Following 3 decades of extensive research into PI3K signaling, it is now evidently clear that the underlying network does not equate to a simple ON/OFF switch. This is best illustrated by the multifaceted nature of the many diseases associated with aberrant PI3K signaling, including common cancers, metabolic disease, and rare developmental disorders. However, we are still far from a complete understanding of the fundamental control principles that govern the numerous phenotypic outputs that are elicited by activation of this well-characterized biochemical signaling network, downstream of an equally diverse set of extrinsic inputs. At its core, this is a question on the role of PI3K signaling in cellular information processing and decision making. Here, we review the determinants of accurate encoding and decoding of growth factor signals and discuss outstanding questions in the PI3K signal relay network. We emphasize the importance of quantitative biochemistry, in close integration with advances in single-cell time-resolved signaling measurements and mathematical modeling. As more and more information has accumulated on the molecular details underlying PI3K–AKT signaling, the field has begun to appreciate that the canonical PI3K pathway is embedded within a larger and highly context-dependent network of protein–protein, protein–lipid, protein–nucleic acid, and protein–metabolite interactions. As such, the intuitive view of PI3K signaling as a deterministic pathway is no longer sufficient to capture the complexity in the network  connections , in stark contrast to the legitimate quest for understanding the emergence of biological function from the dynamic interactions of individual components   . Thus, ssiology) .That systems biology is firmly embedded in quantitative biochemical and molecular processes comes from the fact that cellular function emerges from chemical interactions among diverse molecules. Of these, proteins are the most important molecular machines and are linked functionally through allosteric or other mechanisms into biochemical relays. These relays carry out a range of computational tasks, including amplification, integration, and information storage . Consequ3/PIP2. Moreover, they facilitate the faithful interpretation of the spatiotemporal dynamics of PIP3/PIP2, which can encode the identity of an upstream stimulus, such as growth factor–specific activation of receptor tyrosine kinases (RTKs). Depending on the quantitative properties of the upstream stimulus , the downstream signaling and gene regulatory networks orchestrate an appropriate biological output. Importantly, the underlying biochemical circuits are not only responsible for enabling a cell to sense changes in its environment but also endow it with the capacity to anticipate such changes P2 form part of an exquisitely fine-tuned network of positive and negative feedback loops, including F-actin and members of the Ras and Rho GTPase families  and epidermal growth factor (EGF). This extends beyond binary detection of a signal (present/not present) and includes the capture of dynamic concentration changes. This notion raises the question as to how cells achieve such discrete signaling specificity by using the same set of signaling molecules downstream of a wide array of cell surface receptors. While the field has yet to develop all the relevant context-dependent PI3K signaling models to address this question comprehensively and experimentally, the available evidence suggests that spatiotemporal (de)multiplexing is the basis for signal encoding through PI3K activation .3/PIP2 biosynthesis (phospholipids found in miniscule amounts within cellular membranes), alongside the intracellular activities of key downstream effector enzymes such as AKT, ideally at single-cell resolution. Nevertheless, as discussed later, the currently available data across different signaling systems have provided important insights to build upon. While we focus most of the following discussion on the protein kinase AKT as one transducer of a growth factor–induced PI3K signal, additional PI3K and PIP3/PIP2 effectors exist P2 production in live MCF7 breast cancer cells have revealed quantitatively distinct dynamics of PI3K activation, as a function of both stimulus identity and concentration  as the difference is only apparent in the slope and magnitude of the signal in the first 10 min of growth factor stimulation, with subsequent activity levels converging at a similar quasi steady state –mediated suppression of the AP2 clathrin adaptor protein and an associated delay in IGF1R internalization  . Accordiversus RAS–ERK activation underpins the functional classification of the IGF1R as “mitogenic” compared with the highly homologous INSR, which is classified as “metabolic.” This relative balance appears to be determined by the extent and route of receptor internalization P2 or yet another second messenger, PIP2 P2 , 59. TheIP2 . Endosommulation , 61, witmulation , 62.De novo membraneless RTK-based pathogenic biomolecular condensates have recently emerged as alternative sites of oncogenic activity in cancer cells , inositol polyphosphate 4-phosphatase type II (INPP4), and PH domain leucine-rich repeat protein phosphatase (PHLPP) . This ise.g., ligands, receptors, adaptor proteins, kinases, and phosphatases), initial activity status, reaction rate constants, and diffusion coefficients. In addition to the overall network structure, the subcellular localization, and temporal activity of its individual components, the development of predictive mathematical models of PI3K signaling will require estimates of the key biochemical parameters that determine information flow within the network. These parameters include concentrations of individual components , whereas PDGFRβ has five docking sites including a high-affinity site (KD = 10 nM)  . These b= 10 nM) . If bina= 10 nM) .versus-high concentration of EGFR ligands elicit distinct and sometimes opposite phenotypes   , 77, 78.ormation . Furtherignaling . Notablyenotypes . The undenotypes . Attestienotypes .Collectively, these examples emphasize that mapping key biochemical determinants is essential as part of integrated workflows aimed at generating a quantitative systems-level understanding of the PI3K signaling network.i.e., digital “all-or-nothing”) pulses of ERK activity used by single cells to encode increasing growth factor concentrations   . The latperties) . The tec setting . It is pi.e., molecular attractors) themselves change as a function of environmental inputs, non–cell-autonomous processes, and regulated noise regulatedrsus EGF . Such marsus EGF . This prrsus EGF . Thus, brsus EGF , 106, asi.e., context). Conversely, the constant improvements in weather prediction accuracy rests on the ability of the underlying mathematical models to capture context-dependent changes in noisy processes as a function of time , to be used as pharmacological target  and (2), with the ability to predict therapeutically relevant perturbations capable of stabilizing a desired network configuration and phenotypic function, as determined by the disease context.A biochemical systems view of PI3K signaling may therefore be of great utility for systems pharmacology where the aim is to use prior knowledge and computational modeling to identify intervention strategies that tune a disease network toward a signaling configuration that once again enables normal function , 120. Knl target . The goae.g., an experimental assay) to the broad and complex   to address the challenge of using diverse and typically sparse and noisy experimental data  . Such prtal data . DiffereAchieving a systems biology understanding of the PI3K signaling network calls for an in-depth understanding of and practical proficiency in biophysics, biochemistry, cell biology, physiology, bioinformatics, statistical mechanics, control theory, mathematical modeling, and last, but not least, high-quality data management. Success will therefore depend on interdisciplinary and multidisciplinary collaborations, similar to the approach undertaken by the National Institutes of Health–based LINCS (The Library of Integrated Network-Based Cellular Signatures) . This feversus 3D, coculture, adherent versus suspension)  , 133, mepension) , and oxypension) , to namepension) , 137. Whpension) . Such atpension) , includiBeyond environmental variables, genetic drift may cause even seemingly identical cell lines to behave differently under otherwise similar conditions . AlthougInvariably, signaling studies seeking to establish cause-and-effect relationships rely on perturbation experiments. Most of these feature genetic and/or pharmacological approaches. To some extent, the choice of these is inherent to the choice of biological model(s), and yet key considerations are worth highlighting separately when it comes to quantitative signaling studies.First, it is important to avoid the trap of binary thinking, which often leads to the use of genetic overexpression models even if alternatives are available. In the section “Biochemical determinants,” we alluded to the fact that the nonlinear input–output relationships that govern signal transduction networks make these extremely sensitive to subtle changes in specific biochemical determinants, including molecular concentrations. The system-wide consequences of such changes are difficult if not impossible to predict with unaided intuition alone. Given advances in CRISPR–Cas9 and precision gene editing, it is prudent to minimize the use of overexpression despite the faster time to  results afforded by such systems. This is especially important if the aim is to model the consequences of disease-causing variants in PI3K signaling components . If over50 and IC50 values are context-specific, and, when available, should be used as guiding for the set-up of dose–response assays to determine the optimal concentration for a different system. Moreover, to avoid misleading conclusions of a binary nature , both acute and long-term signaling changes should be evaluated in the relevant model system. For example, dose- and time-dependent proteomics can help decrypt drug actions while also identifying nonlinear signal propagation suggestive of crosstalk and/or feedback regulation  and off-target(s) depends on the parameters of the system under study, including the concentrations of individual target molecules. Consequently, ECgulation . Finallygulation , studiesgulation , 154.e.g., drugs and unique cellular contexts). To avoid astronomical expenses and unmanageable experimental complexity, most -omics measurements of PI3K and other signaling markers are therefore mostly representative of isolated snapshots. It is neither appropriate to discount such data altogether nor is it our intention. It is, however, important to be fully aware of the limitations of snapshot signaling studies and how response variables may or may not be detected as a function of time if this parameter features in the experimental design. A snapshot -omics measurement of the PI3K signaling network may generate a lot of dots, yet without a time component, connecting the dots  is impossible. This approach may also result in erroneous conclusions. This is arguably best illustrated with the time delay between AKT activation and mTORC1 activation upon acute growth factor stimulation. Initial studies of growth factor–induced AKT phosphorylation dynamics revealed a 5- to 10-min delay between AKT phosphorylation and subsequent mTORC1-dependent S6 phosphorylation in PC12 cells stimulated with EGF . A mathematical model may prove particularly useful at the initial stage of experimental design by restricting measurements (and thus cost) to time points that are most likely to be informative for a given question. If possible, a combination of acute and long-term measurements will always yield the most information by enabling disentangling of direct effects from indirect effects as well as nonlinear input–output relationships. How this can be achieved alongside the systematic modulation of signaling dose is illustrated in a study of ERK-induced responses in retinal pigment epithelial cells with inducible expression of the BRAFV600E oncogene . Not all 15 constellations are equally likely in a given setting, depending instead on expression levels, relative affinities, and subcellular localization. There is also evidence that the nucleocytoplasmic distribution of catalytic and regulatory subunits of class IA PI3K exhibits isoform-, lineage-, and stimulus-specific differences  within the canonical PI3K signaling cascade. To avoid the fragmented nature of the current evidence base, however, we suggest a community effort to develop guidelines and standards for subcellular profiling of PI3K signaling, with a strong emphasis on key controls and orthogonal validations that limit the confounding effects of assay-specific artifacts. An example of the latter may include aggregation artifacts and false-positive subcellular enrichments for certain phosphoinositides if researchers are not familiar with the optimal immunofluorescence protocols for phosphoinositide detection.Beyond attention to single- esponses , attentiferences , 174. Monot p85α . Thus, tResolution is also relevant when considering signaling crosstalk, in particular between the PI3K–AKT and RAS–ERK networks given their overlapping upstream activators, downstream effectors, and target phenotypes. Joint experimental measurements of signaling markers for both networks could be critical for predictive modeling of cell fate decisions if the latter depend on nonredundant and highly coordinated activation of both signaling branches , 176. A For accurate and quantitative modeling, much upfront effort is needed to obtain a detailed understanding of key response variables, including biochemical properties and technical details. Such details include dynamic range and signal-to-noise ratio, including how these parameters change as a function of the chosen measurement technique. For example, to use immunoblot measurements for accurate quantitative inference, a dilution series of the lysate to generate a mini standard curve for each marker is best practice , a methoe.g., PRAS40 pthr246). Similarly, while there is a strong correlation between PI3K activation and AKT phosphorylation, it is far from deterministic, and caution is warranted particularly if one is tempted to conclude that lack of AKT phosphorylation equates to lack of PI3K activity. These considerations apply to most if not all readouts within the PI3K and other signaling networks, including exogenous reporters such as commonly used genetically encoded biosensors  is the most reliable method for evaluating proximal PI3K signaling activation. It is therefore worth emphasizing what these and other measurements actually represent, given their wide usage and relevance for accurate signaling inference. In general, a phosphorylation measurement typically reflects the relative balance between the activity of a kinase and that of one or more corresponding phosphatase(s). A change in AKT phosphorylation on thr308 could therefore reflect a change in the activity of 3-phosphoinositide-dependent kinase 1, a change in the activity of the phosphatase PP2A, or both . For AKTosensors , 192.Readers may wonder whether we, too, are not simplifying complexity too much by referring mostly to PI3K signaling in relation to individual cells, and thus seemingly disregarding the additional complexity that emerges from interactions amongst cellular collectives, particularly in the context of tissues, organs, and even whole organisms. Indeed, as already discussed, the “system” in systems biology was originally reserved for physiology. The ultimate goal is therefore to understand how networks of various types and sizes, both within and across cells, tissues, and organs, acquire the capability of performing complex functions that are inaccessible to and beyond the sum of their parts , 194. YeComplexity in signaling, and biology more generally, therefore appears to be a self-perpetuating continuum, operating across scales. Much like viewing the image of a fractal, whether we zoom in or whether we zoom out, we see a self-similar representation of the original, as determined by a set of common rules that govern the interactions of individual units. It is our conviction that the signaling field, including all relevant technologies, is now sufficiently mature to begin the systematic mapping of the context-dependent cellular rules that govern PI3K-dependent information transmission. It will require those working in the field to make the relevant computational and theoretical concepts part of everyday PI3K signaling studies. How a mindset change of this kind can make the impossible possible is perhaps best illustrated by transformational tools like AlphaFold2 and RoseTTAFold in protein structure prediction , 195. WeJournal of Biological Chemistry. The authors declare that they have no additional conflicts of interest with the contents of this article.A. T. is the Editor-in-Chief of the


The extracellular matrix (ECM) is a crucial component of the stem cell microenvironment, or stem-cell niches, and contributes to the regulation of cell behavior and fate. Accumulating evidence indicates that different types of stem cells possess a large variety of molecules responsible for interactions with the ECM, mediating specific epigenetic rearrangements and corresponding changes in transcriptome profile. Signals from the ECM are crucial at all stages of ontogenesis, including embryonic and postnatal development, as well as tissue renewal and repair. The ECM could regulate stem cell transition from a quiescent state to readiness to perceive the signals of differentiation induction (competence) and the transition between different stages of differentiation (commitment). Currently, to unveil the complex networks of cellular signaling from the ECM, multiple approaches including screening methods, the analysis of the cell matrixome, and the creation of predictive networks of protein–protein interactions based on experimental data are used. In this review, we consider the existing evidence regarded the contribution of ECM-induced intracellular signaling pathways into the regulation of stem cell differentiation focusing on mesenchymal stem/stromal cells (MSCs) as well-studied type of postnatal stem cells totally depended on signals from ECM. Furthermore, we propose a system biology-based approach for the prediction of ECM-mediated signal transduction pathways in target cells.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01252-8. Tissue structure homeostasis, function, and renewal depend on cellular composition. Generally, terminally differentiated cells define the structure and normal function of tissue and organs, while the adult stem and progenitor cells determine renewal and regeneration potential –3. Stem Stem cell niches contain cells that regulate the maintenance of stem cells homeostasis and fate through the secretion of various niche components. Almost in all tissues and organs mesenchymal stem/stromal cells (MSC) play this role being the critical regulators of stem cell niche functioning –15. MSC The interaction of cells with the microenvironment, in particular with the components of the ECM, is mediated by specific receptors, leading to the activation of various signaling cascades within the cell and, as a consequence, to changes in its behavior. Looking at the identification of the main receptors and participants in ECM signaling cascades from a historical point of view can help to summarize the knowledge on this issue in the field of matrix biology Table . Even thMany ECM components were found in stem-cell niches, including collagens, laminins, fibronectin, and proteoglycans , and alsIt is presumed that the tissue specificity of the ECM is provided due to differences in the cellular composition of tissue types. Nevertheless, it is known that cells with similar phenotypes and functions isolated from different tissue types differ in the expression profile of ECM proteins. Thus, a comparative proteomic analysis of the ECM, secreted by MSC from bone marrow or adipose tissue, showed the presence of unique sets of proteins produced by each cell type. This allows us to assume that ECM tissue specificity is established at histo- and organogenesis stages and subsequently maintained throughout life . The comThe main class of ECM receptors is integrins, heterodimeric proteins comprising α and β subunits. There are 18 determined α-subunits and 8 β-subunits in humans, and these are responsible for recognizing ECM proteins and their physical properties , and for intercellular communications. Some reviews can provide more detailed information on modern representations of the structure of integrins –55.Integrins realize bi-directional signaling. The high-affinity interaction between integrins and their ECM ligands activates the “outside-in” signaling pathway. Then, focal adhesion kinase (FAK) and Src-mediated phosphorylation of the integrin adhesion complex (IAC) and cytoskeletal components initiate intracellular molecular reorganization and phosphorylation events among many adapters . CrosstaIntegrins are considered crucial receptors for stem cell functioning. Various integrins are widely represented on the surface of different types of stem cells . SubunitThere is no exception in the case of MSC. It has been shown that integrins α2β1 or α11β1 provide adequate interaction of human bone marrow MSC with type I collagen, which ensures cell survival and osteogenic differentiation by activating the protein kinase B (PKB/Akt) survival pathway . SimilarDiscoidin domain receptors (DDRs) (DDR1 and DDR2) are collagen-binding receptors in mammals . SeveralAdditionally, DDR1 demonstrated regulation of collagen transcription by translocating to the cell It appears that DDRs regulate the directed differentiation of progenitor cells. The depletion of DDR1 expression in human adipose-derived MSC suppresses chondrogenic differentiation by decreasing the chondrogenic genes and cartilaginous matrix deposition . FurtherCD44 is primarily a receptor for hyaluronic acid (HA) but can also bind to several ligands, such as ECM components , as well as some cytokines and growth factors . CD44 reCD44 is a participant in multiple ECM-induced signaling pathways , 87 beinProteoglycans are ubiquitous components of the cellular microenvironment, which includes several families. They also act as membrane-bound receptors. These molecules include heparan sulfate proteoglycans (HSPGs), comprising two distinct families: syndecans (4 members) and glypicans (6 members) , 100. NuThere is abundant evidence that syndecans activate different signaling pathways. Studies show that syndecans have no kinase activity, but other kinases can phosphorylate their intracellular domains . The intSDC-1 knockdown in mice leads to the inhibition of canonical Wnt signaling because of deficient levels of β-catenin . TemporaGlypicans are globular glycosylphophatidylinositol (GPI) anchored proteins , 111. TyGlycosphingolipids (GSLs) are amphiphilic membrane lipids of the eucaryotic plasma membrane consisting of glycan chains that are covalently linked to the sphingolipid backbone , 118. GSNumerous in vivo studies have reported that the composition of GSLs in the plasma membrane varies depending on the embryonic stage  Similar Mechanosensitive ion channels in mammals include transient receptor potential channels (TRP) and Piezo channels . TRP chaIn the various stem cell niches MSC are involved in the microenvironmental control process. TRPV4 modulates the formation of collagen fibrils by human bone marrow MSC. The inhibition of this channel in MSC disrupts aligned collagen matrix assemblies. In contrast, TRPV4 activation promotes collagen deposition .Tissue-specific Piezo2 isoforms are formed as a result of alternative splicing, where each isoform may have its own specific function. The diversity of splice isoforms is performed in the neuronal tissue and cells. Only one splice isoform is expressed in non-neuronal tissue . Piezo1 Based on the studied scientific sources, we arrange a general table of known intracellular signaling pathways from ECM receptors in stem and committed progenitor cells with several examples of their realization in MSC Table .Table 2IAmong the promising regenerative medicine strategies involving ECM receptors, two main approaches can be distinguished: 1) blocking of receptors to ECM components and their intracellular signaling pathway, and 2) activation of ECM receptors by functionalization of various surfaces and materials with specific peptides or ECM components.It is well-known that integrins are involved in the tissue regeneration processes. The role of MSC-expressed integrins for each tissue type remains a promising area of research. Thus, following an injury, MSC are recruited from surrounding tissue to the injury site in an integrin-dependent manner . Some reThe role of DDRs in wound healing is still poorly understood. It is known that DDR2 regulated wound healing by activating p38 and ERK1/2 kinases and inducing matrix metalloproteinase (MMP) expression . Recent Previously, it was shown that MSC encapsulated in HA-based hydrogels expressed more markers of cartilage tissue both in vitro and in vivo compared to control samples . This waSyndecans and glypicans regulated the normal regeneration of different tissues –169. SDCCurrently, there are not many publications on the use of MSC glycosphingolipids in the field of regenerative medicine. However, some data support the idea that glycosphingolipids could be a promising target for the treatment of various diseases , 175.Mechanotransduction is also involved in regenerative processes. For example, the hematoma formed after a bone fracture is highly viscoelastic, and such viscoelasticity is necessary to allow infiltration of MSCs and osteogenic stimulation of MSC in vivo . CultivaBiofunctionalization of tissue engineering materials improves cellular interaction and tissue integration. Currently, a large number of functionalization methods of tissue engineering materials are known, ranging from single peptides and components of ECM, including enzymes responsible for its remodeling, to decellularization of tissues and whole organs, which is described in detail in the reviews , 178–180Several approaches of network biology are widely used to study the activation of contributing factors in intracellular signaling cascades, which can subsequently find application in predictive medicine . Nowadayhttp://matrixdb.univ-lyon1.fr/) [https://matrisomedb.org/) [www.matrinet.org) [Subsequently, with the development of omics technology and the rapid evolution of big data analysis, different approaches and specialized databases have been created by the scientific community. This is also relevant to matrix biology. One such database comprising ECM component adhesomes and interactions is MatrixDB (on1.fr/) . Anotherdb.org/) . Recent net.org) .http://www.string-db.org) [For the construction of protein–protein interactions (PPIs), many investigators are using databases that aggregate standard information from large resources . Examples of such PPI databases are STRING and Mint (-db.org) , 197; ST-db.org) , 198.https://www.kegg.jp/kegg/pathway.html). We revealed the signaling pathways for integrins, CD44, and HSPG  [First, we created PPI networks for integrin β1 and CD44 as one of the well-studied surface markers of stem cells and employed DDR1 as an example of a less-investigated receptor but potentially useful for application to mechanosensing. PPI networks were created using the STRING database with the following settings: network type – physical subnetworks; meaning of network edges – evidence; active interaction sources; experiments; minimum required interactions score – 0.4, 2 shell; maximum number of interactors to show – 150. In addition, Cytoscape was used for analysis and network visualization  . As menthttps://reactome.org/) [Next, we analyzed the contributing factors of the signaling pathway for integrin β1, CD44, and DDR1 using ReactomeFIPlugIn in Cytoscape, which accessed the Reactome pathways stored in this database (me.org/) . We seleWe reveal that the signaling pathway members of integrin β1, CD44, and DDR1 are vital for nervous system development Fig. .Fig. 3IlIn addition, we build a prediction network of PPIs for DDR1 pathway members in human adipose-derived MSC. For this step, we normalized the data of PPIs using RNAseq data describing the transcriptome of human adipose-derived MSC obtained in our laboratory Fig. .Fig. 4IlECM-induced signaling pathways within the stem cells may define the functionality and homeostasis of the whole tissue. Trying to understand the complicated molecular interactions between ECM and stem and progenitor cells, scientists are developing new approaches based on a general view obtained from big data analysis. The application of system biology methods for this purpose has been an observable current trend in matrix biology .In this review, we discussed the results of recent studies covering the participation of cellular receptors to ECM components in the maintenance of postnatal tissue homeostasis as well as tissue regeneration after various types of damage through the regulation of MSC functions. We also focused on summarizing the data of the currently known ECM-induced signaling cascades in human stem and progenitor cells. However, literature analysis revealed that very few and only the most common participants of these signal pathways are investigating; moreover, they are involved in multiple cellular processes which makes them not suitable as a potential therapeutic target to modulate stem cell functioning for the purposes of regenerative medicine. Therefore, to detect more specific ECM-induced signaling pathways in stem and progenitor cells as well as to search for novel targets for fine-tuning regeneration processes, we suggest using the approaches of systems biology.Utilizing the established changes in the stem cell profile of ECM receptors (Step 1), it is possible to build predictive networks of PPIs (Step 2). Such networks can be analyzed based on the well-known and available databases described in this review. After receiving the list of participants in the signaling cascade, a normalization step can be added to the data of the proteome or transcriptome of cells of interest to researchers (Step 3). Then, the created network can be compared with a database of known signal pathways to select specific signaling pathways and processes accumulating the most of participants from the previously predicted network of PPIs (Step 4 and Step 5). The resulting theoretical model could be useful in designing the further experimental research exploring the ECM-mediated regulation of stem and progenitor cells .Shortly, we suppose that the suggested approach of generating the predictive networks of PPI could serve as a useful tool which is complementary or even partially replacing the omics experimental work. Combining the known data about ECM receptor on the specific target cells and desirable functional processes one could reveal novel expected or unexpected signal transduction pathways induced by ECM. Few examples are demonstrated in the review. Indeed, these results have limited value until experimental validation. However, the suggested algorithm could intensify the search of ECM-induced signaling pathways in stem and progenitor cells and shorten the time to the potential breakthroughs in the field.Thus, following the current trends in the field of matrix biology, it could allow to identify new promising directions in the study of stem and committed cell behavior within their matrix microenvironment. Importantly, these approaches expand the number of tools in regenerative medicine using to search for the mechanisms regulating tissue and organ renewal and repair.Additional file 1: Fig. 1_Supplement. Predicting the integrin β1 PPIs networks for signal transduction molecules before (A) and after (B) excluded data obtained from cancer cells.Additional file 2: Fig. 2_Supplement. Illustration of contributing factors of the signaling pathway for integrin β1 (A), CD44 (B), and DDR1 (C), which generated using ReactomeFIPlugIn in Cytoscape. The obtained results are presented as Reactfoam using a false discovery rate (FDR) scalebar .Additional file 3: Fig. 3_Supplement. Predicting the DDR1 signaling pathway members participate in semaphorin interactions (A), and regulate the activity of RUNX1, RUNX2, and RUNX3 transcription factors in the case of osteogenic differentiation (B).


Arabidopsis and rice, have long been at the center of plant research. However, technological advances in sequencing have led to a dramatic increase in genome and transcriptome data availability across land plants and, more recently, among green algae. These data allowed for an in-depth study of the evolution of different protein families – including those involved in the metabolism and signaling of phytohormones. While most early studies on phytohormone evolution were phylogenetic, those studies have started to be complemented by genetic and biochemical studies in recent years. Examples of such functional analyses focused on ethylene, jasmonic acid, abscisic acid, and auxin. These data have been summarized recently. In this review, we will focus on the progress in our understanding of cytokinin biology. We will use these data to synthesize key points about the evolution of cytokinin metabolism and signaling, which might apply to the evolution of other phytohormones as well.Angiosperms, especially  Streptophyte algae and more specifically the last common ancestor of Zygnematophyceae and Embryophytes most likely gave rise to land plants , have shown that, although no canonical cytokinin receptor genes are present in the genomes, they respond to treatment with phytohormones, especially auxin, and cytokinin, with increased cell growth and/or metabolic activity  and is believed to be the evolutionarily older pathway of the two (Physcomitrium patens (cis-Zeatin (cZ), which mainly acts in housekeeping, i.e., the maintenance of basic cell function while adenylate IPT, which mainly produces tZ (trans-Zeatin) and IP (Isopentenyl)-type cytokinin, play regulatory roles , which can use either tRNA (tRNA pathway) or ATP/ADP/AMP (adenylate pathway) as a substrate . The tRN the two . Looking the two . Interesm patens . Recentlry roles . This hyry roles .N- or O-glycosylation . It has been speculated that they might have been gained via horizontal gene transfer from bacteria . The classical TCS consists of two components - hence the name - a receptor kinase, which autophosphorylates upon the perception of the signal, and a response regulator, which after activation through phosphorylation by the receptor mediates the downstream response, e.g., cription . In bactcription . The TCScription . Cytokincription . After icription . There acription .via endosymbiosis or horizontal gene transfer  by the CHASE domain, and all known cytokinin receptors possess such a protein domain. However, CHASE domains are also found in a wide variety of other receptors . Therefote clade . At leas. patens . Interesomitrium . Thus, ivolution . The autrception .While the numbers of the members of other protein families of the cytokinin signaling pathway increase steadily during the diversification of vascular plants - probably due to numerous rounds of whole genome duplication (WGD) - the number of CHKs remains near-constant Table 1.In contrast to CHKs, the next type of protein in the signaling chain, the HPts, can be found across many kingdoms . Most alFor the two types of response regulators (RRA and RRB) the evolutionary pattern is more similar to the HPts than the CHKs. But there are also differences between the two types that are quite striking. RRBs are present in most streptophyte algae, and in land plants, but also in chlorophyte algae Table 1.However, the TCS seems to be not the only way that the cytokinin signal can be perceived and transduced. Some species of rice encode CHASE-domain receptor serine/threonine kinases (CHARK). As the name indicates these proteins combine a CHASE domain with a serine/threonine kinase domain . Althougvia regulating key enzymes, even in the absence of a dedicated signaling pathway – in effect functioning as an allosteric regulator. Experiments with brassinolide treatment of Asterionella formosa, a diatom, hinted at such a direct effect on glyceraldehyde-3-phosphate dehydrogenase, a key enzyme in glycolysis and also in the Benson - Calvin cycle  Cytokinins are present before the establishment of their cognate signaling pathway.Cytokinins, like other phytohormones are found in many green algae and plants - regardless of the presence of the dedicated signaling pathway known from angiosperm model plants, e.g., auxin , cytokin2.) Downstream signaling pathways are present before the appearance of receptors.It has long been proposed that new regulatory pathways are made by using existing gene regulatory pathways and connecting them to a novel input (plugin) . Thus, i3.) Receptors are often the last step to completing a pathway.P. patens and interact with the downstream components of the GA signaling pathway but do so without the binding of GA  The specificity of the receptor-cytokinin interaction increases in more recently evolved lineages.Once the complete cytokinin signaling pathway was in place, the receptors displayed a low specificity for the different cytokinins. During evolution, the specificity of the receptor-ligand interaction increased for the different types of the cytokinin found in plants . Similar5.) Different parts of a signaling pathway can be under different evolutionary constraints.Another fascinating aspect of the evolution of cytokinin and other phytohormones is that the different parts of the signaling system are under different evolutionary constraints. It is often found that the number of receptors is relatively constant while downstream components increase in number Table 1 6.) Horizontal gene transfer and whole genome duplications could be major drivers for evolutionary novelties in cytokinin biology.via horizontal gene transfer (HGT). In fact, it has been speculated that the TCS system was delivered to a photosynthetic organism during the primary endosymbiosis of cyanobacteria  originates from a gene duplication of the auxin receptor   . The rolEach of these key points, by itself or in combination with each other, is important for understanding the origin and evolution of cytokinin signaling.Both authors wrote the manuscript. All authors contributed to the article and approved the submitted version.


Our healthcare system consists of 8 acute care facilities, each of which utilizes a Urinalysis (UA) with Reflex to Culture laboratory order. With this order, a urine culture is performed automatically if the UA flags as abnormal, thus resulting in overutilization of urine cultures and subsequent identification of urinary tract infections (UTI), including hospital-associated catheter UTI (CAUTI).The two largest facilities developed and implemented a new urine order to ensure appropriate utilization of urine cultures on adult critical care (CC) patients: the Urinalysis with Hold for Culture (UA/Hold) order. With this order, a culture would not be completed until a clinician reviewed the UA results, evaluated the need for a culture and then placed an add-on urine culture within 48 hours of the original UA specimen. If the UA was not abnormal, an override reason was chosen for the order, i.e. new urgency, frequency, dysuria, fever, altered mental status, hematuria. Logic existed within this order to not allow a urine culture unless a UA had been performed within the last 48 hours or if a patient was neutropenic, pregnant or undergoing a urological procedure.From February 2021 to February 2022, the two facilities who implemented the order avoided a total of 485 cultures (Table 1). These two sites also experienced a decrease in CAUTIs compared to the year prior to implementation. The UA/Hold order resulted in a meaningful reduction in CAUTI rates since urine culture orders were only placed on patients that clinically needed them.Due to the success of the new order, it was expanded to five additional facilities and their CC patient populations in December 2022. Data collected January-March 2023, demonstrates the effectiveness of the implementation, as zero CAUTIs have been reported from CC patients at any of the five hospitals. In the 12 months prior to implementation, 21 CAUTIs had been reported. Overall, 27 cultures were avoided (Table 2).With exceptional results from the first three months since implementation, the months to come show promise for significant culture and CAUTI reduction across the system and provides opportunity to expand the order to non-critical care units as well.All Authors: No reported disclosures


Ultrasound guidance enables direct visualisation of local anaesthetic spread during the performance of regional anaesthetic techniques, thus improving their accuracy and effectiveness while minimising their complications. In human patients receiving thoracic analgesia, a new regional anaesthetic technique, named retrolaminar block, has a lower complication risk when compared with the gold standard, thoracic paravertebral block. The aim of this study was to develop an ultrasound-guided thoracolumbar retrolaminar technique to provide thoracolumbar analgesia in dogs. Another aim was to compare the distribution of contrast solution between the ultrasound-guided and landmark-guided techniques in canine cadavers, using computed tomography. Ten canine cadavers were randomised to receive either an ultrasound-guided or a landmark-guided retrolaminar injection of 0.6 mL/kg of iodinated contrast solution in each hemithorax at the level of the twelfth thoracic vertebra. The injectate spread in a similar manner in the retrolaminar space and the space surrounding the intervertebral foramina, while minimal spread was noted in the paravertebral space. One of the landmark-guided injections failed to spread, thus highlighting the importance of ultrasound guidance in improving outcomes in regional anaesthetic techniques. Further studies are warranted to clarify the role of the ultrasound-guided retrolaminar technique in the management of thoracolumbar pain in dogs.n = 10) and an ultrasound-guided on the other hemithorax . Groups were compared using the Mann–Whitney U test. The median (range) spread of the contrast in the paravertebral space was 0 (0–3) and 1 (0–5) vertebrae in groups B and U, respectively (p = 0.038). The median (range) extent of the spread surrounding the interverbal foramina was 4 (0–5) in group B and 4 (3–5) in group U. The median (range) spread along the retrolaminar space cranial and caudal to T12 was 3 (0–6) retrolaminar segments in group B and 3 (3–4) in group U. The potential of the ultrasound-guided retrolaminar injection to provide analgesia for dogs suffering from thoracolumbar pain should be further investigated.The retrolaminar block was developed in humans as an easier and safer alternative to the thoracic paravertebral block. This study aims to describe an ultrasound-guided thoracolumbar retrolaminar injection in canine cadavers and compare the injectate distribution between a landmark-guided and an ultrasound-guided thoracolumbar retrolaminar technique using computed tomography. Ten canine cadavers were randomised to receive two injections each of 0.6 mL/kg of iodinated contrast at the level of the twelfth thoracic vertebra (T12): a landmark-guided retrolaminar injection was performed on one hemithorax (group B,  Over the past two decades, the benefits of regional anaesthesia have been well established in human patients . Some ofThe key requirement for a successful regional anaesthetic intervention is based on the optimal deposition of the local anaesthetic around the target tissues whether they are nerve structures, plexi, or, more recently, fascial planes ,5. ThereThe utilisation of topographical landmarks to assist anaesthesiologists during procedural care includes various regional nerve blocks, neuraxial techniques, and interventional pain procedures. These methods are based on a thorough understanding of anatomic relationships to effectively deliver local anaesthetics and avoid morbidity and mortality . AdditioMost recently, ultrasonography has emerged as a method to locate peripheral nerves. Ultrasound-guided regional anaesthesia is an exciting and relatively new technique that is currently rapidly developing and allows for the visualisation of the target nerves or structures, the approaching needles, and the deposition of local anaesthetic in close proximity to the nerve in real-time . Thus, tIn an attempt to develop a simpler and safer landmark-guided alternative approach to the thoracic paravertebral block in humans, Pfeiffer and colleagues (2006) described what is known today as the retrolaminar fascial plane block ,13. DuriThe objectives of this study were to describe an ultrasound-guided approach to performing a thoracolumbar retrolaminar injection and demonstrate the feasibility and potential complications of the technique in canine cadavers. A further objective was to compare the distribution of the injectate between the ultrasound-guided and a landmark-guided technique using computed tomography.The hypothesis was that identification of the ultrasonographical landmarks to perform the retrolaminar technique would be easy, and that ultrasound guidance would be useful in optimising needle position and improving accuracy.This study was granted an exemption from full ethical review by the Animal Research Ethics Committee of University College Dublin (AREC-E-20-28-Huuskonen).n = 10) and an ultrasound-guided in the other  using a random sequence generator . A total volume of 0.6 mL/kg iodinated contrast (ioversol)  was injected per site. In all cadavers, the ultrasound-guided injection was performed first, immediately followed by the anatomical landmark-guided retrolaminar injection. The distribution of the injectate was evaluated using computed tomography (CT) immediately after the injections were performed.The cadavers of ten adult greyhound dogs, that had been euthanised for reasons unrelated to the present study, frozen, and subsequently thawed at ambient temperature for 72–96 h, were used. Cadavers with spinal abnormalities that could have hindered the identification of the anatomical or ultrasonographic target landmarks were excluded from the study. All cadavers were randomly assigned to receive an anatomical landmark-guided retrolaminar injection in one hemithorax . The identification of the landmarks was confirmed by two investigators blinded to each other (JP and MV). The spinous processes were then marked with a surgical skin marker . The fasWith the cadaver in sternal recumbency and the T12 already identified, an 18-gauge, 3.5-inch spinal needle  was introduced next to the spinous process through the epaxial muscles while the needle was maintained in a strict parasagittal plane, approximately 1–1.5 cm lateral to the spinous process. The needle orientation was caudoventral, at a 45° angle to the skin, aiming towards the retrolaminar space, and it was advanced until contact with the T12 vertebral lamina was achieved. The advancement of the needle was then stopped, a 20 mL syringe  containing the iodinated contrast was then connected to the needle, and, following a negative air and blood aspiration, the retrolaminar injections were performed by the same investigator (JP). To detect an accidental pleural, epidural, or paravertebral puncture, the loss of resistance technique was used during the injection, in which the injection would be stopped and the needle repositioned if an increased resistance was detected during injection. The depth at which the needle was inserted was approximately 1.5–2 cm, depending on the body condition of the cadaver.All ultrasonographic examinations were conducted using the same ultrasound machine  with a linear ultrasound transducer . The ultrasound probe was positioned in sagittal orientation approximately 1–1.5 cm lateral to the dorsal midline. The transducer was moved in a medial-to-lateral direction in order to identify the hyperechoic spinous vertebral processes, the vertebral laminae, the transverse vertebral processes, and the thoracic pleura. An in-plane technique was used and a 21-gauge, 100 mm insulated needle  was inserted in a cranial-to-caudal direction, through the epaxial muscles, in a 45° angle to the skin, with the goal of having the tip of the needle in direct contact with the dorsal aspect of the T12 vertebral lamina . Once thFollowing the performance of all retrolaminar injections, the cadavers were moved onto the CT scanner table. A CT examination of the thoracolumbar area was performed using a 16-slice CT scanner  with the cadavers positioned in sternal recumbency. The acquisition parameters used to obtain the scans were: 0.75 mm slice thickness at 220 mAs and 130 KVp. Computed tomographic images were then viewed using an image analysis software . One ECVDI and ACVR board-certified veterinary radiologist (SH) and a veterinary radiology resident (BG) evaluated the CT images by consensus.In each injection, the features assessed using the CT images were the following: confirmation of injection at the T12 retrolaminar space (yes/no), contrast location in paravertebral space, extent of contrast surrounding ipsilateral intervertebral foramina, extent of distribution of contrast in ipsilateral retrolaminar space, contrast distribution pattern , epidural contrast migration (yes/no), extent of epidural contrast migration, presence of contrast in pleural space (yes/no), presence of contrast in retroperitoneal space (yes/no), presence of IV contrast (yes/no), presence of pneumothorax (yes/no).To confirm the correct location of the initial injection site, the primary injection site was determined as the region with the highest Hounsfield units (HU) . The HU U test. Results are summarised as median (range). Categorical data are presented as frequencies. All statistical test results were interpreted using a 5% level of significance.Statistical analysis was performed using IBM SPSS 27  for macOS. The data were assessed for normality using the Shapiro–Wilk test. Each hemithoracic retrolaminar injection was considered an independent variable and analysed accordingly. The extent of staining of paravertebral spaces, the intervertebral foramina, and the retrolaminar spaces, and the presence of intravascular contrast were compared between the two groups (group B versus group U) using the Mann–Whitney Ten cadavers were used in total, three of whom were female, and seven were male. Their median (range) weight was 30.75 (26.5–36) kg, with a median (range) body condition score of 4 (3–5) out of 9 . The T12p = 0.038). The median (range) spread of the contrast, cranial and caudal to T12, was 4 (3–5) intervertebral foramina for the ultrasound-guided group and 4 (0–5) for the anatomical landmark-guided group, with statistically significant difference between the two groups (p = 0.038) (p = 0.79). In one cadaver that received an anatomical landmark-guided retrolaminar injection, although contrast was identified in the target T12 lamina, it did not spread into the retrolaminar space or into the surrounding intervertebral foramina, rendering the injection unsuccessful (The median (range) time between injection and the CT scan was 44 (31–56) min. The CT study confirmed that the injection site was always the intended one (T12 retrolaminar space) in all 20 injections. Contrast distribution was linear and intercostal for all cadavers except one that had only linear spread of the contrast. Concerning the presence of contrast in the underlying paravertebral space, the median (range) spread of contrast in the ultrasound-guided injections was 1 (0–5) paravertebral spaces, while for the anatomical landmark-guided injections, it was 0 (0–3), and this difference was statistically significant (= 0.038) . The medccessful .Epidural migration of the injectate was noted in two of the ten cadavers, with the contrast identified locally at the level of T12 in one (cadaver 2) and extending from T11 to T13 in the other (cadaver 10). No contrast was identified in the pleural space or in the retroperitoneal space in any of the ten cadavers.p = 0.93).In 18 out of the 20 retrolaminar injections, intravascular contrast was identified irrespective of the injection technique used, with no difference between the two groups  was small, and a power calculation was not performed prior to the investigation. However, other studies in the past that have investigated regional anaesthetic techniques in canine cadavers have utilised a similar number of dogs [There are several limitations to this study. First, the total number of cadaveric specimens  cadavers would have decreased the incidence of severely compromised tissue integrity that accompanies thawing frozen cadavers, but access to such resources was not available to us. Additionally, the synchronised movement of the chest cavity during normal ventilation is believed to affect the spread of the injectate in clinical retrolaminar injections in human patients , which cFinally, neither the pressure of injection nor the rate of injection was measured or standardised in either of the techniques. This could have also affected the injectate spread, especially since there was a subjective observation of increased resistance to injection during the ultrasound-guided technique. The increased resistance during injection was most likely a result of the increased viscosity of the injectate in combination with the smaller diameter needle used for the ultrasound-guided technique. In fact, the needle used for the ultrasound injections was 21-gauge, while the needle used for the landmark-guided injections was 18-gauge, and, according to Poiseuille’s law, resistance to flow is directly proportional to the fourth power of the radius and the viscosity of the fluid.To conclude, ultrasound-guided thoracolumbar retrolaminar injections were technically simple to perform, and the relevant sonoanatomy was easy to visualise and identify in canine cadavers. All ultrasound-guided retrolaminar approaches were successful, whereas one anatomical landmark-guided injection was unsuccessful, thus supporting the hypothesis that ultrasound guidance provides additional safety and accuracy to regional anaesthetic techniques. The injectate distribution in the intended thoracolumbar retrolaminar space and intervertebral foramina area, where dorsal branches of the spinal nerves are located, should allow for analgesic provision in clinical scenarios. Further clinical studies should assist in a better understanding of the potential indications of this technique.


Wnt signaling is one of the main evolutionarily conserved developmental pathways needed to instruct embryo formation and maintenance of organ tissues. Decades of research also clearly recognize the fundamental role of Wnt signaling during carcinogenesis. Indeed, dysregulation of Wnt pathway components has been suggested as a relevant hallmark of several neoplastic malignancies. In this review, we summarize the main molecular mechanism through which the Wnt pathway exerts its intracellular effects, with a specific focus on brain development and brain tumors, and how Wnt interacts with the surrounding brain environment. In this context, we review the latest anti-cancer therapeutic approaches employed to specifically target Wnt signaling in cancer, and their potential application in the brain tumor context. Moreover, we discuss the additional efforts that will be needed to define the real clinical impact of Wnt modulation in different types of brain tumors and even how to overcome the unsolved concerns about the potential systemic effects of such therapeutic approaches.The involvement of Wnt signaling in normal tissue homeostasis and disease has been widely demonstrated over the last 20 years. In particular, dysregulation of Wnt pathway components has been suggested as a relevant hallmark of several neoplastic malignancies, playing a role in cancer onset, progression, and response to treatments. In this review, we summarize the current knowledge on the instructions provided by Wnt signaling during organogenesis and, particularly, brain development. Moreover, we recapitulate the most relevant mechanisms through which aberrant Wnt pathway activation may impact on brain tumorigenesis and brain tumor aggressiveness, with a particular focus on the mutual interdependency existing between Wnt signaling components and the brain tumor microenvironment. Finally, the latest anti-cancer therapeutic approaches employing the specific targeting of Wnt signaling are extensively reviewed and discussed. In conclusion, here we provide evidence that Wnt signaling, due to its pleiotropic involvement in several brain tumor features, may represent a relevant target in this context, although additional efforts will be needed to: (i) demonstrate the real clinical impact of Wnt inhibition in these tumors; (ii) overcome some still unsolved concerns about the potential systemic effects of such approaches; (iii) achieve efficient brain penetration. Vertebrate developing embryos are extremely complex entities. Every single embryo develops from one single cell through the instructions provided by peculiar signaling pathways able to guide cellular specification in both space and time. Most importantly, during adulthood, the same pathways are fundamental to maintain the structure and functionality of specific organs and, consequently, control the homeostasis of the entire organism. This means that during the entire life of a vertebrate organism, the disruption, imbalanced dosages, or altered activation patterns of these fundamental pathways can lead to the onset of various diseases, including cancer, which may occur at different life stages. Among the fundamental molecules responsible for the development of several organs and the maintenance of their homeostasis, the Wnt (Wingless-related MMTV integration site) family of secreted lipid-modified glycoproteins and its downstream effectors is key. In 1980, the Wnt cascade was identified as an early segmental patterning signal for the correct development of Drosophila larva . Since tIt is now evident that Wnt signaling is linked with a variety of human diseases ,14 spannIn this review, we summarize the role played by Wnt signaling in normal brain and brain cancers. In particular, we focus on the determinant role played by Wnt during brain development and specification and in brain cancer physiology, with an important inset in the reciprocal stimulation (Wnt-dependent) occurring between cancer cells and their microenvironment. Moreover, we extensively report on and discuss the most promising pharmacological tools available for the inhibition of Wnt signaling in cancer and, possibly, in brain tumors, considering the key obstacles still present on the way to their clinical translation and administration to patients.Wnt ligands comprise a family of 19 secreted hydrophobic glycoproteins that exert their function by binding to Frizzled (FZD) receptors and transmembrane low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors located on the target cell surface  Figure . The ligTCF7, TCF7L2, TCF7L1, and LEF1) encodes for specific transcription factors  that bind to DNA through a SRY-Box Transcription Factor (SOX)-like high mobility group domain, which recognizes a specific DNA consensus around the core “CTTTG” sequence, known as the Wnt Response Element (WRE) [The cytoplasmic-nuclear shuttling of β-catenin is the most importantly regulated molecular mechanism which modulates the Wnt/β-catenin pathway activation. Indeed, additional tyrosine phosphorylation Tyr142/Tyr654) of β-catenin probably dictates peculiar interactions with α-catenin and E-cadherin and results in its dissociation from cadherin-containing adherens junctions, cytoplasmic accumulation, and nuclear translocation to promote gene transcription . Once rent (WRE)   . Despitent (WRE) , the basnt (WRE) , variousnt (WRE) , and mannt (WRE) . This in/Tyr654 ont (WRE) , bone rent (WRE) , but alsnt (WRE) , diabetent (WRE) , and oxint (WRE) ,47.2+)  and the Wnt-Calcium (Wnt/Ca2+) .Wnt/PCP signaling is an evolutionarily conserved pathway, both in vertebrates and invertebrates, whose function is to control polarized cell behavior, a process that involves the asymmetric distribution of a set of core proteins within the cell and the subsequent cell polarization across the tissue plane ,49. Gene2+ signaling, the Wnt-FZD binding and, in particular, the binding of the Wnt-5a ligand to the FZD5 receptor and the Receptor Tyrosine Kinase Like Orphan Receptor (ROR1/2) family of co-receptors, leads to the activation of the DVL family of transducers  in Xenopus. In particular, they found that the Wnt signaling gradient is higher in the posterior and lower in the anterior Xenopus body axis, thus properly specifying the anterior–posterior polarity of the neural plate during development  Figure A. As sooNGN1), which participates in stimulating cortical neuronal differentiation [The neural tube is made up of pluripotent precursor cells that proliferate, commit to a specific cell fate, and then migrate to their final destination to arrange the different layers of the CNS, including neuronal ganglia and nuclei, and the cerebral cortex. The Wnt/β-catenin pathway has been associated with both proliferation and specification of neural stem cells (NSCs) during CNS development, in cooperation with other vestigial developmental pathways such as Notch, Sonic Hedgehog (SHH), Bone morphogenetic protein (BMPs) and Fibroblast Growth Factor (FGF) signaling. Indeed, the dorsal–ventral/rostral–caudal gradients of these morphogens and their relative receptors, together with specific cell–cell contacts between NSCs, finely specify the differentiated cell types that compose the nervous system, including mature neurons, astrocytes, and oligodendrocytes . The Wntntiation .Besides substantial evidence for the role of Wnt/β-catenin in controlling neurogenesis through the promotion of neuronal differentiation, some studies have demonstrated that this signaling can also stimulate neural progenitor cell proliferation, since the β-catenin-dependent signal induces the expansion of proliferating precursors in the sub-ventricular zone ,81. TherAs suggested by the above considerations, it is clear that any dysregulation occurring at the level of both canonical and non-canonical Wnt signaling components may severely affect the fine equilibrium existing in the cellular composition of the CNS, eventually contributing to the onset, progression, and peculiar behavior of different brain tumors. Accordingly, in the following sections, the major known roles played by Wnt signaling in a series of brain cancers are described, thus providing the rationale for considering Wnt signaling as a relevant therapeutic target against brain malignancies.The involvement of the Wnt signaling pathway in brain tumors has been extensively reported, with its functionality found to be largely contingent upon the tumor subtype ,82,83. TThe role of Wnt signaling in the onset and progression of GBM has been extensively studied and characterized, resulting in complex and sometimes contradictory evidence ,91,92,93PLAGL2) expression has been linked to the upregulation of FZD2-9 receptors, thus promoting Wnt pathway activation and contributing to GSC self-renewal and maintenance [As discussed in previous sections, Wnt pathway activation requires the participation of several, and sometimes redundant, molecular transducers, co-activators, repressors, transcription factors, etc., thus making its study very complex and challenging. In GBM, it is well-known that the binding of Wnt ligands to FZD receptors activates the canonical β-catenin-dependent signaling pathway, which then promotes the expression of target genes through the formation of a large molecular complex together with TCF/LEF transcription factors and CREB Binding Protein (CBP)/p300 transcriptional co-factors ,98,99 F. In normntenance . Moreoventenance . Further confirming a prominent Wnt signaling activation in GBM cells, through large-scale whole genome approaches it has been demonstrated that they exhibit epigenetic-dependent decreased expression of several Wnt pathway inhibitors, such as Wnt Inhibitory Factor 1 (WIF1), Dickkopf inhibitors (DKKs), and Secreted Frizzled Related Protein 1 (SFRP1) ,107,108.Data presented so far seem to delineate a quite shared consensus on the fundamental role played by the Wnt pathway in GBM, by promoting cell motility and invasion through increased EMT, sustaining the growth and maintenance of GSCs, and contributing to intrinsic chemo- and radio-resistance . On the Since we provided examples that Wnt signaling may serve both as a pro-cancerous and a pro-differentiation stimulus in GBM, a better comprehension of the complex molecular interactions contributing to Wnt signaling modulation is a mandatory goal to be achieved soon. This will allow us to increase our molecular knowledge of GBM biology and even identify relevant intracellular signaling nodes with potential therapeutic impact. Nevertheless, despite the reported dichotomous effect of Wnt signaling activation in GBM tumors, several studies still consider Wnt inhibition as a promising therapeutic strategy in these tumors.MRP-1) expression, eventually promoting EC stemness and chemoresistance , with a focus on how GBM tumors, through activation of the Wnt pathway, can manipulate neighboring cell behavior and growth. GBM tumors evolve within an intricate and interdependent microcosm of diverse cellular components, such as immune cells, normal astrocytes, and blood vessels, as well as a dense extracellular matrix ,127. Thesistance . AccordiRecently, the Wnt signaling pathway has also emerged as a key player in the regulation of immune cell behavior in GBM tumors. The composition of immune cells infiltrating the GBM TME is highly variable during tumor progression. These include tumor-associated macrophages (TAMs), neutrophils, dendritic cells, plasmacytoid cells, lymphocytes, natural killer cells, mast cells, and a significant amount of microglia ,138. In Cerebellar medulloblastoma (MB) is a highly malignant (grade IV) and invasive brain tumor with a preferential manifestation in the pediatric age. MBs are classified into four molecular groups: Wnt-activated, Shh-activated, group 3, and group 4 MBs . These sTP53), Patched 1 (PTCH1), and β-catenin (CTNNB1) gene mutations [APC mutations have also been found in a fraction (4.3%) of sporadic MB [CTNNB1 mutations, considered the main hallmark of Wnt-driven MB, are found in 86% of patients, clearly suggesting the molecular dependency of Wnt MB onset and progression on the canonical Wnt/β-catenin signaling [The best characterized is the Wnt subgroup, due to its very good long-term survival of patients, exceeding 90% . Severalutations . It is nutations , but APCradic MB . Moreoveignaling .+/lox(Ex3); Trp53flx/flx) in which a conditional stabilized allele of Ctnnb1 is targeted to neural progenitor cells of the lower rhombic lip [Ctnnb1 transgene in a Trp53-deleted cellular context, develop classical MB tumors [Pik3caE545K), previously identified in human MB [+/lox(Ex3); Trp53+/flx; Pik3caE545K mice, which develop Wnt-like MB tumors with 100% penetrance within 3 months [In 2012, Gibson et al. generated a mouse model of Wnt-driven MB (brain lipid-binding protein (Blbp)-Cre; Ctnnb1mbic lip . These mB tumors , clearlyhuman MB , thus ge3 months . These tThere is limited understanding of the impact of Wnt signaling on the development, growth, and aggressiveness of other brain tumors. However, previous research has explored the involvement of the Wnt pathway in meningiomas and pituitary adenomas.CTNNB1, Cyclin-Dependent Kinase 5 Regulatory Subunit 1 (CDK5R1), Cyclin D1 (CCND1), and Ectodermal-Neural Cortex 1 (ENC1) in atypical and anaplastic MGs, relative to benign tumors [TCF3, SFRP3, SFRP1, Cadherin 1 (CDH1), and FZD7, when comparing atypical, anaplastic, and benign MGs [Meningiomas (MG) are usually considered benign tumors originating at the level of brain meninges, more precisely from meningothelial arachnoid cells . They arn tumors . Similarn tumors . Supportnign MGs ,163. Finnign MGs ,165.WNT4 has been linked to the over-activation of β-catenin-dependent and independent pathways, eventually increasing PA invasiveness [SFRP2 is associated with the development of corticotrophic adenomas [SLC20A1), which may be linked to the Wnt pathway, has been associated with larger tumor size, invasive behavior, and tumor recurrence in somatotroph adenomas [Pituitary adenomas (PA), which are typically benign tumors originating from the pituitary gland , are thesiveness ,169,170.adenomas , while oadenomas .In this section, we will try to provide a comprehensive description of the most promising approaches developed in the last years to achieve a therapeutic anti-cancer targeting of the Wnt/β-catenin pathway, and some examples of their application to treat brain tumors. In this context, it is worth investigating the potential employment of Wnt inhibition on certain brain cancers, particularly MB. Indeed, although aberrant Wnt signaling activation is the recognized oncogenic driver of Wnt MBs , its oveIn general, Wnt signaling inhibitors may be classified into four quite broad groups: (1) non-steroidal anti-inflammatory drugs (NSAIDs), (2) vitamins, natural compounds, and their derivatives, (3) small molecule chemical inhibitors , and (4) antibodies against Wnt pathway components .NSAIDs are drugs of common use and some of them, including aspirin, indomethacin, sulindac, and celecoxib, have been proposed for the treatment of different types of cancer. Besides their anti-inflammatory properties that may represent by themself an adjuvant strategy during cancer prevention and therapy , NSAIDs From a clinical point of view, celecoxib is the only NSAID approved by the Food and Drug Administration (FDA) for the reduction in the number of colorectal polyps in FAP patients. However, despite apparent effectiveness, reports of COX-2 inhibitor-dependent cardiotoxicity now limit their use in FAP patients , supportNatural compounds are a major source of drugs with anti-cancer properties. Indeed, at least one-third of drugs FDA-approved for the treatment of cancer are composed of natural products or their direct derivatives . VitaminRhodiola crenulata [In addition to the above-mentioned pro-differentiating compounds, several other drugs of natural origin have shown promising Wnt signaling inhibition-dependent, anti-cancer (including GBM) effects, although their proposed mechanisms of action would benefit from further clarification, due to lack of specificity. Nevertheless, curcumin ,197 and renulata  and resvrenulata  have beerenulata  and heavrenulata . Wnt-deprenulata  or by prrenulata . Certainrenulata . Moreoverenulata .However, due to a general lack of specificity and poor knowledge of their molecular mechanisms of action, many of these natural products have failed to proceed to the clinal phases of experimentation, with only a few of them, such as vitamins and resveratrol, being included in clinical trials, essentially based on their pro-differentiation activity on various cancers .Several small chemical inhibitors of the Wnt/β-catenin signaling have been identified/developed during recent years through in silico and in vitro high throughput screening (HTS) approaches. Nevertheless, before providing a representative description of these discovery process-derived compounds, at least a few drugs, characterized by a repurposed action against Wnt signaling and a reported anti-cancer effect in multiple GBM models, should be described. The anthelmintic compound niclosamide, in addition to a prominent anti-migratory action on human cells ,209, disIn addition to repurposed drugs, several other compounds with the ability to interfere with various Wnt pathway components have been identified and developed during the last 20 years. A brief description of these compounds, subgrouped according to their suggested mechanism of action, is reported here below.A small series of Wnt signaling inhibitors, acting as β-catenin/TCF interaction disrupting agents, was first identified in 2004 by Lepourcelet et al. through a HTS approach of a large library of compounds . HoweverICG-001 exerts its Wnt signaling inhibitory properties by selectively binding the β-catenin transcriptional co-factor CBP, but not its homologous p300, thus only suppressing a CBP-dependent transcriptional program , with reThe more recent inhibitors of the β-catenin-containing transcriptional complex  are BC20This class of compounds is designed to inhibit the capability of DVL to intracellularly transduce Wnt ligand-dependent FZD receptor activation. NSC668036, FJ9, and 3289-8625 have been reported to interact with the PZD protein–protein interaction domain of DVL and to block Wnt signaling activation in vivo in Xenopus embryos and lung cancer mouse models ,230,231.One of the first examples of compounds affecting Axin protein stability were the Intracellular Wnt Response (IWR) inhibitors. IWRs are targeted at blocking Axin destruction, thus favoring the suppression of Wnt signaling through the increase of β-catenin proteasomal degradation ,234. ComThe HTS approach implemented by Chen and colleagues in 2009, besides identifying IWRs, uncovered a subset of Inhibitors of Wnt Production (IWPs) . Wnt sigROR receptors may participate in Wnt signaling by serving as co-receptors for FZDs and enhancing specific Wnt-5a/ROR/FDZ non-canonical intracellular signaling , which iAnother promising agent is ONC201, generally indicated as a dopamine receptor D2 antagonist, which has been shown to reduce the expression of several Wnt pathway components, including peculiar Wnt ligands, receptors, and co-factors . In addiWe and others previously suggested that certain HDAC inhibitors of the hydroxamate class, including suberoylanilide hydroxamic acid , could be indicated as Wnt signaling antagonists since they were able to deplete TCF4-dependent Wnt pathway activation in colon carcinoma cells  and to cFinally, small molecule inhibitors such as proteolysis-targeting chimeras (PROTACs) have recently attracted interest for their potential application against several cancers by targeting protein degradation. As an example, a novel PROTAC β-catenin has demonstrated efficacy in inhibiting Wnt signaling in colorectal cancer cell lines and patient-derived organoids . These aSeveral therapeutic antibodies against Wnt signaling pathway components have been developed in recent years. Indeed, based on the relevance of ROR1 expression in human cancers, cirmtuzumab was developed as a humanized antibody inhibiting Wnt-5a-ROR-induced signaling, which then entered a phase I clinical trial for CLL patients . Along t90Y labeled OTSA101-DPTA-90Y monoclonal antibody (mAb) [Additional antibodies have been mainly developed to trap Wnt ligands or target FZD receptors. Antibodies against Wnt-1, Wnt-2, Wnt-5a (pAb5a-5), and secreted frizzled receptor protein 2 (SFRP2) have all been demonstrated to induce prominent apoptosis and cell death in several solid tumor models ,259,260.dy (mAb) , TT641 pdy (mAb) , and MAbdy (mAb) , all desdy (mAb) .Despite increasingly promising results being reported for the use of these therapeutics against several cancers, we still have no data on the possible implementation of Wnt-targeting antibody-based therapies in the brain tumor context.Although we provided evidence that emerging compounds targeting Wnt signaling (or its ancillary modulators), may represent an effective therapeutic strategy against several cancers, inhibition of Wnt signaling in brain tumors still remains a challenging deal, due to its recognized role in brain vascularization and blood–brain barrier (BBB) integrity. Indeed, Wnt-7 ligands produced by neural progenitors activate canonical Wnt signaling through FDZ binding, thus stimulating ECs ,291. In It is mandatory to also consider the tight epigenetic regulation that acts upstream of the Wnt pathway function. In particular, it has been reported that hundreds of non-coding RNAs are able to regulate (positively or negatively) several Wnt signaling components. Although these have not been mentioned within the previous sections, an exhaustive summary of the main micro-RNAs and long non-coding (lnc)RNAs able to affect Wnt signaling was recently reported by Daisy Precilla et al. . https://clinicaltrials.gov/, accessed on 21 April 2023), with most of them withdrawn early due to bone and gastrointestinal toxicity [Collectively, the factors to examine and the problems to overcome are multiple and could be also related to the mechanism of action of the proposed drugs/therapeutics and the knowledge that Wnt signaling is fundamentally involved in the homeostasis of nearly all adult tissues. Indeed, there are few clinical trials verifying the use of Wnt inhibitors in brain tumors (summarized in toxicity . As an etoxicity . Indeed,toxicity .Based on these considerations, the blockade of the Wnt system, which in normal tissues controls vital functions, is thus limited in its clinical applications. Consequently, it is necessary to identify additional pharmacological strategies to restrict Wnt inhibition exclusively, or mainly, at the level of cancer cells.One proposed approach could be the specific targeting of defined Wnt branches, rather than the whole Wnt system. Indeed, as described above, the non-canonical signaling downstream of the Wnt/ROR cascade is more associated with cell migration and invasion. For this reason, besides potential concerns regarding its impact on the wound healing process, this pathway may be considered less toxic systemically, but nonetheless endowed with therapeutic efficacy . In agreThe Wnt pathway is a complex, multifaceted, and tightly regulated signaling pathway involved in vertebrate development and tissue homeostasis. The role of Wnt is also pivotal during adulthood, thus impacting on the functionality and structure of several organs. In this review, we focused on the role of Wnt in determining brain tumor onset, aggressiveness, and microenvironment. Moreover, we evidenced that Wnt signaling also depends on, and modulates, several other pathways to control cell differentiation and sensitization to therapies. In this context, the modulation of the Wnt signal achieved through specific compounds or additional epigenetic mechanisms must be finely tuned for its activation level and correct time of intervention during cancer patient treatment. Despite several clinical trials concentrating on Wnt pathway inhibition as a promising anti-cancer therapy, data collected so far suggest that targeting multiple signaling pathways, aberrantly activated in tumor cells, may be the only reliable strategy to include Wnt signaling inhibitors in the future treatment of multiple cancer types, with a clear anticipated benefit for patients.