was used to compare changes across the patient population. Regions of subcutaneous fat and muscle tissue were examined as control ROIs.T2-weighted, T1 mapping, and oxygen-enhanced T1 maps were acquired before and after 8–10 fractions of chemoradiotherapy and examined whether the oxygen-enhanced MRI response relates to clinical outcome. Patient response to treatment was assessed 3 months following completion of chemoradiotherapy. A mean T1 was extracted from manually segmented tumour regions of interest and a paired two-tailed t-test, p < 0.001, n = 7). At baseline, prior to receiving chemoradiotherapy, there were no significant changes in T1 across patients from breathing oxygen (n = 9). In the post-chemoRT scans (8–10 fractions), there was a significant decrease in T1 of the tumour ROIs across patients when breathing 100% oxygen . Out of the 12 patients from which we successfully acquired a visit 1 T1-map, only 1 patient did not respond to treatment, therefore, we cannot correlate these results with clinical outcome.There was a significant increase in T1 of the tumour ROIs across patients following the 8–10 fractions of chemoradiotherapy (paired These clinical data demonstrate feasibility and potential for T1-mapping and oxygen enhanced T1-mapping to indicate perfusion or treatment response in tumours of this nature. These data show promise for future work with a larger cohort containing more non-responders, which would allow us to relate these measurements to clinical outcome. Magnetic resonance imaging (MRI) is routinely used in the diagnostic process and could provide a non-invasive method for extracting this biomarker information. In this study, we hypothesize that patients with poor vascularity or hypoxia will correlate with those more likely to be at risk of relapse, and that a technique known as oxygen-enhanced MRI (OE-MRI) can be used to provide indicators of tumour perfusion In this study, we quantify the changes in mean T1 between a T1-map acquired at diagnosis and again on fraction 8–10 of chemoradiotherapy and examine whether this oxygen-enhanced MRI response relates to clinical outcome in patients with anal squamous cell carcinoma.22.1Patients included in this study were 23 trial participants (mean age = 60 years) who underwent radical chemoradiotherapy for anal cancer in Oxford University Hospitals NHS Trust between 2014 and 2017 . All patients had newly diagnosed, histologically confirmed ≥T2N0 anal SCC with no prior treatment and were suitable for radical CRT. Exclusion criteria were contraindications to MR imaging and previous pelvic radiotherapy. This study was approved by National Research Ethics Service Committee South Central Oxford (14/SC/1130) and all patients provided written informed consent.2.22 on day 1 and Capecitabine 825 mg/m2 orally twice a day on radiotherapy treatment days. Post CRT tumour response assessment was undertaken three months following completion of CRT using clinical, radiological and, where appropriate, histological analysis. At this point all patients were defined as having had a complete response or having persistent disease.CRT was delivered according to UK-based guidance 2.2.1Patients were scanned on a GE Discovery MR750 3T MR scanner with a GE 8-channel Torso Array  using a flat-topped couch to reproduce the radiotherapy treatment position. Following the acquisition of a T2-weighted image , the Modified Look-Locker Inversion Recovery (MOLLI) 2.2.2A tumour region of interest (ROI) was delineated by an experienced oncologist for all primary tumours and any lymph nodes >2 cm on high-resolution T2-weighted images and nonlinearly registered to the T1-map. Due to the resolution and slice thickness differences between the MOLLI T1 maps and T2-weighted images, as well as mild spatial distortions in the MOLLI due to the high speed acquisition, there is not a direct 1:1 mapping between the T2-weighted images and T1 maps and thus registration was needed. Registration was implemented using MATLAB’s intensity-based registration algorithm . Patient response to treatment was assessed 3 months following completion of chemoradiotherapy. For use as control ROIs, regions of fatty tissue and muscle tissue were also delineated (examples shown in t-test (a = 0.05) considering 3 cases: the change in T1 (1) from visit 1 to visit 2, (2) breathing air versus oxygen on visit 1, and (3) breathing air versus oxygen on visit 2. The tumour ROIs contained an average of 265 ± 187 voxels, and patients with tumour ROIs containing <50 voxels were excluded from the analysis. Out of the 54 total MOLLI images acquired, 5 were excluded from analysis due to containing <50 tumour voxels within the MOLLI slice. The largest tumour ROI was 728 voxels. If the T1 map was of insufficient quality due to excessive patient motion or scanner artefacts, the patient was excluded from the analysis.For each region of interest, the histogram distribution of T1 values was examined, checking whether there was a normal distribution or, for example, a bimodal distribution possibly representing two populations of tissue in the ROI. In all cases, the distribution was normal, thus indicating that a comparison of means was suitable to describe changes in the ROIs. The changes in mean T1 was assessed with a paired two-tailed air − T1oxygen), the tumour T1 while breathing air in visit 2 and % change in T1 in visit 2 /T1air), and the tumour T1 while breathing air in visit 1 and either of these measures in visit 2.In addition, we investigated the possibility that the native T1 could correlate to the amount of change seen from breathing oxygen in either visit. Pearson correlation was used to estimate the correlation between: the tumour T1 while breathing air in visit 2 and the decrease in T1 from oxygen in visit 2  the T1 on visit 1 on air and (5) the change in T1 from oxygen on visit 1. However, there was only one patient whose data passed the quality metrics who did not respond to therapy, so it was not possible to perform these final two analyses.3Out of the 23 patients included in the study, a T1 map was successfully acquired on visit 1 for 11 patients. By visit 2, following chemoradiotherapy, some tumours were too small to include in the analysis, resulting in a visit 2 T1 map for 7 of those 12 patients. In total, oxygen-enhanced T1 maps were successfully acquired for 9 patients on visit 1 and 8 patients on visit 2. t-test, p < 0.001, n = 7), while there were no significant changes in T1 in the control ROIs of fat and muscle tissue. The largest change in mean T1 in the tumour ROI seen was an increase of 200 ms, and the mean change across patients was an increase of 115 ms.The change in the mean T1 in the tumour, fat and muscle ROIs were compared across the two visits . There wt-test, p < 0.11, n = 9). In visit 2, after receiving 10–12 fractions of chemoradiotherapy, there was a significant decrease in T1 of the tumour ROIs across patients when breathing 100% oxygen . In all cases, there were no significant changes in T1 in the control ROIs of fat and muscle tissue. In visit 2, the largest change in mean T1 in the tumour ROI from breathing oxygen was a decrease of 207 ms, and the mean change across patients was a decrease of 78 ms.The mean T1 in all ROIs taken while the patient was breathing air versus 100% oxygen were compared, in each visit separately (patient example shown in air − T1oxygen), the tumour T1 while breathing air in visit 2 and % change in T1 in visit 2 /T1air), or the tumour T1 while breathing air in visit 1 and either of these measures in visit 2.A Pearson correlation showed no significant correlation between: the tumour T1 while breathing air in visit 2 and the decrease in T1 from oxygen in visit 2  T1 or oxygen-enhanced MRI T1 maps relate to clinical outcome in patients with anal squamous cell carcinoma. Out of the 12 patients from which we successfully acquired a visit 1 T1-map, only 1 patient did not respond to treatment. We demonstrate feasibility and potential for T1-mapping and OE-MRI to indicate perfusion in future work with larger cohort containing more non-responders, which would allow us to relate these measurements to clinical outcome. As the numbers are small a clinical correlation cannot be made.Following chemoradiotherapy, there was a statistically significant increase in tumour T1 across the 7 patients , all of Since the tumours were often smaller at visit 2 due to treatment response, it is possible that the tumour remaining in visit 2 is just the more aggressive section, and perhaps always had a different T1, which could explain this change. However, we hypothesize that this is not the case, since no bimodal distributions or multiple populations were seen in histograms when analyzing all of the ROIs, suggesting this change in T1 does represent a true change in the tumour biochemistry and/or physiology.In addition, the changes seen from oxygen-enhancement changed greatly following chemoradiotherapy. Before chemoradiotherapy, OE-MRI showed no significant changes in tumour T1, however after receiving chemoradiotherapy, OE-MRI showed a significant decrease in tumour T1 (p < 0.001) . The larAs shown in While this study offers promising results, the technique has some limitations which warrant discussion. First, due to loss of data from a combination of patient motion, trial withdrawal, and tumour shrinkage, this study had a relatively small sample size, and no results could be correlated to clinical outcome. Secondly, the T1-mapping method used, MOLLI, currently provides only single-slice data, limiting the analysis to one region of the tumour. As tumours can be highly heterogeneous, it is possible that this analysis misses large changes that were occurring in other regions, and in addition, although care was taken to select the same region of the tumour on the second visit, it is possible that the slices are analyzing slightly different slices of the tumour on each visit. This limitation could be mitigated by the use of a 3D T1 mapping method such as the variable flip angle method A final limitation that warrants discussion is that the T1 contrast provided by increased fractions of inspired oxygen tends to decrease as magnetic field strength increases Lastly, we investigated the possibility that the native T1 could correlate to the amount of change seen from breathing oxygen, however there was no significant correlation between these values on either visit. This is not unexpected, since tumours are highly varied and heterogeneous between patients and the mean T1 will be affected by a variety of factors, not only the perfusion of the tumour.In conclusion, this clinical trial data demonstrates the feasibility and potential for T1-mapping and oxygen enhanced T1-mapping to indicate perfusion or treatment response in tumours of this nature. The data show promise for future work with a larger cohort which would likely result in more non-responders, allowing us to relate these measurements to clinical outcome.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of  Natural killer (NK) cells display spontaneous cytotoxic activity without prior sensitization in a process known as natural cytotoxicity. Accordingly, this subset of lymphocytes plays a key role in early immune defense within innate immunity ,2. In paNatural cytotoxicity receptors (NCRs), namely the natural killer cell proteins NKp30, NKp44, and NKp46 (numbers refer to their molecular weights), stand out for their role in activating NK cells and initiating tumor targeting . NCRs arNatural killer protein 30  is an Ig-like activating receptor of NK cells ,6. SimilFurther, three specific cellular ligands of NKp30 have been identified. Consistent with the other members of CD28 family, NKp30 binds a member of B7 family, in particular B7 homolog 6 (B7-H6), that is constitutively expressed on the surface of some tumor cells . AnotherSince the B7-H6:NKp30 interaction was first discovered, considerable research efforts have been made to define B7-H6 expression specificity in tumor cell lines and its underlying clinical potential ,16,17. S+ and CD161+ pro-inflammatory monocytes. In contrast, the blood of patients presenting with sepsis caused by Gram-negative bacteria contains soluble or exosomal forms of B7-H6 [B7-H6 is expressed in several tumor cell lines, both ex vivo and in vivo, but remains undetected in healthy cells thus far, and is therefore an excellent tumor marker ,12. In aof B7-H6 .N-glycosylation sites of NKp30 are crucial for its signal transduction and for B7-H6 but not BAG-6 ligand binding [Escherichia coli) [N-glycosylation; glycosylation at Asn42 is essential for ligand binding, and glycosylation at Asn68 also has a substantial effect, whereas glycosylation at Asn121 does not play a key role, as determined by site-directed mutagenesis [Although crystal structures of NKp30, both unbound  and in a binding . Moreove binding . The sta binding . Interesia coli)  but not ia coli) . Neverthia coli) . Greateragenesis .Escherichia coli [N-glycosylation pattern). In the latter case, the affinity of NKp30 to B7-H6 additionally increased with longer stalk region [Escherichia coli was two times higher [Furthermore, the analysis of NKp30 ligand binding has highlighted differences in affinity as a function of the protein expression system used. NKp30 affinity to B7-H6 ranged from 2.5 to 3.5 μM with both recombinant proteins expressed in hia coli . Howeverhia coli  and evenk region . Even hik region . Interess higher .N-glycosylation. NKp30 produced in HEK293S GnTI− cells lacking N-acetylglucosaminyl-transferase I activity forms oligomers but disassembles to pure monomers after enzymatic deglycosylation. We have further characterized the binding affinity of B7-H6 to NKp30 and its dependence on glycosylation and oligomeric state using proteins expressed in human cell lines, that closely mimic the natural post-translational modifications of this receptor. Finally, we have determined the crystal structure of the NKp30:B7-H6 complex with the glycosylated receptor. Our results suggest that dimerization may be a necessary step for NKp30 oligomerization and stable signal transduction upon B7-H6 binding.In this study, we show that NKp30 oligomerization depends on its N-glycans, or in HEK293S GnTI− cell line that provides uniform, simple Asn-GlcNAc2Man5N-glycans, in the latter case allowing also for a possibility of efficient enzymatic deglycosylation, when required [For our studies, we cloned the extracellular domains of NKp30 and B7-H6 into a mammalian expression vector with a C-terminal histidine tag. Both proteins were expressed either in HEK293T cell line that provides the protein with complex wild-type required ,25,26. PN-glycosylation), but in non-reducing buffer, a band with a size corresponding to the B7-H6 dimer was identified  ; therefoN-glycosylation sites , B7-H6 deglycosylation caused its precipitation, most likely because of the loss of all its glycans. For this reason, we sought to improve the buffer conditions for deglycosylation and the storage of this protein by differential scanning fluorimetry. Twenty-five conditions were screened by analyzing protein melting point temperatures . However, at 20% glycerol, the mT increased similarly (56 °C) and B7-H6 aggregation was completely prevented during deglycosylation. Therefore, glycerol was added to a final concentration of 20% (v/v) for convenience and used for further B7-H6 deglycosylation and storage at low temperatures (−20 °C). Glycerol was always removed by buffer exchange on desalting columns prior to subsequent experiments.Under standard conditions  completely depletes the sample of oligomers  showed a more homogeneous oligomer profile with one dominant species of ca 8 units and with secondary species of ca 16 units, based on their calculated molar masses of 180 and 320 kDa, respectively. These findings corroborate the molar masses calculated from the MALS signal . This suggests that not all NKp30 binding sites in the oligomeric species are accessible for B7-H6 binding and that NKp30 molecules might sterically block the access to the neighboring binding sites within the oligomer itself or with the B7-H6 molecules bound to it. The modest increase in MALS-calculated molar mass for the oligomeric NKp30:B7-H6 complex  and 480 nm (B7-H6 only) wavelengths c. The bi complex a support complex c. FinallN-glycosylation sites, five of which were confirmed by our MS analysis  reduces the affinity for the B7-H6 ligand, whereas the N42Q mutation of the Asn42 glycosylation site almost completely abrogates binding . For thimaxB was higher, as expected for oligomer binding  was solved at 3.1 Å resolution, and the refinement parameters are summarized in When viewing neighboring symmetry-related molecules, surrounding the asymmetric unit of the crystal, a dimer of two NKp30_LBD:B7-H6 complexes (LBD_A:B7-H6_C and LBD_B:B7-H6_D) becomes apparent . The intN-acetylglucosamine at NKp30_LBD asparagine residues 42 (chains A and B), and B7-H6 residues 208 (chains C and D) and 43 (chain E). Furthermore, low-quality peaks in the electron density map, corresponding to glycans, were observed near NKp30_LBD asparagine residues 68 (chain A) and 121 (chain B), and near B7-H6 residues 43 , 57 , 174 , 208 (chain E), and 242 . In the case of NKp30_LBD Asn42, the glycosylation changes the orientation of the Asn42 side chain and main chain and consequently, the placement of residue Ala43 in comparison with NKp30_LBD in PDB 3NOI and 3PV6. Noticeably, the glycosylation site at Asn42 of one NKp30_LBD molecule (LBD_A) localizes in the proximity of the C-terminus of the other NKp30_LBD molecule (LBD_B) and vice versa  , whereas+ hematological and solid tumors. More recently, T cells expressing B7-H6-specific human single-chain fragment variable (scFv) as chimeric antigen receptor (CAR) have been shown to induce potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7-H6 [The first key stage of this study was the preparation of the stable, soluble, extracellular part of the B7-H6 protein. Members of the B7 family have moved to the forefront of cancer research for their underlying involvement in tumorigenesis  and tumoof B7-H6 .+ tumors [Although B7-H6 was the target molecule in these approaches, other strategies have been developed exploring the potential of B7-H6 as a natural ligand for the activating receptor NKp30. Kellner et al. ,32 gener+ tumors . In our N-glycans, similar to those present in HEK293S GnTI− cell lines. When recombinantly expressing these constructs, the authors found that both NKp30_Stalk and NKp30_LBD oligomerized in their study, although the latter to a lesser extent [Oligomerization of the NKp30 ectodomain has been previously characterized using constructs expressed in Sf9 insect cells providing uniform, simple paucimannose r extent . HoweverDK, as assessed by SPR and ELISA (both surface-based interaction methods), and hypothesized that the increase in apparent ligand binding affinity of the oligomers is caused by the increase in the avidity of higher-molecular-order NKp30 complexes under these conditions. We noted a similar trend in our SPR analysis when using immobilized B7-H6, observing a higher affinity for NKp30_Stalk than for NKp30_LBD, and an even higher affinity for the NKp30_Stalk oligomeric fraction. In contrast, when analyzing the same system by AUC and ITC (solution-based techniques), we found that both monomeric and oligomeric fractions of NKp30_Stalk, and NKp30_LBD, showed similar thermodynamic parameters and affinity to soluble B7-H6, but the oligomers exhibited significantly lower binding stoichiometry. This suggests that interaction data on immobilized B7-H6 and oligomerizing NKp30_Stalk construct collected using surface methods might not correctly express DK values for interactions of individual binding sites and that the NKp30 oligomers might not be completely biologically active species, at least when present in solubilized form and not on the cell surface.In addition, the ability of the NKp30_Stalk construct to oligomerize is lost upon its deglycosylation with endoglycosidase F1, thereby highlighting the unappreciated but key role of glycosylation in NKp30 oligomerization. Hermann et al.  observed+ reporter cell lines. This key role of glycosylation and of the stalk region in signal transduction was further corroborated by using various NKp30-transfected CD3ζ reporter cell lines stimulated by B7-H6+ target cells [N-glycans, or deglycosylated, independent of the method that we used to measure these interactions. The deglycosylated proteins used in our study still had a single GlcNAc residue at each glycosylation site, in contrast to the disruptive Asn-Gln mutations used in the previous study, which may account for the differences in the results. Nevertheless, our findings indicate that glycosylation most likely does not directly affect the NKp30:B7-H6 interaction and instead primarily affects the ability of NKp30 to oligomerize. Impaired oligomerization would then translate into lower apparent binding affinities, when using surface-based methods such as SPR and ELISA, and into impaired signal transduction in cell-based experiments. The role of the stalk domain in CD3ζ-mediated activation of NK cells was thoroughly characterized by Memmer et al. [DK of B7-H6-Fc whereas BAG-6 binding, again, remained mostly unaffected, although NKp30-Fc IgG fusions and SPR detection were used in these experiments. Subsequent reporter cell-based assays showed that Arg143  alignment with the aspartate of CD3ζ is required for signal transduction and that this alignment might be achieved by ligand-induced receptor clustering and/or stalk-dependent conformational changes [Another important aspect of the NKp30:B7-H6 interaction is the glycosylation of the NKp30 ectodomain, especially on Asn42 and Asn68 sites, as shown by Hartmann et al.  in bindiet cells . In our  changes ,35.Although NKp30 constructs artificially dimerized through IgG-Fc fusion may not be the best tools to describe the natural behavior of this receptor at the plasma membrane of NK cells, their thorough characterization should be useful for developing immunotherapeutics. Over ten years ago, Arnon et al.  demonstrThe original motivation for our work was to understand better how NKp30 oligomers are formed and structured. To this end, we used multiple techniques, but not all of them produced conclusive results, such as structural mass spectrometry (cross-linking and H/D exchange) or cryo-electron microscopy. We also tried to crystallize the NKp30_Stalk construct and thus solve its oligomeric structure, albeit to no avail. Neither this protein nor its complex with B7-H6 formed crystals. Nevertheless, we solved the structure of the glycosylated NKp30_LBD:B7-H6 complex, which is somewhat similar to the previously known structures of NKp30_LBD  and of Most interestingly, our crystal structure shows the formation of an NKp30_LBD dimer in the bound state with B7-H6. The symmetrical arrangement of Asn42 glycosylation sites, near the C-terminal stalk region beginnings on both sides of the dimer, strengthens our hypothesis of glycosylation-supported, stalk region-mediated NKp30 oligomerization. In contrast to the structure of the glycosylated NKp30_LBD dimer, in the dimer of the refolded NKp30_LBD observed in the PDB 3NOI crystal structure, the Asn42 residue is in close contact with neighboring side chains of other amino acids of the dimerization interface. With such a bulky glycan chain bound to the Asn42, this type of dimer is unlikely to exist. Therefore, we may assume that glycosylation induces the observed dimer arrangement and that this arrangement is further stabilized by the interaction between the glycan moiety and the stalk region of full-length NKp30.Moreover, for soluble NKp30_Stalk, the stalk regions that extend on both sides of the dimer would be free to interact with stalk regions of other NKp30_Stalk dimers, thereby forming linear oligomers composed of such dimers. To acquire at least some low-resolution data on the structure of these oligomers, we have also performed SEC-SAXS analysis of the NKp30_LBD, NKp30_Stalk monomeric and oligomeric fractions, and their complexes with B7-H6 . RepreseAnother interesting aspect of our crystal structure is the overall topology of the dimer of the NKp30_LBD:B7-H6 complex observed in the crystal lattice. Its arrangement is compatible with both NKp30 molecules inserted within the same NK cell membrane and B7-H6 in the cell membrane of a target tumor cell . Such arThese results support the proposed close interaction mediated by the NKp30 dimer ligated with two B7-H6 monomers, as observed in the presented crystal structure. However, further studies are required to confirm such an arrangement directly on the membrane of living NK cells, as well as the oligomeric arrangement of NKp30. Considering the short length of the stalk region, it is unlikely that NKp30 forms octameric, decameric, or even larger oligomers through its stalk region on the cell surface, in contrast to its oligomerization in solution. Therefore, data on NKp30 receptor oligomers collected in solution, using its solubilized ectodomains interacting in three-dimensional (3D) space, should not be directly extrapolated to its natural habitat within the plasma membrane and to a 2.5D space on the cell surface. Moreover, all future immunotherapeutic strategies and reagents designed to target or trigger this NK cell receptor:ligand system should also allow enough flexibility with respect to the B7-H6 moiety, thus ensuring its proper orientation and interaction with NKp30.− cells [6/mL) were maintained in ExCELL293 serum-free medium  supplemented with 4 mM L-glutamine in square-bottom glass DURAN flasks with gas permeable caps  using 30–40% of the nominal volume at 135 rpm on orbital shaker placed within the incubator at 37 °C and 5% CO2 [− cell lines was performed using the plasmid pTW5sec, a derivative of the pTT5 plasmid backbone [HEK293T cells were kindly provided by Radu A. Aricescu . HEK293T− cells  (0.3–6 ×d 5% CO2 . Transiebackbone  modified6 HEK293 cells were centrifuged for 5 min at 90× g. Cells were resuspended in 34 mL of ExCELL293 medium in an empty flask and 800 μg of DNA (1 μg/106 cells) were diluted in 6 mL of phosphate-buffered saline (PBS) and filtered through a 0.22 μm filter into the flask with cells. Subsequently, linear 25 kDa polyethyleneimine (lPEI) was added in a 1:3 DNA:lPEI (w/w) ratio. Cells were incubated in high density on a shaker for 1.5–4 h at 37 °C. Afterwards, up to 400 mL ExCELL293 medium and 0.5 M valproic acid [g at 20 °C. The supernatant was filtered through a 0.2 μm filter, diluted two-fold with PBS buffer, and loaded onto a pre-equilibrated 5 mL HiTrap TALON column using an ÄKTAprime FPLC system . Protein was eluted with 250 mM imidazole in PBS, concentrated using Amicon Ultra  concentrators, filtered using spin filter and subjected to size-exclusion chromatography on Superdex 200 Increase 10/300 GL column  as a final purification step with HEPES buffer  as the mobile phase. For NKp30_Stalk, fractions were collected separately for oligomeric and monomeric species, and immediately frozen in liquid nitrogen. Protein glycosylation and disulfide bonds pairing were characterized by mass spectrometry  using NHS labelling chemistry according to the manufacturer’s instructions. Nine volumes of B7-H6 solution in HEPES buffer were mixed with one volume of 1 M bicarbonate buffer, pH 8.5. ATTO 488-NHS ester was added to the protein solution at a molar ratio of 1:3 (protein:ATTO488-NHS). Reaction mixture was incubated for 60 min in the dark at room temperature on a roller shaker at 35 rpm. The labelled protein was purified on a HiTrap Desalting column  connected to an ÄKTA basic HPLC system.B7-H6 expressed in HEK293S GnTI− cell lines, the proteins in HEPES buffer at 1 mg/mL concentration were mixed with recombinant GST-tagged endoglycosidase F1 (Endo F1) [w/w) ratio (target protein:Endo F1). For B7-H6, the buffer was supplemented with 20% glycerol to increase protein stability. The mixture was incubated for 3 h at 37 °C or at 4 °C overnight while mixing. Then, the solution was loaded onto a 5 mL GST-trap column  connected to the fast protein liquid chromatography (FPLC) system to capture the Endo F1, while collecting the flow-through containing deglycosylated protein. The solution was concentrated to 200 µL and loaded onto a Superdex 200 Increase 10/300 GL  column for further purification and characterization of the deglycosylated proteins. Samples from collected fractions were used for SDS-PAGE analysis.For deglycosylation of NKp30 and B7-H6 expressed in HEK293S GnTIEndo F1)  in a 200− cells with 10 μL of 2× concentrated screen buffer and 1 μL of 50× concentrated stain solution and complemented by HEPES buffer up to 20 μL. The sample without protein was used as a blank, and the sample with no additive was used as a reference. Data were collected on a Rotor-Gene 2000 Real Time Cycler  at an excitation wavelength of 300 nm. Fluorescence was measured at 570 nm. The temperature increased stepwise, 0.5 °C each 30 s from 25 °C to 95 °C. The data were evaluated using OriginPro 8 software (version 8.500161). Melting points can be extrapolated by finding the inflection point of sigmoid melting curves. By depicting the data as a negative derivative of relative fluorescence (−d(RF)/T), the melting points correspond to the minima of the melting curves.Differential scanning fluorimetry was performed using SYPRO Orange Protein Gel Stain . Its 5000× DMSO stock solution was diluted to 50× concentrated solution. The samples were prepared by mixing 4 μL of B7-H6 expressed in HEK293S GnTI3) as a reference. Sedimentation velocity experiments were conducted with 20 μM protein samples using double sector cells and An50-Ti rotor at 20 °C and 36,000 or 48,000 rpm for oligomeric or monomeric samples, respectively. Absorbance scans were recorded at 280 or 480 nm at 3–7 min intervals. Buffer density, protein partial specific volume, and particle dimensions were estimated in Sednterp (www.jphilo.mailway.com). Data were analyzed in Sedfit [Sedimentation velocity measurements were performed on an analytical ultracentrifuge ProteomeLab XL-I . Protein samples were purified by gel filtration, using the mobile phase buffer .SEC-MALS experiments were performed using an FPLC station equipped with miniDAWN Tristar light scattering  and Shodex RI-101  refractive index detectors with a Superose 6 Increase 10/300 GL column  equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM NaN3 buffer. First, control heat was determined by buffer–buffer titration. For protein interaction measurements, 200 µL of 20–30 µM NKp30 variants were loaded into the cell and 40 μL of 200–300 µM B7-H6 into the syringe (these concentrations varied slightly in different experiments). An initial injection of 0.4 µL of B7-H6 was followed by 25 injections of 1.5 µL. Injection duration was 2 s, in 120 s intervals, performing the measurements at 25 °C and stirring the cell solution at 750 rpm. The data were evaluated using NITPIC [Thermodynamic parameters of NKp30:B7-H6 interactions were determined using the PEAQ-ITC instrument . All measurements were performed in the 10 mM HEPES pH 7.5, 150 mM NaCl, 10 mM NaNg NITPIC , Sedphatg NITPIC .N-glycosylation states , to B7-H6, also in three glycosylation states. Measurements were performed using the Biacore T200 system  in a buffer composed of 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mg/mL dextran, and 0.05% Tween-20. Three glycosylation variants of B7-H6 were biotinylated  according to the manufacturer’s instructions and immobilized to the streptavidin sensor. Binding experiments were performed in single cycle kinetics mode with 15 sequential injections of NKp30 samples in each cycle with concentrations ranging from 1 nM to 16.3 μM. Data from a reference flow cell with an empty channel were subtracted and fitted using the Biacore T200 evaluation software (version 3.0). Maximal fitted responses from each cycle were analyzed using Sedphat [Surface plasmon resonance (SPR) experiments were performed to measure NKp30_Stalk and NKp30_LBD binding, in three different − cells; the mixture was concentrated to 10 mg/mL before the drop set-up. Initially, needle-shaped crystals of NKp30_LBD:B7-H6 complex were obtained in 0.1 M sodium citrate pH 5.0, 20% PEG 8000. These crystals were crushed and used for seeding in optimization performed using the sitting drop method in 300 nL . The number of crystals grew in the drops that were seeded, and diffraction data were collected for a few of them. 25% glycerol was used as cryoprotectant and data were collected at 100 K. The best diffraction data were collected from crystals grown in 0.1 M sodium citrate pH 6.7, 11.7% PEG 6000.Protein crystallization was performed at the Division of Structural Biology of the Wellcome Centre for Human Genetics, University of Oxford. Initial screening was performed using the sitting drop method in 300 nL  using the Hydra and Cartesian instruments. All crystallization plates were stored in Rock Imager  at 21 °C. Four commercially available screens  were used for the equimolar mixture of deglycosylated B7-H6 with NKp30_Stalk or NKp30_LBD, both expressed in HEK293S GnTIC2 (recommended by POINTLESS and ZANUDA) and alternatively in P1. Finally, C2 was selected as the correct space group based on the phase problem solution and refinement. Data suffered by strong anisotropy, with effective resolution in the direction of axis a being the lowest, ca. 4.4 Å. Anisotropy corrections were not applied to the data. Final data processing statistics are shown in Eleven diffraction data sets were collected at the Diamond Light Source  at beamline I02 using a wavelength of 0.97949 Å and a PILATUS 6M-F detector . The resolution of all data sets was 3–4 Å and the crystals degraded during the data collection. Eventually, three data sets from three crystals (set B7x1: images 1 to 800 (80°), set B8x5:1 to 800 (80°), and set B8x3:1 to 450 (45°)) were merged to process the data for optimal results. The data were integrated in XDS  and mergC2, using MORDA [C2 contains three B7-H6 protein chains and two NKp30 chains. After refinement trials in REFMAC5 [Rfree set). Manual editing was performed in COOT [The phase problem was solved in space group ng MORDA  and PHASng MORDA , data cu REFMAC5 , conside REFMAC5 ), using  in COOT . Structu in COOT . There aDiffraction data have been deposited in the SBGrid Data Bank under code 753 (doi:10.15785/SBGRID/753). The crystal structure has been deposited in the Protein Data Bank under code 6YJP.N-glycosylation. Furthermore, we solved the first crystal structure of a glycosylated NKp30 ligand binding domain, in a complex with its tumor ligand B7-H6, highlighting why glycosylation is crucial for the NKp30 oligomerization and for signal transduction. Furthermore, our results indicate that glycosylation should not be overlooked when planning or conducting structure-function studies. In conclusion, our structure of the complex between glycosylated NKp30 and B7-H6 provides a template for designing molecules to stimulate NKp30-mediated cytolytic activity for tumor immunotherapy.Glycosylation is an essential post-translational modification of cell surface proteins, not only stabilizing them in the extracellular environment but also providing new functional modalities. Moreover, glycosylation is crucial for immune recognition and self-non-self discrimination, especially when involving receptors of the innate immune system, which often bind to their ligands with weak affinities. Accordingly, their ability to transduce the signal into the cell should significantly vary as a function of their glycosylation presence and its type. Glycans may help these receptors to create stable signaling complexes through dimerization and oligomerization or to organize them within complex dynamic structures of the immune synapse. In this study, we exemplified this behavior for the NK cell activation receptor NKp30, whose oligomerization depends on its