The tumour microenvironment (TME) determines vital aspects of tumour development, such as tumour growth, metastases and response to therapy. Cancer-associated fibroblasts (CAFs) are abundant and extremely influential in this process and interact with cellular and matrix TME constituents such as endothelial and immune cells and collagens, fibronectin and elastin, respectively. However, CAFs are also the recipients of signals—both chemical and physical—that are generated by the TME, and their phenotype effectively evolves alongside the tumour mass during tumour progression. Amid a rising clinical interest in CAFs as a crucial force for disease progression, this review aims to contextualise the CAF phenotype using the chronological framework of the CAF life cycle within the evolving tumour stroma, ranging from quiescent fibroblasts to highly proliferative and secretory CAFs. The emergence, properties and clinical implications of CAF activation are discussed, as well as research strategies used to characterise CAFs and current clinical efforts to alter CAF function as a therapeutic strategy. The stromal components of a tumour have thus graduated in our appreciation from bystanders, to enablers, to indispensable and determinant players with respect to disease evolution and response to therapy.3 Thanks mainly to more sophisticated in vivo models and analysis techniques,5 a focused effort is currently underway to consider the tumour-cell phenotype as an emergent property of both the tumour microenvironment (TME) and the activity of stromal cell populations that surround and infiltrate the tumour.Conceptualising cancer as an exclusively tumour cell-centric disease is no longer an effective approach for identifying novel therapeutic targets.8 Tumour stromal tissue comprises fibroblasts, endothelial and immune cells, and extracellular matrix (ECM) components, which all differ markedly from their quiescent counterparts in homoeostatic tissues. Fibroblasts are of particular interest owing to their extensive matrix-synthesising and matrix-remodelling capacities, both of which are pivotal for establishing an invasion-permissive TME.9 In fact, levels of fibroblast markers within tumours are known to be predictive of disease outcome, with the number and properties of fibroblasts in the tumour stroma influencing overall survival, time to recurrence and resistance to therapy.11 Targeting fibroblast function for therapeutic benefit is therefore an area of intensive research.11The stromal content of solid tumours can vary significantly, both in amount and composition; in certain tumour types, such as pancreatic, stomach, colon and breast cancer, the stroma contributes 60–90% of the total tumour mass.12 Fibroblasts that have become activated during wound healing are referred to as ‘myofibroblasts’, while those activated in the context of tumours are called carcinoma-associated fibroblasts (CAFs) or peritumour fibroblasts.13 Though different in nomenclature, activated fibroblasts display analogous marker expression and very similar phenotypes, with the important distinction that in contrast to acute inflammation, fibroblasts in chronic inflammation and tumour tissue fail to inactivate.10 A great number of studies have identified the signalling mechanisms by which CAFs influence tumour cell properties ,14 and it has become clear that CAFs play a pivotal role in the TME. However, our understanding of the diverse properties of CAFs throughout the different stages of tumour progression remains incomplete. The function of CAFs has remained especially elusive in light of their heterogeneity and different effects on tumour development; while their pro-tumorigenic role has been well documented across tumour types,15 CAFs have also been reported to restraining tumour growth in several studies.18The term ‘fibroblast’ describes a heterogeneous group of spindle-shaped cells of mesenchymal origin with a spectrum of phenotypes characterised by varying degrees of contractility, ranging from the resting fibroblast to the activated myofibroblast observed in wound healing.In this review, we describe the dynamics of fibroblast activation in acute and chronic wounds, focusing on solid cancers. Most studies investigating the CAF–tumour cell relationship concentrate on carcinomas at the invasive stage, but we attempt to look before and after this stage to provide insights into the wider life cycle of fibroblasts located in the tumour stroma. Specifically, we describe the properties of fibroblasts as they progress from their resting state in tissue homoeostasis to the different magnitudes of activation occurring during early, invasive and metastatic tumour growth. Finally, we consider the clinical relevance of the activated fibroblast phenotype and opportunities for targeting fibroblast activation therapeutically.19 by the basement membrane (BM), a specialised ECM structure that presents the biophysical cues necessary for cell attachment, polarity and cell movement within and across tissue compartments.21 Resting or quiescent fibroblasts are dispersed as single cells within the stromal tissue compartment and are found proximal to the BM, where they orient themselves in the direction of matrix fibres, but have no direct association with the BM.22 During homoeostasis, quiescent fibroblasts show minimal metabolic and transcriptional activity but play a pivotal role in defining the differentiation status of adjacent epithelia via the secretion of specific signalling factors, including among others Wnt and bone morphogenic proteins,24 and the production and organisation of matrix proteins, including collagens, fibronectin and elastin, at a minimal rate, to maintain a compliant, yet tension-resistant BM.4During tissue homoeostasis, epithelial cells are strictly segregated from fibroblasts in the adjacent connective tissue25 as well as being expressed on macrophages26); other markers include vimentin, filamin A and Thy-1/CD90.27 Consequently, the quiescent fibroblast is generally considered to be a fibroblast that does not express the markers observed in activated fibroblasts, as defined primarily from studies of fibrosis and wound repair. As such, perhaps the most apt characterisation of a quiescent fibroblast is that of a mesenchymal cell with the potential to become activated by growth factors, and drastically increase its contractility, proliferation and secretory phenotype in response to an appropriate stimulus.4 Note that the lack of data specific to resting fibroblasts might be attributable to two factors. First, the focus is typically placed on their tissue-specific, activated counterparts (myofibroblasts or CAFs).4 Second, the inability to establish fibroblast cultures with a truly ‘resting’ phenotype has hampered the investigation of these cells in vitro; although fibroblasts can readily be cultured in vitro,4 they become at least partially activated owing to the properties of culture plastic, which is reminiscent of stiffened, fibrotic ECM.29Although the structural characteristics of quiescent stroma are relatively well defined, the molecular characteristics of quiescent fibroblasts have remained elusive. From a collection of several markers that are not exclusive nor obligate to fibroblasts across tissues, fibroblast-specific protein 1 (FSP-1), a cytoplasmic calcium-binding protein, has been proposed as the most accurate marker for resting fibroblasts to date  to perform a number of functions. They regulate the de novo deposition of matrix proteins and remodelling of the existing matrix via the secretion of matrix-degrading enzymes  and through enhanced contractility.31 Their contractile apparatus also enables them to regulate interstitial fluid pressure.24 Through a plethora of secreted factors, fibroblasts can also interact with other cell types,32 in order to re-vascularise the healing tissue, and furthermore they secrete and absorb metabolites to rebalance the tissue niche.12 A key functional difference between fibroblasts and myofibroblasts therefore involves the cell secretome, with myofibroblasts demonstrating an increased production of proteins, including signalling molecules. Furthermore, on a molecular basis, while resting fibroblasts are characterised by the absence of activation markers and markers typical for other cell types,12 myofibroblasts acquire, in addition to FSP-1, and among others, the expression of α-smooth muscle actin (α-SMA), desmin, fibroblast activation protein (FAP), discoidin domain-containing receptor 2 (DDR2), collagens (e.g. collagen 1a1) and fibronectin, as well as numerous other growth factors and receptors, cytokines and ECM components,10 many of which are listed in Fig. The acute inflammation that occurs during wound healing is a classic example of the loss of the homoeostatic ‘separation’/sorting behaviour of epithelial and stromal-cell populations and is characterised by the appearance of activated fibroblasts (myofibroblasts); indeed, much of what we know about fibroblast activation was first observed in the context of acute inflammation. During wound healing, the goal of fibroblast activation is to re-establish the pre-injury tissue architectural and homoeostatic states,33 A crucial difference between chronic wounds/tumours and acute inflammation however, is the failure to abate the fibrotic response in the former. Whereas the properties of myofibroblasts lead to the re-establishment of tissue architecture in acute inflammation, the acquisition of these same properties over prolonged periods in chronic wounds/tumours leads to further disorganisation of tissue architecture and tissue desmoplasia, a term that specifically denotes an increase in stromal cells and matrix components within an injured tissue.3CAFs in tumours acquire the same features as do myofibroblasts in inflammation, and no systematic stratification or differences have been identified between these fibroblast populations. Figure 35 which represent the most immediate pool of cells to be activated into highly proliferative CAFs and recruited to the site of tumour growth. It is unclear whether these different CAF sources give rise to different CAF subpopulations that maintain distinct properties within the tumour over time.During both inflammation and tumour growth, the number of activated fibroblasts at the injured tissue site increases via recruitment of progenitor cells from different sources and proliferation of these cells, as well as tissue-resident fibroblasts,36 Indeed, conditions of chronic inflammation, such as inflammatory bowel disease, hepatic fibrosis or prostatitis, can be pre-emptive of tumour development at these anatomical sites.37 Furthermore, because of the similarities between chronic wounds and tumours in establishing fibrotic tissue niches, certain signalling pathways have been identified in both conditions as potential therapeutic targets for decreasing fibrosis and CAF-driven tumour aggressiveness, including the platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) pathways.39Tissue desmoplasia is common in cases of chronic inflammation and tumours, highlighting the parallels between the two states, and positions the activated fibroblasts as an important mechanistic link between cancer and fibrosis.The above discussion has compared the two best-known states of fibroblasts—quiescence versus activation in chronic wounds/tumours. In this section, we focus on the transition that occurs between these states during tumour growth, which is concurrent with the breakdown of local tissue structures and tumour cell invasion. In addition to highlighting the properties of CAFs in invasive disease, we emphasise studies that have provided insight into what precedes and what follows the establishment of a mature CAF and this timeline is illustrated in Fig. 40 and the mechanisms and timing of fibroblast recruitment and activation at the preinvasive stage are poorly understood. The local dysregulation of epithelial signalling and proliferation in an otherwise homoeostatic tissue potentially result in altered chemical signalling to the surrounding tissue stroma and mechanical changes to the BM. Other than paracrine signalling from epithelial cells,41 potential sources of fibroblast-activating signals include cues from immune cells that are recruited to the site of epithelial hyperproliferation,42 microRNAs (miRNAs) produced by the growing mass of transformed epithelial cells43 and mechanical cues arising from a local increase in tissue tension.44 These activating signals potentially permeate through the ECM and BM structures of certain porosities (BM pore diameter ranges from 5 nm to 8 µm21); alternatively, tumour-derived exosomes carrying, for example, miRNAs or TGF-β family members, have been implicated in establishing tumour-promoting signalling in tumour niches and fibroblast activation.46 Immune cells, especially macrophages, B cells and regulatory T cells, can directly activate fibroblasts, and increased densities of these cells are found in tumour stroma,47 where they accumulate in response to the release of inflammatory mediators by hyperproliferative, transformed epithelial cells.49 Fibroblast activation might also result from an external insult responsible for initiation of the neoplastic growth itself: one study showed that dermal fibroblasts could be primed by UV radiation, which resulted in an epigenetic loss of Notch signalling, increasing their synthesis of growth factors and capacity to remodel the matrix.50To date, it is unclear whether changes in stromal properties precede or are a result of the hyperproliferation of transformed epithelial cells,51 and identified a 66-gene signature common to fibroblasts from invasive breast cancer and a subset (40%) of DCIS tissues, suggesting that fibroblast activation occurs at the preinvasive tumour stage. Analogously, a comparison of the expression of angiogenic genes between the stroma of non-malignant, preinvasive, invasive and metastatic breast tissues revealed a clustering of these genes in all but the healthy tissues.52 Furthermore, a study specifically investigating the appearance of α-sma+ myofibroblasts in normal, benign and malignant breast tissue observed a shift from the presence of exclusively CD34+ fibrocytes in normal tissue to the emergence of α-sma+-reactive myofibroblasts in benign lesions and invasive tumours concurrent with the loss of the CD34+ cell populations.53 These results further suggest that stromal-cell activation can occur at the preinvasive stages of the disease, and that the tumour stroma as a whole is then ‘primed’ for the subsequent stages of tumour development.A few specific cancers, such as breast ductal carcinoma in situ (DCIS), offer the opportunity to explore fibroblast status prior to BM breaching. For example, one study examined the gene expression signatures of stromal cells in DCIS versus invasive cancer54 and exposes these cell populations to each other, allowing for direct contact and increased short-range signalling between them. CAFs are capable of digesting BM components and might therefore be responsible for mediating significant breaching of this structure.2 Previous studies, for example, have revealed the capacity of CAFs to break down BM via matrix-metalloproteinase (MMP)-mediated digestion to generate ‘tracks’ that were subsequently occupied by invading carcinoma cells.55 Moreover, CAFs that were presented with murine mesentery (a BM substitute) induced cell permeability in the membrane via an MMP-independent mechanism; they remodelled the matrix by applying contractile forces, creating gaps between BM fibres that allowed cancer cells to pass through.56 Importantly, a caveat of such studies is the use of fully mature CAFs sourced from tumours that were already invasive and our appreciation of the contribution of stage-matched CAFs to BM breaching during the transition to locally invasive tumour growth is limited.The transition from in situ to locally invasive disease is a crucial step for tumour growth and requires breaching of the BM. This process is carried out actively by the tumour epithelium, immune cells and CAFs57 In inflamed tissue, fibroblasts increase their production of stress fibres (composed of cytoplasmic actin) and organised fibronectin on their cell surface, due to the mechanical tension present. These features promote the transition of a normal fibroblasts to an intermediate stage known as a protomyofibroblast. This cell type is characterised by an enhanced contractile capacity compared with quiescent fibroblasts, and has been observed in 2–4 day-old wounds within the granulation tissue .58 Protomyofibroblasts have not yet incorporated α-SMA into their stress fibre network in order to generate the potent traction forces on the surrounding matrix that are characteristic of a mature myofibroblast.31 However, continued mechanical tension induces the production of specific splice variants of fibronectin, mainly ED-A and ED-B, which have been shown to guide transdifferentiation of protomyofibroblasts into myofibroblasts through poorly understood mechanisms.58 Concurrently, autocrine or paracrine signalling through the TGF-β pathway results in the synthesis of α-SMA and collagen type 1, ultimately giving rise to fully mature myofibroblasts.57As a consequence of the changes in tissue structure (ECM composition and organisation), tissue architecture/composition (intermingling of CAFs with other cellular fractions) and activated fibroblast secretome during local tissue invasion see Fig. , the par10 For example, the production of TGF-β and stromal-cell-derived factor-1 , by CAFs results in further fibroblast activation.59 Similarly, CAF-mediated expression of leukaemia inhibitory factor (LIF) is responsible for initiating, within CAFs themselves, the constitutive activation of Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) signalling via p300-mediated histone acetylation, resulting in enhanced CAF contractility and consequent ECM remodelling. These properties lead to further CAF activation, consequent tumour-cell invasion and further breakdown of tissue architecture.61In contrast to the process described above, the temporal dynamics of fibroblast activation during tumour progression have yet to be firmly pinpointed. However, it is reasonable to surmise that a fibroblast entity equivalent to the protomyofibroblasts found in acute wounds might exist, which can be thought of as an intermediately activated fibroblast with lower contractility that arises at some point during the early stages of tumorigenesis. The further activation of such intermediates could then be achieved by the several CAF-mediated signalling pathways known to perpetuate further CAF activation.62 First, fibroblast transition from the quiescent to the activated state is not based on the acquisition of genetic errors . Second, the epigenetic status is heritable and microenvironmentally induced, and is therefore likely to fit the dynamics of fibroblast activation.64 For example, TGF-β and other growth factor signals result in global changes in the methylation status of fibroblast genes.65 In lung cancer stroma, SMAD3 was one of the genes found to be silenced by hypermethylation, which elicited a hyper-responsiveness of CAFs to TGF-β signalling, leading to an increased expression of wound-response genes such as COL1A1, thereby linking TME methylation to increased ECM deposition by CAFs.66As exemplified above, epigenetic regulation is considered, for a number of reasons, to be a major contributor to CAF activation within the TME.2 These mixing zones attract a continuous supply of fibroblast progenitors from different cell pools (see above), which differentiate/become activated, proliferate and infiltrate the tumour mass.67 Such interface zones of tumour cells and tumour stromal-cell populations are arguably sites in which intense TME remodelling occurs, and which are characterised by unique functionalities and marker expression, including markers of invasion or EMT in epithelial cells.69 Utilising a clonal tracing strategy, a recent study found that tumour cells with cancer stem cell properties were located along the tumour margin and regulated by CAF-secreted osteopontin.70 With respect to CAFs, a study investigating the differences between fibroblasts at increasing distances from the surgical tumour margin identified increased pro-proliferative and EMT-inducing capacities between all CAFs compared with fibroblasts taken at least 10 mm from the tumour margin.71 More importantly, however, CAFs sourced from the interface zone were more potent inducers of tumour progression than CAFs present within the tumour core, as assessed in vitro, indicating that interface CAFs possessed a pronounced tumour modulatory capacity.71During local invasion, the physical interaction and bidirectional movement between epithelial and stromal tissue components that follows BM breakdown creates a ‘zone of mixing’, from which metastatic cells eventually emerge.73 The consequent rise in tissue tension continues to catalyse the activation of CAFs, which enter the feed-forward loop of contractility, ECM stiffening and TGF-β production.31 Analyses of CAFs present in invasive carcinomas have revealed the presence of diverse activation markers and associated functions, which might reflect differences in CAFs across tissues, differences in the proportions and origins of CAF progenitor cells recruited, and a possible spectrum of CAF ‘maturity’ within the TME.74The complete CAF response to the presence of tumour epithelia and remodelled matrix takes place in tumour–stroma mixing zones. The local tissue architecture becomes highly distorted by CAF infiltration, with aberrant de novo fibrinogenesis and matrix cross-linking (via CAF-secreted lysyl oxidase).75 The tumour-promoting activities classically identified in CAFs across tumour tissues include promoting tumour cell survival, growth and stemness, as well as metastatic capacity.10 Furthermore, CAFs have been implicated in affecting chemoresistance via specific CAF paracrine signalling (e.g. interleukin [IL]-6 76 or plasminogen activator inhibitor [PAI-1] 77), as well as CAF-mediated drug scavenging and the establishment of a biophysical ECM barrier (the latter two were more commonly identified in highly desmoplastic solid tumours such as PDAC). These CAF activities hamper drug delivery and reduce drug efficacy.79 CAFs with tumour-restraining activity, on the contrary, have been named the ‘F1’ subtype, and a landmark study has shown their tumour-restraining qualities in the pancreatic tumours in a transgenic mouse model, where depletion of α-SMA+ CAFs resulted in tumours with enhanced aggressiveness via an upregulation of CD4+ regulatory T cells.16 Meflin, a marker of perivascular mesenchymal stromal cells, was recently proposed as a marker of tumour-restraining CAFs in pancreatic cancer.18 By contrast, the ‘F2’ CAF phenotype denotes pro-tumorigenic fibroblasts, which have been the focus of most studies and described above.75 It remains unclear whether a choice exists in different organs between an F1 and F2 fate, or how the F1 phenotype persists in certain cases, and whether a switch from an F2 phenotype to an F1 phenotype is possible. Further stratification between CAF subtypes has been provided, again in pancreatic cancer, by Tuveson et al., who revealed the presence of CAF populations termed ‘iCAFs’, ‘myCAFs’ and antigen-presenting CAFs, which have distinct roles in direct interactions with tumour cells, paracrine signalling and modulation of the immune niche, respectively.81 Other studies have further assessed CAF heterogeneity at the single-cell level.83 Based on the expression patterns of specific sets of genes in breast cancer, distinct but overlapping clusters of ‘protomyofibroblasts’, ‘ECM-regulating myofibroblasts’ and ‘secretory myofibroblasts’ were identified and a fibroblast differentiation model could be constructed, which suggested a hierarchical organisation of fibroblast populations.82 RNA-Seq revealed further CAF heterogeneity with CAF subgroups expressing independent prognostic biomarkers and arising through EMT from distinct cellular sources, including tumour cells, resident fibroblasts and perivascular cells.83 Overall, this heterogeneous impact of CAFs on tumour progression might depend on the properties of the tumour cells themselves, or be reflective of tumour stage.35A number of classifications of CAFs have been recently proposed. At a minimum, and based on the most polarising effects of the CAF phenotype, a stratification between tumour-promoting and tumour-restraining CAFs has been identified.85 In terms of the positioning of senescence along the CAF life cycle timeline, no information is available; however, in vitro, senescent CAFs are encountered after several culture passages—particularly rapidly when the cells were obtained from older tissues.4 Senescence is a process associated with cellular ageing that results from telomere dysfunction and senescence stimuli, such as the accumulation of reactive oxygen species and DNA damage,86 eventually leading to growth arrest in G1, an enlarged morphology in vitro, non-responsiveness to mitogens and resistance to apoptosis. Importantly, apoptosis and senescence are viewed as alternative cell fates, and the balance between them appears to be cell specific.87 In CAFs, senescence has a reported dual effect: it arrests the proliferation of CAFs in the TME, which could therefore prove beneficial; however, senescent CAFs secrete inflammatory mediators and growth factors, such as IL-888 and MMP-2,89 which have pro-tumorigenic effects.90 SDF-1/CXCL12, a chemokine present in the secretome of senescent CAFs, was found to mediate tumour cell survival and proliferation both directly and through the recruitment of other TME components.91Another stage in the life cycle of CAFs that has been identified is senescence—that is, the decline of the proliferative and differentiation capacity of CAFs and CAF progenitors, with a concurrent increase in secretory capacity.86 However, the question of whether senescence is the obligate fate of a CAF in desmoplasia remains open. Notably, senescence was observed in fibroblasts cultured in the presence of doxorubicin and paclitaxel, and therapy-induced senescence is a well-known phenomenon,90 which highlights an important issue: although it is desirable to therapeutically induce senescence in cancer cells, the senescence of stromal-cell components might induce further inflammation, and ultimately, tumour relapse.90Because senescent fibroblasts accumulate in ageing tissues, it stands to reason that they could be responsible for conditioning stromal tissue over time, transforming it into a premetastatic niche.92 Spontaneous myofibroblast inactivation was specifically described in a model of regressing liver fibrosis, which demonstrated that half of the myofibroblast population is inactivated while the remainder undergoes apoptosis.93 However, very little is known about the role of inactivation or apoptosis in regulating CAF abundance and function within the TME. CAF loss of function, or at least partial inactivation, has been observed in hypoxic tumours, in which CAFs lose their capacity for contraction and matrix deposition owing to the inhibition of propyl hydroxylase domain protein 2 (PHD2) and stabilisation of hypoxia-inducible factor (HIF)-1α,94 which leads to a reduced metastatic potential. The apoptotic sensitivity of CAFs in vivo has also been described and found to be analogous to myofibroblast apoptosis in wound resolution or activation-induced T-cell death, although the underlying basis and timing of the process is unclear.95 It remains to be investigated under which circumstances apoptotic sensitivity, rather than senescence, is engaged, and questions surrounding CAF apoptosis, its role in regulating CAF abundance in the TME and its implications in tumour progression are yet to be answered. The addition of all-trans retinoic acid (ATRA)96 or the vitamin D analogue calcipotriol97 has been shown to inactivate CAFs in genetically engineered mouse models of pancreatic cancer. Beyond these studies however, CAF inactivation and significant levels of apoptosis are not reported in tumours in vivo, most likely as a result of the perpetual activating signals delivered to CAFs by the TME and its dysfunctional matrix,92 and possibly also due the induction of a senescent CAF phenotype after a certain, unknown, point in the CAF life cycle. These observations raise the question of whether treatments that aim to achieve CAF inactivation will succeed while the TME ‘wound’ persists.During wound resolution, the reconstituted ECM eventually takes over the mechanical load of the healing tissue, allowing a large portion of the now stress-released myofibroblast population to undergo apoptosis or inactivation.99 In this section, we summarise the attempts made at targeting or modulating the activated CAF phenotype within the context of the CAF life cycle. Included are compounds in preclinical and clinical stages, which can be roughly categorised, based on the point of activity within the life cycle that they target, as ‘blocking CAF activation’, ‘blocking the CAF hyperproliferative phenotype’, ‘blocking mature CAF function’, ‘inactivating the CAF phenotype’, and ‘directly depleting the CAF population’. Compounds that target CAF-mediated chemoresistance are classified under sections relevant to the mature CAF life cycle and its function. Of note, all five methods ultimately result in a reduced number of CAFs with pro-tumorigenic activity within the TME.While therapies targeting cancer cells directly encounter issues with drug resistance, due to the cells’ genetic instability, the concept of targeting CAFs therapeutically has received growing attention, since CAFs are characterised by relative genetic stability.101 such as the Rho/Rho kinase (ROCK) pathway and STAT3-mediated activation of insulin-like growth factor type 1 receptor (IGF-1R). As the hyperproliferative phenotype is also observed in fibrosis, anti-fibrotic treatments, such as pirfenidone and tranilast, which target CAF hyperproliferation in maturing and mature CAF populations, have actively made their way towards applications in anticancer therapies.104 Blocking mature CAF function is currently the focus of a large research area for anti-cancer, CAF-centric therapies that target the cells’ secretome and inhibit growth factor signalling, such as IL-6 and PDGFR signalling.106 Inactivation of the CAF phenotype attempts to transdifferentiate CAFs towards fates reminiscent of their quiescent counterparts, by leveraging mechanisms involved in apoptosis and/or inactivation, which have been observed to spontaneously occur in fibrotic models, as well as in acute wound healing, and include, for example, calcipotriol (a vitamin D receptor ligand) and minnelide (which de-regulates the TGF-β pathway).107 Finally, directly depleting the intratumoural CAF population has been accomplished by targeting cell surface markers, such as FAP108 and by inducing cell death through inhibition of the Bcl-2 pathway.95 Selected drugs and compounds within each category are summarised in Table 109 However, besides compounds in preclinical and clinical stages, there is currently no specific CAF-based adjuvant therapy on the market, which is undoubtedly a result of the inability to specifically target CAFs, due to a lack of CAF-exclusive surface markers and the functional heterogeneity within the CAF population.11Blocking initial and further CAF activation has been attempted by intercepting signalling pathways between CAFs and/or between cancer cells and CAFs,The growth and phenotypic changes that occur in tumour cells during disease progression are concurrent with, and enabled by, a co-evolving tumour stroma. In this review, we have outlined the properties of fibroblasts at different stages in their life cycle within this stroma, from fibroblast quiescence in homoeostasis to CAF senescence, and highlighted the parallels between activated states in acute injury and in chronic wounds/tumours. Based on this life cycle, we have also highlighted the therapeutic opportunities available to leverage CAF inactivation to achieve CAF depletion from tumours. Similar to the situation in chronically fibrotic tissue, in which a critical mass of myofibroblasts is responsible for extreme levels of matrix deposition (which ultimately leads to the loss of organ function), understanding how to interfere with the recruitment and proliferative mechanisms that lead to increased fibroblast densities in the TME has the potential to be hugely beneficial, as proliferation of CAFs leads to an exponential amplification of the amount of pro-tumorigenic interactions present within the TME.The overview presented here highlights that the life cycle of a CAF is strewn with knowledge gaps, particularly in its temporal trajectory Fig. . Studies110 These views imply the convergence of multiple progenitor populations towards a specific set of functionalities within the TME, such as enhanced contractility and protein synthesis. They also raise the question of whether current research efforts are merely capturing snapshot data from tumours, which in reality harbour a much wider hierarchical network of CAF maturation stages. Future studies aiming at revealing the ‘differentiation hierarchy’ of CAFs during tumour evolution must focus on stage-specific and stage-matched in vitro models of CAF–cancer cell co-cultures and untangle intratumoural heterogeneity on a single-cell or CAF subpopulation basis, as well as answering the question of whether in vitro culture approaches are supporting the full spectrum of CAFs present in vivo, and if so, which of these types of CAF might be clinically relevant.Our review also emphasises that CAF heterogeneity represents another challenging aspect of tumour biology. The concurrent existence of CAFs with different properties within the TME might depend on the origin of the precursor cell or the evolutionary stage or clonal composition of the TME. At the same time, myofibroblasts/CAFs have been described as cellular entities of a particular functional state, rather than as a singular cell type.The unknowns scattered throughout the CAF life cycle represent challenges for therapeutic intervention. Understanding these aspects could have implications for therapies that aim to reduce the presence of activated, pro-tumorigenic CAFs in tumour stroma. Such therapies could act on CAFs during their maturation phase, halting their differentiation into fully and irreversibly activated fibroblasts, and thereby cutting off further supply of CAFs for the growing tumour. Finally, a paramount consideration for any CAF-centred therapy will be the question of which part of the CAF spectrum remains unaffected by, or is specifically induced by, a given treatment. Given both the pro- and anti-tumorigenic potential of CAFs, understanding how to appropriately modulate these phenotypes at a given tissue/cancer stage will probably be critical in determining the failure or success of a fibroblast-targeting therapy.


We introduced and validated a method to miniaturize graded-index (GRIN) fiber-based optical coherence tomography (OCT) probes down to  For example, it can enable integration with minimally invasive medical needles. Note that although 41G needles  and inexpensive telecom splicing/cleaving equipment  for this work.We etched the probes by immersing them in a plastic [resistant to hydrogen fluoride (HF)] container filled with 48% HF solution  for 15 to 18 min at room temperature. The length of the etched part of each fiber probe was 2 cm.For the beam profiling measurements, we coupled an infrared laser  to the probes. We used a microbeam profiler setup, composed of a OCT measurement was carried out using a common path configuration. We used a homemade spectral-domain OCT (SD-OCT) setup, composed of the laser source described above, an optical isolator , a 90:10 fiber coupler , and a compact spectrometer . The spectrometer was equipped with an We used freshly enucleated porcine eyes to acquire comparative retinal B-scans. Prior to imaging, we placed the porcine eyes on a holder and removed the cornea and iris by dissecting a circular segment of the sclera. We also removed the lens but not the vitreous. The probes were immersed into the vitreous during the measurement. For both porcine eye and scotch tape imaging, we mounted the probes on a metallic post and used a motorized translation stage to scan the samples with a 33.1To achieve extreme miniaturization, we sought for a probe design allowing controllable size reduction with HF wet etching. We opted for a widely used probe configuration, an assembly of SM, CL, and GRIN fibers,diameter , composecladding . Note thcladding . Note thhickness . To illuNote that HF etching has been shown to increase the surface roughness of the etched fiber.3.2We routinely fabricated miniaturized probes by varying the CL and GRIN fiber length, to evaluate experimentally whether the miniaturization process affects their lensing performance. We used previously published analytic expressions for the working distance (WD) and beam waist (BW) of the probesWe etched the probes following the protocol given at the experimental part. The etching process affected only the diameter of the probes and the length of protective CL, while the splicing points remained intact  and 2. Wnsets in . We alsonsets in . The WD ,These results indicate that the miniaturization process does not alter the optical performance of the probes. In fact, the focusing ability of the probes remains predictable using analytic expressions.3.3We further investigated the optical performance of the miniaturized probes by performing comparative OCT measurements. me probe . To evale probes . We used–We also performed comparative B-scan imaging of a Scotch tape roll and tissue using the probes prior and after miniaturization. Given that intraoperative guidance of intralocular tools  is a promising field of applications of miniaturized probes,3.4Overall, the miniaturization technique presented in this work requires two additional steps compared to the well-established technique of preparing 4In conclusion, we fabricated and characterized extremely miniaturized focusing GRIN-based OCT probes having approximately half the diameter of previously reported probes based on the same focusing principle . The developed miniaturization process is simple and controllable and does not compromise their optical performance. Similar to their thicker counterparts, the miniaturized probes have tunable and highly predictable focusing capabilities. Due to their small size, they can be encased within 41G cannulas, the smallest devices used in clinical practice as for today for minimally invasive injections. Ophthalmic, brain, and gastrointestinal tract system endoscopic tools can be benefited by this approach as the use of miniaturized probes can significantly reduce their footprint and invasiveness.


Cancer cells differ in size from those of their host tissue and are known to change in size during the processes of cell death. A noninvasive method for monitoring cell size would be highly advantageous as a potential biomarker of malignancy and early therapeutic response. This need is particularly acute in brain tumours where biopsy is a highly invasive procedure. Here, diffusion MRI data were acquired in a GL261 glioma mouse model before and during treatment with Temozolomide. The biophysical model VERDICT (Vascular Extracellular and Restricted Diffusion for Cytometry in Tumours) was applied to the MRI data to quantify multi-compartmental parameters connected to the underlying tissue microstructure, which could potentially be useful clinical biomarkers. These parameters were compared to ADC and kurtosis diffusion models, and, measures from histology and optical projection tomography. MRI data was also acquired in patients to assess the feasibility of applying VERDICT in a range of different glioma subtypes. In the GL261 gliomas, cellular changes were detected according to the VERDICT model in advance of gross tumour volume changes as well as ADC and kurtosis models. VERDICT parameters in glioblastoma patients were most consistent with the GL261 mouse model, whilst displaying additional regions of localised tissue heterogeneity. The present VERDICT model was less appropriate for modelling more diffuse astrocytomas and oligodendrogliomas, but could be tuned to improve the representation of these tumour types. Biophysical modelling of the diffusion MRI signal permits monitoring of brain tumours without invasive intervention. VERDICT responds to microstructural changes induced by chemotherapy, is feasible within clinical scan times and could provide useful biomarkers of treatment response. This need is particularly acute in brain tumours, in which grading and diagnosis with biopsy can be costly and invasive2, with risk factors including bleeding, functional loss and increased metastatic potential3.Noninvasive techniques for monitoring changes in cell size, and other microstructural parameters 4. Ongoing treatment strategies differ substantially for patients that respond and those that progress5, yet traditional imaging methods of response that use measurements of enhancing tumour on T1-weighted MRI6, can be confounded by pseudoprogession,  and pseudoresponse, .Gliomas are the most common and most deadly type of primary brain tumour in adults, with an annual incidence of 4–5/100,000 people. For newly diagnosed glioblastoma (GBM), the most common and malignant of the gliomas, no treatment has yet been shown to be more effective than surgical resection followed by chemoradiation and adjuvant chemotherapy with Temozolomide (TMZ)14 can play a role, as its sensitivity to the restriction of water diffusion by tumour microstructure can be used to probe structures below the resolution of the image acquisition. It does not require the administration of a contrast agent and does not require any specialist equipment, not already present in almost all clinical MRI scanners. Various quantitative DWI models have been investigated for assessment of response in cancer, including the apparent diffusion coefficient (ADC)16, bi-exponential and stretched exponential modelling18, statistical diffusion modelling19 and diffusion kurtosis imaging21.A further challenge is that invasive biopsy cannot be performed repeatedly in the brain in order to unambiguously confirm true progression or true response with histology. Here, diffusion weighted imaging (DWI)23: restricted diffusion, which is expected to be greatest inside tumours cells, is represented by an impermeable “sphere”; isotropic hindered diffusion, which is expected to be greatest in the extracellular space, is represented by a “ball”; and fast, anisotropic pseudodiffusion, such as in vasculature, is represented by a “stick”. This configuration of compartments has been applied for modelling tumour microstructure in various tissues24, however, its ability to characterise response to chemotherapy in gliomas has not been investigated so far.In this work, we implement a three-compartment biophysical model applied to DWI data to provide clinically useful biomarkers of glioma microstructure. VERDICT (Vascular Extracellular and Restricted Diffusion for Cytometry in Tumours) uses multiple MRI diffusion gradients to probe the microscopic motion of water molecules in tumours at a range of length scales. The compartments aim to represent different water pools within the underlying tissue25, both to examine pre-therapy microstructure and to measure response to Temozolomide therapy. We imaged mice bearing gliomas using diffusion MRI at three post-therapy timepoints. Our hypothesis was that chemotherapy would cause the tumour cells to shrink as a consequence of cell death, which would alter the diffusion within the different compartments of the biophysical model. The aim was to compare the VERDICT parameters with other diffusion models, including ADC and diffusion kurtosis, to examine which was the earliest biomarker of treatment response. In the second part of this study, we applied VERDICT in a clinical set-up to image patients with gliomas. The aim here was to assess the feasibility of applying the VERDICT model in a range of different human gliomas to examine how the parameters vary with different tumour microstructure environments.In the first part of this study, we have investigated the use of VERDICT in a GL261 GBM mouse model2) where the tumour had a lower signal than the rest of the brain . Following 9-days of treatment, tumour volumes diverged in the two groups, increasing to 89 ± 22 mm3 in the control cohort, compared with 61 ± 22 mm3 in TMZ-treated mice.Tumour growth increased monotonically with time in both treated and control cohorts Fig. . MeasureADC = 28 ± 6, BICkurtosis = 19 ± 5, BICVERDICT = 13 ± 4.The diffusion models were fitted on a voxel-wise basis across mouse brains Fig. . VERDICTfsphere, Fig. dstick) of 10.8 ± 4.0 ×10–9 m2/s, sphere fraction (fsphere) of 0.54 ± 0.05, ball fraction  of 0.39 ± 0.06, stick fraction (fstick) of 0.07 ± 0.02 and sphere radius of 10.6 ± 0.6 µm. Mean ADC prior to therapy was 0.99 ± 0.06 ×10–9 m2/s. Mean kurtosis prior to therapy was 0.85 ± 0.03.ADC maps Fig.  and meanfball parameter also significantly increased by day 6  Fig.  and meany 6 Fig. . Of all y 6 Fig.  and sphere, Fig.  decreasere, Fig.  and ballll, Fig.  which stick Fig. , remaineick Fig.  decreasefsphere , histology was performed for comparison with VERDICT parameter maps Fig. . Coronalath Fig. .Figure 4fsphere, which was also significantly lower in the TMZ-treated group, compared to the control group .Quantitative analysis of H&E stained sections was performed to the estimate cellular volume fraction within the tumour, for comparison with the sphere fraction in the VERDICT model. A k-means clustering-derived threshold was used to mask the stained tissue . However, quantitative MRI techniques can potentially provide noninvasive biomarkers of tumour structure and function, which could be used as noninvasive biomarkers of tumour grade and/or response to therapy27. Multi-compartment models of the diffusion MRI signal32 can provide parameters linked to the properties of tissue microstructure, which have the potential to be useful for clinical assessment.The current radiological standard for assessing brain tumour response to therapy with MRI is to make two-dimensional measurements of tumour size, based on structural T1- and T2-weighted imaging (as recommended in the Response Assessment in Neuro-Oncology criteria23) and isotropic diffusion outside of these compartments . The aims of this study were to characterise the microstructure of brain tumours using the VERDICT MRI parameters, both in a mouse GBM model and in humans; to assess its ability to detect acute changes induced by chemotherapy  compared with traditional models; and to examine the feasibility of applying the VERDICT model to a range of different human gliomas.Of particular interest in the assessment of cancer therapy is the use of VERDICT, which broadly models three levels of diffusion in tumour tissue: restricted diffusion in cells (sphere), fast diffusion in vasculature  within 6 hours of treatment with gemcitabine and was detectable in vivo in the sphere compartment of VERDICT22.Prior to TMZ treatment in GL261 mouse orthotopic models, we measured a mean sphere fraction of 0.54 ± 0.05, ball fraction 0.39 ± 0.06, stick fraction 0.07 ± 0.02 and radius 10.6 ± 0.6 µm, each of which is physiologically feasible for tumour tissue and was reflected in our histological measures. With TMZ treatment, gross tumour growth was reduced although the effect was only clear between day 6 and day 9 Fig. –i. VERDI35. Based on structural MRI, the tumour phenotype for human GBM was most similar to the GL261 mouse model at day 9. In both mice and patients, a bulk tumour mass was observed containing regions of T2-weighted hyperintensity. However, mouse GBMs enhanced with gadolinium contrast agent across the whole tumour mass, whereas only a patch of solid enhancement or rim of enhancement was observed in the GBM patients. There was also no evidence of a peri-tumour region in mice.Given these promising results in mice, VERDICT was trialled in a cohort of patients with brain tumours, on a 3 T MRI scanner. A reduced diffusion MRI protocol with fewer diffusion shells and directions was implemented to permit a total imaging time of 12 minutes. This acquisition time is relatively long for practical clinical usage, but could be reduced further using efficient diffusion acquisition methodsfsphere was 22% compared to 17% in treated mice; fball was 72% compared to 76%; and fstick was 6% compared to 7%. The similarity in these values probably reflects the fact that patients with GBMs tend to present late into development of the tumour, whereas the tumours in the untreated mice at Day 9 have yet to progress to this stage. Moreover, it seems that treatment with Temozolomide has accelerated cell death in the treated cohort to the extent that the volume fractions are comparable with late-stage GBM patients.Based on ADC and VERDICT parameters, human GBMs were also most similar to GL261 mouse tumours at day 9 compared to the other tumour subtypes. The parameters were actually more comparable with the TMZ-treated cohort of mice rather than the control mice, despite the GBM patients receiving no treatment. Averaged across the equivalent region in GBM patients (tumour core and rim), fsphere, lower fball and higher radius parameter values, indicating an increase in restricted diffusion, typical of increased cellular density. It is possible that this simply reflects a greater mix of neuronal and tumour tissue compared to the bulk of the GBM, however, the lack of uniformity across the peri-tumour region indicates pockets of unusual tissue microstructure. Evidence of localised tumour progression within the peri-tumour region is highly desirable for pre-operative surgical planning36, therefore further imaging with VERDICT in more GBM patients is required to thoroughly interrogate this finding.VERDICT revealed interesting sub-regions of heterogeneous tissue microstructure within the peri-tumour zone of the GBMs, which were largely homogenous on structural images and ADC maps. These regions contained higher fsphere was increased and fball was decreased. Estimates of the sphere radius parameter were also considerably higher (15–16 µm), which was caused by many voxels within the astrocytomas and oligodendrogliomas hitting a prescribed upper boundary of 20 µm. The implementation of the VERDICT model used in this paper appears to be less appropriate for modelling these tumour subtypes, partly because of their diffuse phenotype which infiltrates the normal brain parenchyma, which the VERDICT model was not designed to represent. Calcification in oligodendrogliomas is also common and known to introduce susceptibility artefacts38, which may have affected the diffusion signal. Alternative biophysical models could be investigated and implemented to better approximate these tumour types22.Tissue microstructure in astrocytomas and oligodendrogliomas was markedly different to the GBMs. The VERDICT parameters indicated increased restricted diffusion  compared to GBMs: 40. For example, the constraints imposed on the models, such as which compartments  are chosen, can affect the parameter specificity42. For the present implementation, a stick compartment was used to approximate the vasculature, but another compartment type (e.g.: astrosticks: uniformly oriented sticks28) may have provided different but plausible results. There are other limitations of the present VERDICT model to consider as well. The sphere compartment only fits a single radius value within each voxel, which is not reflective of variable in vivo cell sizes or shapes. The relaxation times and proton densities of the different model compartments were assumed constant, but recent work shows that compartment-specific T2 values affect the final parameter values in biophysical models43. Furthermore, each tissue compartment will also have a distribution of diffusivities, however for VERDICT the diffusivity parameters were fixed to facilitate stable model fitting. If the diffusivity is inaccurate, then the error will affect the other parameters in the model. For example, this effect contributes to the radius parameter hitting the upper boundary in the non-GBM gliomas. Nonetheless, despite these shortcomings, VERDICT was the model which best captured the data based on BIC scores, and, showed a significant difference at the earliest timepoint when compared to ADC and kurtosis models. In both cohorts of mice, ADC increased and the mean kurtosis decreased as the tumours grew. These effects were likely due to the onset of necrosis in both treated and control groups, but was even greater in the TMZ-treated group, because of additional Temzolomide-induced cell death. A significant difference in mean ADC and mean kurtosis values was observed between the two cohorts of mice after 6 days of treatment. The VERDICT fball parameter also showed a significant difference after 6 days, whilst the sphere radius parameter showed a significant difference earlier still at 3 days post-therapy. Furthermore, the difference in effect sizes between the control and TMZ-treated animals observed between day 0 and day 3 were largest for the fball and fsphere parameters compared to the tumour growth, ADC and kurtosis measurements.More generally, whilst biophysical diffusion models attempt to provide insight into tissue microstructure, they are a simplistic representation. There are various factors to consider when constructing these models which affect the resulting parameter estimatesIn summary, VERDICT MRI appears to perform better than structural MRI, ADC or kurtosis measurements as a biomarker of tumour response in GL261-bearing mice. The clinical part of this study demonstrated that further optimisation is required when applying VERDICT to new tumour types. The VERDICT parameters in the patient GBMs appeared broadly consistent with the measurements in mouse GBMs, however, the model was less stable when applied to astrocytomas and oligodendrogliomas. Further studies to optimise VERDICT so that it can be generalised to all tumour subtypes are required.44.All animal studies were approved by the University College London Biological Services Ethical Review Committee and were performed in accordance with the UK Home Office Animals Scientific Procedures Act, 1986 and United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines4 GL261 mouse glioma cells (Supplementary Methods). At 13-days post inoculation (day 0), mice were randomly assigned to control or TMZ-treated groups (n = 12 for each), and baseline imaging was carried out. Immediately after scanning, mice in the TMZ group were administered with a first dose of Temozolomide  by oral gavage (130 mg/kg in vegetable oil). Two further doses were given on consecutive days, to a total dose of 490 mg/kg. Mice in the control group received sham doses of vegetable oil according to an equivalent regimen. MRI was performed (Supplementary Methods) every three days on a 9.4 T horizontal bore scanner , to a final timepoint at 22-days post tumour injection (day 9).Female, 8-weeks old, C57BL/6 mice were implanted with 2×102). Specific gradient combinations are detailed in Table 2 (B0) image was acquired for every combination of diffusion gradients. Total imaging time for VERDICT was 70 minutes.Tumours were localised using a structural T2-weighted spin-echo sequence. For VERDICT, diffusion-weighted images were acquired in a coronal orientation using a 3-shot spin-echo echo planar imaging (EPI) sequence, which included the following parameters: TR = 3 s, TE = min, data matrix = 64 × 64, FOV = 20 × 20 mm, shots = 3, slice thickness = 0.5 mm, slices = 5, averages = 2. In total, 46 diffusion weightings (each of 3 directions) were acquired in addition to a 42 direction DTI acquisition b = 1000 s/mm000 s/mm2Following acquisition of the diffusion weighted images, mice were injected with 0.6 mmol/kg of gadolinium-DTPA. After 10 minutes to allow for the contrast agent to circulate, slice-matched T1-weighted spin echo EPI images were acquired. Tumour regions of interest (ROI) were drawn based on these images and used during the quantification of the diffusion data. The total imaging time for all scans, including planning and shimming, was 100 minutes.22. In brief, a BallSphereStick model  models isotropic hindered diffusion; the “sphere” compartment (Ssphere) models restricted water in impermeable spheres; and the “stick” compartment (Sstick) models maximally anisotropic pseudodiffusion. The compartments are intended to capture the properties of diffusion in distinct water pools, namely the extracellular-extravascular space, the intracellular space and the intravascular space, respectively. However, given the complexity of tissue microstructure, this compartmentalisation may not always hold.VERDICT models the water signal from three non-exchanging tissue compartments within tumoursfball, fsphere and fstick correspond to the fractions associated with each compartment. Sball is a simple monoexponential with one parameter, dball, which represents isotropic diffusivity. Ssphere contains two parameters: dsphere, which represents diffusivity within a sphere45 of radius R. Sstick represents diffusion along a unidirectional tensor, with diffusivity dstick, and orientation given by the angles θ and ϕ in spherical coordinates.The total signal within a voxel is the weighted sum of the contributions from each compartment, according to:46 (the open-source Java code can be downloaded from: http://camino.cs.ucl.ac.uk/) using a similar iterative optimization procedure described by Panagiotaki et al.23 which accounts for local minima and Rician noise. VERDICT was fitted on a voxel-wise basis within the tumour ROIs and mean parameter values were calculated by averaging across the ROIs. For the mouse study, a total of six parameters were fitted: fball, fsphere, R, dstick, θ and ϕ. The fitted parameters were constrained so that the range of parameters were limited to biologically relevant values. The volume fractions were constrained between 0 and 1 and to sum to 1, the sphere radius was constrained to 0.1–20 μm and dstick was constrained to be larger than free water diffusion (dstick ≥ 3.05 μm2/ms47). For model stability, the ball and sphere diffusivities were fixed to: dsphere = 1 × 10–9, dball = 2 × 10–9 m2/s. The stick volume fraction was not fitted and instead was calculated as dstick = 8 × 10–9. These parameter choices were informed by previous applications of VERDICT to preclinical and clinical tumours using similar imaging protocols23.Model-fitting of the DWIs was carried out in MATLAB  with the Camino toolboxADC (apparent diffusion coefficient). The normalised signal is given by:To compare VERDICT with standard approaches, the diffusion data was also fitted with ADC and diffusion kurtosis models. The ADC model is a simple monoexponential signal decay with b-value, with one fitted parameter: Dk, which represents diffusivity corrected for kurtosis, and K, the mean kurtosis parameter which quantifies the level of dispersion from a Gaussian distribution. For instance, if K = 0, then the diffusion is purely Gaussian. When diffusion is impeded as the water molecules encounter cells, vessels and other structures, and this is reflected by a higher K. The normalised signal in the diffusion kurtosis model is given by:The diffusion kurtosis model assumes non-Gaussian water dispersion. It has two parameters: In the mouse study, parameter maps were generated by model-fitting of the diffusion-weighted images. For the ADC and kurtosis models, whole-brain parameter maps were generated as these models are generaliseable to multiple tissue types. However, as the VERDICT model is an unsuitable descriptor of normal brain tissue , only voxels within tumour ROIs were used for analysis. For statistical comparison between the different models, the same tumour ROIs were applied to the ADC and kurtosis parameter maps.For a similar reason, in the human study, modelling was only applied to tumour regions. In the GBM patients, ROIs were drawn around three separate tumour regions: these were the tumour core, the tumour rim and the peri-tumoural zone. Analyses were performed independently on each of these regions. Any regions of haemorrhage (hypointensity on T2-weighted images) within the tumours were excluded from parameter analysis because there was little-to-no signal available for fitting.in vivo MRI data.Optical projection tomography (OPT) of complete brains was used to quantify the blood volume of tumour tissue (Supplementary Methods), in a subset of mice , for comparison with kmeans function in MATLAB) was applied to the H&E slices to estimate the ratio of the stained tumour cells to unstained extracellular-extravascular space, for comparison with the VERDICT fsphere parameter, which is the parameter most comparable with intracellular volume fraction.Histological slices were used to quantify intracellular volume fraction for comparison with VERDICT parameters using an in-house MATLAB script. After the final MRI scan, mouse brains were extracted , immersion fixed in 4% PFA, and then sliced in a coronal orientation and stained with hematoxylin and eosin (H&E). A k-means clustering threshold  UCL/UCLH (REC: 07/Q0502/15). All experiments were performed in accordance with JRO and UK Good Clinical Practice guidelines, and all patients provided written informed consent.2) in a protocol that minimised scan time whilst maximising the range of diffusion times covered . Total acquisition time for VERDICT was 12 minutes. DWIs were normalised to b = 0 s/mm2 (B0) images acquired with the same TE. Five patients immediately underwent a second set of same-session scans for assessment of repeatability.Nine patients , enhancing rim (GBM Rim) and GBM perilesional T2-weighted signal abnormality (GBM Peri).Prior to VERDICT analysis, diffusion images were registered and corrected for eddy current effects using the ECMOCO toolboxfsphere) (Supplementary Methods). Nucleus volume fraction was estimated, rather than cellular volume fraction, due to the difficulty in delineating cell boundaries on biopsy samples. In principle, if it is assumed that nuclei volume is proportional to cellular volume, then nucleus fractional volume is a valid analogue of intracellular volume fraction.Biopsies were retrieved from seven of the brain tumour patients. Image processing and analysis was performed on H&E biopsy samples to estimate nuclei volume fraction for comparison with the VERDICT sphere fraction (ρ) was used to assess the strength of correlation between ADC and VERDICT and kurtosis parameters. For repeatability measurements of VERDICT parameters in patients, significance was assessed using a Wilcoxon matched-pairs signed rank test. The repeatability coefficient (RC) was also calculated for each parameter, which represents the 95% confidence interval of the difference in the two trials. RC is given by:ΔP is the change in parameter value between trials and RC provides an indication of the change required to observe a difference above variation.Mean parameter values are reported ± standard deviation (SD). For all model parameter comparisons between control and TMZ-treated groups, multiple t-tests with Holm-Sidak multiple comparisons correction were performed to assess significance using GraphPad Prism v6.01 . For mean parameter values, there were 32 comparisons across 8 parameters and 4 timepoints. For effect sizes there were 28 comparisons across 8 parameters and 3 time periods. Spearman’s coefficient (n is the number of data points fitted, k is the number of fitted parameters in the model and L is the maximum likelihood for the model. A lower BIC score corresponds to a better model fit.For model fitting comparison, voxel-wise BIC maps were calculated for all animals according to:Supplementary Material.


In this paper, the rate of heat transfer of the steady MHD stagnation point flow of Casson fluid on the shrinking/stretching surface has been investigated with the effect of thermal radiation and viscous dissipation. The governing partial differential equations are first transformed into the ordinary (similarity) differential equations. The obtained system of equations is converted from boundary value problems (BVPs) to initial value problems (IVPs) with the help of the shooting method which then solved by the RK method with help of maple software. Furthermore, the three-stage Labatto III-A method is applied to perform stability analysis with the help of a bvp4c solver in MATLAB. Current outcomes contradict numerically with published results and found inastounding agreements. The results reveal that there exist dual solutions in both shrinking and stretching surfaces. Furthermore, the temperature increases when thermal radiation, Eckert number, and magnetic number are increased. Signs of the smallest eigenvalue reveal that only the first solution is stable and can be realizable physically. Some applications of non-Newtonian fluids can be seen in polymer engineering, manufacturing of foods, petroleum drilling, certain separation processes, and papers4. It is very hard to convey all properties of numerous non-Newtonian fluids in a single momentum equation due to non-linearity among the rate of strain and stress of the fluids. In this regard, many non-Newtonian models have been proposed based on variation of physical characteristics9. Among these models, Casson fluid model which can be defined as “a shear-thinning fluid in which zero viscosity at an infinite rate of shear and an infinite viscosity at zero rates of shear”10 is the most popular one. Ullah et al.11 inspected Casson fluid over a non-linear stretching cylinder and found that the shear stress is directly proportional to suction/blowing and porosity parameter. Dual solutions have been obtained by Yahaya et al.12 in Casson fluid with the effect of homogeneous-heterogeneous reactions. Moreover, the analysis of the stability was carried out to determine which solution is stable. They found that only the first solution is stable and can be easily utilized in different applications. Khan and Husain13 explored Casson liquid on the circle disk where the system of governing ordinary differential equations was solved by utilizing homotopy method. Meanwhile, Hamid et al.14 considered Casson liquid encased in the cavity. Further, double solutions were found by Hamid et al.15 within the sight of thermal radiation. Stability analysis was additionally performed by utilizing of BVP4C in the MATLAB programming. Some ongoing improvements on Casson fluid can be found in these articles20.Two significant classes of fluids have gotten a lot of consideration from researchers and mathematicians in the previous few years, specifically Newtonian and non-Newtonian fluids. A fluid in which the rate of change of deformation is directly proportionate to viscous stresses is known as Newtonian fluid. On contrary, a fluid wherein properties of the fluid are not the same as Newtonian fluid is known as non-Newtonian fluid. The important property of non-Newtonian fluid is viscosity. In many industrial problems, the comportment of non-Newtonian fluids is more important as compared to Newtonian fluids21 proposed a method for solving incompressible steady-state 2-D viscous fluid on which later extended to many diverse aspects. Some of its recent important directions over-stretching flows can be seen in these articles26. Vajravelu27 and Cortell28 considered viscous fluid on a non-linear stretching sheet. In this paper, we also considered a stretching surface with the shrinking surface due to its extensive utilizations in different fields.The study of boundary layer Casson fluid flow on stretching and shrinking surfaces was widely investigated for single solution cases. Stretching surface has many applications in many manufacturing such as the extrusion of the molten polymers due to the slit die in productions of plastic sheets, paper productions, wires as well as in the fibers coating process of the food stuff. The qualities of final products in such processes depend heavily upon the cooling rate in the process of the heat exchange. Therefore, a MHD parameter is an important element to be considered so that the rate of the cooling can be controlled in order to obtain the desired quality products. Crane32. These multiple solutions exist due to several physical parameters impacts such as suction parameter, mixed convection parameter and so forth. Furthermore, the past researches demonstrated the probabilities of the occurrence of multiple similarity solutions of fluid flow over a shrinking sheet are more than over a stretching surface31. The possibilities of non-uniqueness solution of fluid flow on a stretching surface are probable when the flow is stagnation point flow or opposing flow. Similarly, multiple solutions for Newtonian fluids can be gotten easily as compared to non-Newtonian fluids. It is stated in the previously published literature that non-uniqueness of the solutions occurs because of the existence of non-linearity in the fluid equations30. However, the models of non-Newtonian fluids contain many non-linear terms and thus leads to non-existence of multiple solutions.From the previous couple of years, multiple similarity solutions of fluid flow problems have been considered on the stretching and shrinking sheets in the presence and absence of bouncy effect by numerous scholars32. In real situations, multiple solutions cannot be visualized in the boundary layer problems and difficult to be detected. Hence, many researchers fail to notice multiple solutions34. Multiple solutions of MHD fluid flow problems have been examined theoretically as well as numerically by numerous researchers. Dero et al.35 obtained triple solutions during the investigation of micropolar fluid with thermal radiation effect. Further, Dero et al.36 inspected the unsteady flow of nanofluid on the shrinking sheet and found double solutions in deaccelerated case. It seems that Ridha and Curie37 are the pioneers who found dual solutions in the opposing flow. The motivation behind this study is to consider multiple similarity solutions of the MHD stagnation point flow of Casson fluid on a permeable exponentially stretching and shrinking surfaces unanimously with viscous dissipation and thermal radiation effect.The boundary layer flows of fluid and their similarity solutions are gotten much attention due to their vast applications in many industrial fields10):x-axis is supposed alongside the shrinking/Stretching surface and the y-axis is normal to it. Further, B=There has been studied as steady incompressible 2-D stagnation point flow of Casson electrically leading fluid on an exponentially shrinking and stretching surfaces with the impact of thick viscous dissipations and thermal radiation. There has additionally been supposed that rheological equation of the state for the isotropic and the incompressible progression of the Casson fluid which are reported as allude0:1\documS is the suction/injunction parameter, and along the following boundary conditionsFollowing similarity variables are used to get the similarity solutionsBy employing Eqs.  and 7)  7) in EqPhysical quantities of interests are coefficient of skin friction, and the local Nusselt number, which are reported as:15, the upper branch solutions always show stability and physically reliability. On the other hand, the second solutions are unstable and consequently physically unreliable. The same remarks were reported by many researchers [refer to42].In this study, we found dual solutions of Eqs.  along wiIn order to accomplish the solutions’ temporal stability, the unsteady form of Eqs.  must be  of Eqs. . Equatio of Eqs.  can be eThe new dimensionless time-dependent variable By substituting Eq.  in Eqs. With corresponding boundary conditions43, Ismail et al.44, and Naganthran et al.45, the stability of the dual solutions is tested by perturbing the steady solution by using following functionsAccording to Lund et al.ting Eq. –17, we gSubject to boundary conditionsSystem of linearized eigenvalue problem Eqs.  along boThe solution is said to be stable flow if and only if the sign of the 46 for different values of the Casson parameter In this segment of the article, we discuss about the effect of numerous physical rising parameters on temperature, velocity, rate of heat transfer, and coefficient of skin friction profiles. In order to validate the results of our numerical technique, the numerical results of mentclass2pt{minimFigure 47. Figure Figure Figure mentclasspt{minimaThe rate of heat transfer of the steady MHD stagnation point flow of Casson fluid on the shrinking/stretching surface in the existence of thermal radiation and viscous dissipation has been examined. By using similarity transformation, the self-similar nonlinear ODEs have been gotten and then solved by using the shooting method in MAPLE software. Double solutions occur for the different ranges of the velocity ratio parameter and mass suction parameter. Stability analysis is done for the solutions by using BVP4C solver in the MATLAB software and the results suggest that only the first solution is the stable. It is found that the velocity and its boundary layer thickness decrease for the greater


Staphylococcus aureus superinfection. We report an exceptional, not-yet described complication of chickenpox with hypopyon-forming superinfection in an atopic child. In addition, our case nicely underscores the necessity of early VZV vaccination, which has been available and recommended now for more than 10 years in pediatric vaccination programs to avoid severe complications.Varicella-zoster virus (VZV) infection, also known as chickenpox, is a common childhood affliction. Generalized small itchy single-standing vesicles on erythematous skin are typical. Both cutaneous and systemic complications of the VZV infection may commonly occur. A three-year-old girl with a previous history of mild atopic dermatitis presented in our Pediatric Dermatology Clinic in poor general condition, with a skin rash predominantly consisting of generalized large blisters with hypopyon sign and erosions. On a closer look, scattered erythematous papules and vesicles were also visible. A positive Tzanck smear from an intact pinhead-sized vesicle and VZV PCR confirmed the clinical diagnosis of chickenpox. Cultures from hypopyon material revealed  Varicella-zoster virus (VZV) is the causative agent of varicella (chickenpox) as primary infection and herpes zoster (shingles) as re-activation of the virus . The priS. aureus [S. aureus superinfections and AD deterioration [Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease, typically starting in early infancy in genetically predisposed persons . In pati. aureus ; this adioration .A three-year-old, otherwise healthy girl suffering from mild atopic dermatitis (AD) (SCORAD index of <25) presented in our Pediatric Dermatology Outpatient Clinic in reduced general condition with high-grade fever and a new skin rash. Her skin exhibited generalized blisters of 2–5 cm in diameter, partly translucent, partly with pus accumulation in the dependent part of the flaccid bullae forming a transverse fluid level (hypopyon sign) , 4. ErosStaphylococcus aureus without methicillin resistance.Rapid diagnosis was made by Tzanck smear from a small intact vesicle revealing multinucleated giant cells and acantholytic keratinocytes. Herpes polymerase chain reaction (PCR) from both serum and blisters were positive for varicella-zoster virus (VZV). VZV serology demonstrated a recent infection . In addition, bacterial culture from the skin smear revealed superinfection with Staphylococcus aureus superinfection on the basis of atopic skin and VZV infection, the patient was isolated in the pediatric ward and received acyclovir 130 mg TID (10 mg/kg/day) and amoxicillin/clavulanic acid 650 mg TID (50 mg/kg/day) intravenously for 5 days. She also received parenteral fluid substitution, paracetamol infusions, and oral antihistamines. Subsequently, treatment was switched to oral therapy for an additional 5 days. She was discharged after 10 days. The topical treatment consisted of neomycin sulfate powder on intact blisters and methylprednisolone aceponate 0.1% cream plus sterile paraffin gauze dressing of the erosive skin. She recovered completely, only some eczema plaques persisted from the known AD  strains [VZV infection is a common, airborne droplet-transmitted primary, highly contagious, acute childhood infection with typical morphology and distribution of skin lesions—easy to diagnose for the trained clinician . While t strains . In our Hypopyon sign is rarely reported in secondarily infected vesiculobullous autoimmune disorders , but hasIn several European countries and the United States, VZV vaccination is a fixed component of vaccination programs for children between the 12th and 18th month of life . Since 2The peculiarity of our case is that i) the typical distribution of chickenpox was missing, as palms, soles, scalp as well as oral mucosa were disease free; ii) predominant large blisters and unusual hypopyon sign almost masked the acute VZV infection, leading to the disorder being overlooked; iii) although bacterial superinfections are common in chickenpox, hypopyon sign has, to the best of our knowledge, not yet been reported. In addition, our case nicely underscores the necessity of early VZV vaccination in children to avoid severe complications and hospitalization.


Pleomorphic adenomas are benign salivary gland tumours that mostly affect the superficial lobe of the parotid gland. They are commonly found incidentally as rigid, unilateral, painless masses, growing gradually. Ninety percent of pleomorphic adenomas occur in the parotid gland, while 10% appear in minor salivary glands. The incidence of the parotid tumour is 2.4 in 10,000 per year. We describe the case of a 53-year-old woman presenting with a gradually growing retroauricular mass on the left side of the ear. Radiographic imaging and histopathological findings suggested a pleomorphic adenoma of the parotid gland. The tumour was removed and the patient recovered well upon follow-up. Pleomorphic adenoma is well recognized as a benign mixed tumour. It is the most frequently encountered salivary tumour in the clinic, accounting for two-thirds of all salivary gland neoplasms . In geneA 53-year-old woman presented with a posterior left retroauricular mass, that was discovered 10 months ago and resected respectively at a local hospital. Ten days after this operation, a pruritic, painless lesion of a size 2 cm x 2 cm was observed at the incision area . The patWorld Health Organization has described pleomorphic adenoma as clear-defined metastasis distinguished by its chondroid, mixed and mucoid appearance . PatientMRI is preferred for its exceptional illustration of soft tissue and its characterization of tumour border and relationship with enclosing structures . The hisPleomorphic adenoma is the most frequent tumour that originates from the parotid gland. Diagnosis may prove challenging in a setting of unusual findings, which can lead to blind alleys when recommending treatment. This was a rare case of pleomorphic adenoma that presented as a mere retroauricular mass. This case is an example that accurate history, diagnostic imaging, and satisfactory tissue sampling is crucial to exclude other lesions associated with the salivary glands. Appropriate diagnosis led to an excellent prognosis with less chance of recurrence.


Adrenal myelolipoma is a rare, non-functional, benign neoplasm which is constituted of mature haematopoietic elements and adipose tissues in various proportions. It is diagnosed accidentally and frequently with the widespread use of imaging modalities.We report a 63-year-old lady with incidental findings of adrenal tumour on computed tomography (CT) scan during a routine medical check-up. She underwent tumour resection in view of a large tumour of 7 cm in size.CT scan is sensitive to diagnose adrenal myelolipoma in view of its fat-laden property and useful to monitor the tumour progress. Even previously she opted for conservative management; the decision for surgery was made in view of enlarging tumour and risk of surrounding tissue compression.With increased awareness, the detection rate of this tumour is improving, hence able to prevent the complications of a large tumour such as compression, bleeding and tumour necrosis. It is localized in the retroperitoneum, mostly involving the adrenal glands. In the past, it was discovered at autopsy but nowadays is usually diagnosed accidentally and frequently with the widespread use of current imaging modalities. The use of ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) has enhanced the detection rate up 15% of incidental adrenal masses [Adrenal myelolipoma has an equal gender distribution and is commonly found between the 5th and 6th decades of life with a mean age of 62 years . We revi2A 63-year-old female was noted to have an adrenal mass during her medical check-up 7 years ago. She has underlying hypertension with one anti-hypertensive drug. Otherwise, she does not have endocrine disorders or associated comorbidities in the patient and relatives. During the initial finding, it was 4.5 cm in size. Since it was asymptomatic, she was decided for routine imaging follow up. She underwent a two-yearly computed tomography (CT) scan to monitor the growth of the tumour. It remained stable initially until recently it started to grow to a size of 7 cm. The CT features were in favour of right benign adrenal tumour but in view of the risk of compressive symptoms, she was decided for surgical resection of the tumour via laparoscopic retroperitoneal approach. The surgery went well without any intraoperative and postoperative complications. She was discharged after day 2. There was no further treatment or follow up required.Grossly the tumour was firm with a lobulated surface measuring 95 mm × 85 mm × 40 mm and weighing 140 g . It was Histologically, it is a well-circumscribed tumour composed of lobules of mature univacuolated adipocytes separated by fibrous septae and rimmed by a thin capsule A. There 3Adrenal myelolipoma is a very rare variant of adrenal lipoma. In our case, she is 63 years of age, which falls to the mean age group and the tumour size is 7 cm which is larger than the baseline of 4 cm. It is usually asymptomatic, but it can cause flank pain and abdominal discomfort from compression to the surrounding structures, tumour necrosis, rupture, or bleeding leading to haemorrhagic shock . Due to Routine imaging modalities such as ultrasonography, CT, and MRI are all effective in diagnosing adrenal myelolipoma. Among all, CT is the most sensitive modality on the basis of the fat-laden tumour . Since iIn cases when the diagnosis can not be made radiologically or in any doubt of malignant potential, the use of fine needle aspiration cytology (FNAC) can be considered . FNAC miHistological assessment can be made on the resected specimen. Thorough gross and microscopic evaluation is needed for confirmatory diagnosis and to exclude malignancy. Specimens should be sampled 1 cm apart for microscopic analysis in order not to miss any malignancy. Microscopically, a typical myelolipoma showed the presence of a mixture of mature adipocytes with foci of trilineage hematopoietic elements .Management of adrenal myelolipoma should be per individual basis. In a lesion of less than 4 cm in size, conservative treatment can be applied with an imaging technique. Surgery is indicated in symptomatic tumours, rapidly growing lesions, or tumours more than 6 cm in size . This is4Adrenal myelolipomas are clinically silent tumours and mainly benign in nature. Patients with adrenal myelolipoma are usually presented with incidental findings. This type of tumour can be definitively diagnosed with CT imaging. With increased awareness, the detection rate of this tumour is increasing, hence able to prevent the complications of a large tumour.The authors have no conflict of interests to declare.The study did not receive any funding.No ethical clearance required as it only involves case report.Written informed consent was obtained from the patient.Case design and writing – NA, FH.Case collection – OM.Literature review – AAW, TTT, NASNL.Acquisition of histology figures – NA.Manuscript editing – FH.No ethical clearance required as it only involves case report.Firdaus Hayati.Not commissioned, externally peer-reviewed.