Although substantial progress has been made over the past 40 years in treating patients with cancer, effective therapies for those who are diagnosed with advanced metastatic disease are still few and far between. Cancer cells do not exist in isolation: rather, they exist within a complex microenvironment composed of stromal cells and extracellular matrix. Within this tumour microenvironment exists an interplay between the two main stromal cell subtypes, cancer-associated fibroblasts (CAFs) and immune cells, that are important in controlling metastasis. A complex network of paracrine signalling pathways between CAFs, immune cells and tumour cells are involved at multiple stages of the metastatic process, from invasion and intravasation at the primary tumour site to extravasation and colonisation in the metastatic site. Heterogeneity and plasticity within stromal cell populations also contribute to the complexity. Although many of these processes are likely to be common to a number of metastatic sites, we will describe in detail the interplay within the liver, a preferred site of metastasis for many tumours. A greater understanding of these networks provides opportunities for the design of new therapeutic approaches for targeting the metastatic disease. As such, the majority of cancer-related deaths are associated with metastatic spread.3 To initiate the first step of the metastatic cascade—invasion into the local stroma—tumour cells must become motile and invasive, which requires changes in cell–cell and cell–extracellular matrix (ECM) contacts as well as reorganisation of the ECM. Tumour cells can then intravasate, breaking through the basement membrane of the vasculature or into lymphatic vessels. Once in the circulatory or lymphatic system, the newly defined circulating tumour cells (CTCs) must survive exposure to mechanical force and the immune system. CTCs can become trapped in small circulatory beds in the lungs and liver, for example—common metastatic sites for multiple cancer types—where they can adhere to endothelial cells and extravasate into the surrounding tissue. Tumour cells in metastatic sites are known as disseminated tumour cells (DTCs) and can either proliferate to form micrometastases or enter a dormant state, from which they can be ‘awakened’ after long periods of latency. The final step in the metastatic cascade, colonisation, occurs when DTCs proliferate to form clinically relevant metastases  has been suggested to occur during metastasis in multiple solid carcinomas, and the pattern of expression of 22 so-called ‘matrisome’ genes was shown to be predictive of disease outcome.13 The primary tumour can also direct the formation of a ‘pre-metastatic niche’ via the systemic release of tumour-derived factors and extracellular vesicles that result in the restructuring and ‘priming’ of the metastatic site prior to the arrival of tumour cells (reviewed in ref. 14). The TME within these metastatic niches provides survival cues for the tumour cells, including immune-suppressive signals that determine whether DTCs form metastases or remain dormant, and can also direct the reactivation of dormant cells to mediate late recurrences in patients .ses Fig. . Whereas16Understanding the relationship between the various elements of the TME is key to fully elucidating the biological processes that drive the metastatic cascade, which, in turn, should provide opportunities for therapeutic intervention. In this review, we will introduce the interplay between different cellular components of the TME, with a focus on CAFs and immune cells, and describe significant advances made over the past 5 years detailing the role of these components in mediating metastatic spread. Although many of these processes are likely to be common to a number of metastatic sites, we will describe in detail the metastatic microenvironment in the liver, a preferred site of metastasis for many tumours, including colorectal, pancreatic tumours, melanomas and sarcomas.11Due to the lack of cell markers that are unique to fibroblasts and CAFs, the accurate definition of fibroblasts and CAFs remains elusive, as does the origin of CAFs. Classically, fibroblasts are defined as spindle-shaped cells that are embedded within the ECM and lack epithelial, endothelial and leucocyte markers. In the normal physiological stroma, fibroblasts exist in a quiescent state; however, in the reactive tumour stroma, multiple signals can push resting fibroblasts to an activated state. These signals include inflammatory cytokines and growth factors such as transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) secreted from tumour cells and other stromal cells within the TME. In addition, mechanical changes and remodelling of the ECM can drive CAF activation. Although, in most cases, CAFs originate from pre-existing quiescent fibroblasts, in some tissues, activated CAF-like populations can also derive from additional tissue-resident precursors, such as hepatic stellate cells , bone marrow-derived mesenchymal stem cells, adipocytes and pericytes.18 while the release of circulating factors such as TGF-β can also promote metastatic outgrowth.19 Importantly, CAFs have a role in shaping the immune environment at primary and metastatic sites. Moreover, CAFs can facilitate the systemic delivery of soluble factors and extracellular vesicles to prime the metastatic niche in an organ-specific manner.14 Within the metastatic niche, CAFs can also directly influence DTCs to promote their growth and colonisation. Unsurprisingly, therefore, a number of studies have attributed pro-metastatic functions to CAFs in various tumour models.23A plethora of studies have demonstrated that CAFs are critical regulators of tumour growth and metastasis, acting via multiple mechanisms Fig. . They ca25 An increased understanding of heterogeneity within the stromal compartment (below) might help us to shed light on the multifaceted roles of CAFs in balancing pro- and anti-tumour effects.Despite the well-established pro-tumorigenic activity of CAFs, a tumour-suppressive CAF phenotype has also been reported in mouse models of pancreatic ductal adenocarcinoma (PDAC).28 These markers include fibroblast activation protein (FAP), fibroblast-specific protein 1  and PDGF receptor-β. However, the characterisation of CAFs is further complicated by reports that these markers can also be expressed in tumour cells that are undergoing EMT.29Several studies have revealed the existence of distinct CAF subtypes—as defined by their expression of surface markers—that display functional heterogeneity and might also be tumour type-dependent.30 Furthermore, in breast cancer, a CD146+-fibroblast population could preferentially increase metastasis associated with the deposition of a number of ECM components found in clinically aggressive disease.31 An analysis of lymph node metastases from breast cancer patients identified divergent CAF subsets that were similar to those found in primary tumours. These subsets were shown to promote metastases through complementary mechanisms: either through their highly contractile phenotype and activation of NOTCH signalling or through the induction of EMT resulting from C-X-C motif chemokine ligand 12 (CXCL12) and TGF-β signalling.32 Education of subsets of CAFs by tumour cells to drive a pro-metastatic niche suggests that, in addition to the spatial context of CAFs with respect to tumour cell proximity,33 the tumour genotype plays an important role in the regulation of CAF heterogeneity.27 In this context, the p53 status in pancreatic tumours can reprogramme a subset of CAFs to establish a metastatic-permissive environment.34Distinct CAF subtypes also affect tumour invasion and metastasis. For example, a subset of CAFs is able to drive tumour progression through integrin α11-mediated promotion of CAF invasion and CAF-induced tumour cell invasion via the production of the pro-invasive matrix protein tenascin C.36 This additional level of CAF spatial heterogeneity has been associated with unique metastasis-enhancing phenotypes. For example, CAFs generated from breast cancer brain metastasis were able to induce the migration of patient-derived tumour cells through the expression of CXCL16 and CXCL12.35 In addition, the selective upregulation of the insulin-like growth factor 2 gene IGF2 and interferon-related genes in CAFs from different breast cancer metastatic tissues compared with primary-site derived CAFs facilitated CAF-dependent tumour formation and metastasis in vivo, and immunosuppression via regulation of T cells.36Several reports have identified CAF populations within metastatic sites that exhibit distinct gene expression patterns compared with those in the primary tumour.37 Furthermore, the horizontal transfer of entire mitochondrial genomes from CAF-derived extracellular vesicles to breast cancer cells treated with hormone therapy restores oxidative phosphorylation, promoting metastasis and reawakening therapy-induced dormant cells.38 The exchange and hydrolysis of intracellular lipids provide another approach by which CAFs support tumour progression. PDAC-derived CAFs secrete abundant amounts of lysophosphatidylcholines (LPCs), which can be taken up by PDAC cells, leading in turn to the production of lysophosphatidic acid (LPA), a growth-permissive phospholipid, via the activity of autotaxin. This stroma-derived LPC–autotaxin–LPA axis was found to fuel PDAC growth and migration, although any effects on metastatic progression have yet to be established.39 Metabolic differences among patient-derived melanoma xenograft models driven by differences in the levels of monocarboxylate transporter 1, a lactate transporter, are associated with differing metastatic potential,40 highlighting the importance of metabolic alterations in regulating metastatic progression and the potential for new therapeutic opportunities. It will be important to consider how dysregulated metabolic signalling in the CAFs might also influence such treatments.Other studies have also highlighted the importance of CAFs in providing metabolic support to drive metastasis. In an ovarian cancer co-culture model, bi-directional signalling between CAFs and tumour cells leads to the mobilisation of glycogen as an energy source in cancer cells, which facilitates metastatic outgrowth.9The immune system can influence all stages of the metastatic cascade, from the regulation of tumour cell migration and invasion at the primary tumour site through to priming of the metastatic niche and supporting the outgrowth of DTCs Fig. .8,942 Immune cells also secrete factors that promote tumour cell intravasation at the primary tumour site.43 Further along in the metastatic process, the recruitment of inflammatory monocytes at metastatic sites promotes vascular permeability and extravasation of CTCs.44 Moreover, metastasis-associated macrophages derived from these inflammatory monocytes can further enhance metastatic outgrowth through the secretion of cytokines, and several studies have identified specific subsets of macrophages that are involved in organ-specific metastatic outgrowth.46 Neutrophil-derived factors also promote the extravasation of CTCs, and the release of neutrophil traps further acts to enhance the trapping of CTCs at distant metastatic sites, leading to increased metastatic spread.47 Neutrophils also play an important role in promoting metastatic colonisation: they help to create an immunosuppressive environment by secreting nitric oxide, which inhibits T cell functions.48 However, anti-metastatic effects of neutrophils have also been described. Work published by Li et al.49 in 2020 attempted to address these differences and highlighted the importance of natural killer (NK) cells in dictating whether neutrophils exert pro- or anti-metastatic effects. In NK cell-competent mice, neutrophils facilitate metastatic colonisation, while in NK cell-deficient mice, neutrophils have an inhibitory effect on metastatic colonisation.In primary tumours, immune cells are important regulators of the ECM and secrete a number of pro-tumorigenic and pro-metastatic proteases, such as metalloproteinases, that remodel the ECM.50Immune cells also play a pivotal role in establishing a permissive pre-metastatic niche by secreting a number of chemoattractants, while tumour-derived factors stimulate the mobilisation and recruitment of bone marrow-derived myeloid cells to the metastatic niche to remodel the ECM, promote angiogenesis and drive a pro-inflammatory environment—all of which contribute to metastatic seeding and colonisation.51 Important bi-directional crosstalk between tumour cells and CAFs controls the balance between the recruitment of pro-tumorigenic and anti-tumorigenic immune cell populations, which influences both primary tumour growth and metastatic progression. A number of studies have provided further insight into the complex signalling pathways involved. For example, CAFs act as sensors of damage-associated molecular patterns produced within primary tumours, which activates the NLRP3 inflammasome pathway and induces the subsequent secretion from CAFs of the pro-inflammatory cytokine interleukin-1β (IL-1β). IL-1β, in turn, promotes metastases through multiple mechanisms, among which is a reduction in the recruitment of immunosuppressive CD11b+Gr1+ myeloid cells in the metastatic environment.52 Conversely, DTCs can induce a pro-inflammatory phenotype in fibroblasts within the metastatic niche by secreting IL-1α and IL-1β, which trigger the production by fibroblasts of CXCL9 and CXCL10 to subsequently support the outgrowth of the DTCs.53CAFs also play a key role in regulating the inflammatory tumour environment through the secretion of cytokines and deposition of ECM proteins that are involved in the recruitment and activation of immune cells.54 Detailed review of the role of the ECM in metastasis is beyond the scope of this article and has been reviewed elsewhere.13 Here, we highlight the role of CAFs and immune cells in regulating ECM remodelling.The non-cellular component of the TME, the ECM, is a network of macromolecules, including collagens, fibronectin and elastin, which provides structural support in a tissue-specific manner. It is a highly dynamic structure that responds to external cues, while also providing biochemical and biomechanical signals to surrounding cells within the tissue, thereby playing a fundamental role in normal tissue homeostasis and disease processes including metastasis formation.55 This reciprocity is predominantly mediated via cell-surface ECM receptors, which include integrins and collagen-binding discoidin domain receptors (DDRs), and both CAFs and macrophages play a key role in this process leading to remodelling of the ECM in the metastatic niche to promote cell adhesion and survival.The concept of dynamic reciprocity, whereby cells process signals from their environment, which can then, in turn, regulate the ECM, was first introduced in the early 1980s in the context of epithelial morphogenesis and wound healing, and has since been identified as a crucial process regulating the behaviour of tumours, especially the invasive and metastatic potential of tumour cells.56 In a model of melanoma, a novel role for fibroblast-secreted factors in altering the lymphatic ECM has been identified. Secretion of hyaluronic and proteoglycan link protein (HAPLN1), which crosslinks hyaluronan to the ECM, leads to changes in the permeability of lymphatic endothelial cells, thereby enabling melanoma cells to escape from the lymphatic system to distant metastatic sites.57 Bertero et al.58 have also shown that changes in ECM stiffness induce changes in amino acid metabolism in both CAFs and tumour cells, and that these changes drive a complex crosstalk whereby CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodelling. Targeting aspartate and glutamate transporters either genetically or pharmacologically reduced metastatic spread.A number of new players in the CAF-dependent remodelling of the ECM that control mechanotransduction and ECM stiffness to drive metastasis have been identified. The deletion in CAFs of DDR2, a critical regulator of integrin-based mechanotransduction and collagen fibre organisation, results in a significant reduction in tumour stiffness and the extent of pulmonary metastasis.The interaction between CAFs, immune cells and remodelling of the ECM is of particular importance in the fibrotic liver environment, and we will now discuss in detail the interplay between metastatic tumour cells, the immune environment, and HSCs (the resident fibroblasts within the liver) in the liver environment, a common site of metastasis.61 Why the liver is such a privileged site is a topic of much debate. Nevertheless, for a metastatic cell to take hold, the recipient organ requires an extensive vascular network to provide ready access for the cancer cell. Moreover, to establish a secondary tumour, the cancer cells must be able to take over a dynamic local immune environment that it can utilise to facilitate its growth.Following metastatic spread, cancer cells from a number of primary malignancies find themselves in the liver, which appears to be a preferred site of metastasis for many tumours.62 Within the lumen of hepatic sinusoids, CTCs have the potential to extravasate to initiate a metastasis; however, relatively few do. Although a number of CTCs might pass through the hepatic sinusoids, many will be killed by a complex sinusoidal immune surveillance network.64 Importantly, the sinusoids function as active cellular suppressors of metastasis, with cytokine and chemokine crosstalk between sinusoidal endothelial cells, Kupffer cells, and circulating lymphocytes acting together to identify CTCs and mark them for cellular killing.65 This multicellular approach involves the release of pro-apoptotic factors, such as reactive oxygen species, nitric oxide and interferon-γ, by sinusoidal endothelial cells, thereby inducing the direct apoptosis of CTCs.66 In tandem with this, Kupffer cells lining the sinusoids are able to detect the presence of cancer cells and, through the release of a number of pro-inflammatory cytokines, recruit a transient lymphocyte niche that senses and targets metastatic cells, thereby clearing them from the microvasculature and preventing them from establishing a new metastatic tumour.68The vasculature of the liver is highly specialised, with blood entering through the portal circulation and passing through sinusoids. These narrow vessels, which are lined with sieve-like fenestrated endothelial cells , form a highly permeable barrier that facilitates the rapid exchange of biological and xenobiotic compounds between the blood and the metabolic hepatocytes. As blood passes through this highly efficient filtration system, it is surveyed by local populations of immune cells adjacent to the LSECs, including Kupffer cells (resident hepatic macrophages) and other circulating lymphocytes Fig. .62 Withi71The initiation of metastasis in the liver requires cancer cells to cross the immunologically defensive barrier of endothelial cells and immune cells, and establish themselves within the space of Dissé, a region adjacent to the sinusoid which, in healthy individuals, accommodates HSCs—liver fibroblasts that are central for establishing scar formation following injury Fig. . Somewha+ T cells, thereby promoting colonisation of the liver with cancer cells.72 However, the formation of micrometastases in the space of Dissé is limited by the availability of a vascular network. Evidence suggests that, in the liver, these DTCs can promote the formation of a local vasculature from the sinusoidal endothelial cells or from the larger vessels in the liver; whether one of these vascular origins is favoured is not clear.74 However, the dynamic recruitment of a vasculature necessitates a change in the local immune microenvironment, and again, high levels of TGF-β (produced by the cancer cells) can promote the polarisation of metastasis-associated macrophages to an M2-like phenotype  while suppressing M1  tumour necrosis factor-α (TNF-α)-producing macrophages.77 This immunological switching of immune cell phenotypes is considered to be an essential trait of tumour initiation and, as a consequence, M2-like macrophages drive remodelling of the ECM through the production of matrix metalloproteinases, as well as supporting fibroblasts to produce pro-angiogenic signals such as vascular endothelial growth factor (VEGF) to facilitate further vascular recruitment.79Once in the space of Dissé, tumour cells are able to recruit myeloid-derived suppressor cells, which can then modulate the tumour cell immune microenvironment by, for example, suppressing CD880 and portal fibroblasts surrounding the bile duct.81 Fibroblasts are normally quiescent but, in response to injury, both of these cell types are capable of activation and express classical markers of activated fibroblasts such as α-smooth muscle actin (α-SMA), tissue inhibitors of metalloproteinases and ECM components. In premalignant, fibrotic disease, HSCs are activated following exposure to a number of extracellular cytokines (reviewed in ref. 82) and through a number of cell-autonomous processes.83 The plasticity of these cells and their relative contribution to premalignant disease, however, might be more dynamic than previously thought, and only since the application of single-cell RNA-sequencing to these populations has their complexity been appreciated (reviewed in ref. 84).In addition to a complex immune microenvironment, the liver also contains two distinct populations of fibrogenic cells: HSCs in the space of Dissé85 This stiffening of the liver is thought to make the tissue permissive to cancer cell proliferation and, in primary liver cancer, HSCs are a known source of many mitogens and cytokines that are required for cancer growth.87 In metastatic disease too, HSCs appear to play a reiterative role in metastatic priming and tumour progression.Activated HSCs  contribute to scar formation and fibrosis in chronic liver disease by depositing collagen and other components of the scar.88 reported in 2020 that activated fibroblasts isolated from liver metastases, which express α-SMA, phosphorylated myosin light chain and collagen-1, are highly contractile and increase the local rigidity surrounding the metastasis, thereby supporting angiogenesis and metastatic growth. Furthermore, the altered ECM promotes pro-survival signalling through the engagement of ECM receptors such as integrins,71 thereby conferring a survival advantage on tumour cells within the space of Dissé over those that remain within the sinusoid.The de novo production of highly crosslinked collagen in this new metastatic environment has two principal effects. Initially, it promotes survival by stiffening the local environment, and a number of studies have linked increased stiffness of the ECM to enhanced tumour cell survival in the liver and increased progression through the cell cycle. Shen et al.78 and are sufficient to promote a stem-like phenotype in pancreatic cancer cells by downregulating E-cadherin and inducing the expression of mesenchymal markers and nestin. This stem-like phenotype is partially reversed by inhibiting TNF-α signalling between the HSCs and tumour cells.89The transition of quiescent HSCs into activated fibroblasts also controls PDAC quiescence in the hepatic niche. Whereas cancerous epithelial cells from PDAC are forced into IL-8-induced quiescence by quiescent HSCs, activated HSCs fail to induce cancer cell quiescence, but rather support cancer cell proliferation by expressing growth factors such as VEGF,90 placing HSCs and activated macrophages at the centre of a microenvironment that can modulate both the growth of metastases and also their immune surveillance.The quiescent HSC to active HSC transition is clearly important in allowing the liver to be permissive to colonisation by pancreatic cancer cells. Similar mechanisms are likely to play a role in the colonisation of the liver by other tumour cells, too. Importantly, should the process by which these different tumour cells colonise the liver be a common one, then developing methods to intervene in this would be far-reaching. Relaxin, an endogenous peptide hormone, has anti-fibrotic properties and has been shown to be effective in ameliorating scar formation in the liver and reversing systemic syndromes associated with liver stiffening in cirrhosis. Interestingly, in mouse models of liver colonisation by pancreatic, colorectal and breast cancer cells, the production of ectopic relaxin reduces the activation of HSCs at the metastatic site and inhibits metastatic colonisation. Moreover, when the activation of HSCs is reduced, metastases are more susceptible to treatment with immunotherapy,91 The exosomes, which contain high levels of macrophage migration inhibitory factor (MIF), are taken up by Kupffer cells, which are induced to express TGF-β. These TGF-β-expressing macrophages subsequently activate local HSCs to produce fibronectin, thereby generating a local microenvironment that is permissive for the recruitment of bone marrow-derived macrophages that is required for the formation of the pre-metastatic niche and metastatic outgrowth. Inhibition of exosomal-derived MIF leads to reduced colonisation of the liver with metastatic cancer cells, highlighting the importance of distant cues in priming the liver to receive metastatic cells. Subsequent studies have indicated that the population of bone marrow-derived monocytes that is recruited to the site of metastasis comprises pro-inflammatory monocytes that secrete high levels of the glycoprotein granulin, which promotes the production of periostin by HSCs and results in the formation of a fibrotic microenvironment that supports tumour growth.92As mentioned, the liver appears to be a privileged site for cancer metastasis. As such, there has been a significant focus on how a metastatic microenvironment is established within the liver, whether this microenvironment is essential for the seeding of metastatic cancer within the liver, and whether primary tumours themselves are able to remotely alter the liver metastatic microenvironment to make it receptive to colonisation. In PDAC, tumour-derived exosomes mediate the formation of a pre-metastatic microenvironment in the liver, which then facilitates colonisation by metastatic cancer cells.93 Such changes include the reprogramming of the fibroblast secretome, which can enhance metastatic spread by mediating physical alterations in the ECM that promote enhanced migration and invasiveness94 Interestingly, age-related changes were also associated with a reduction in the motility of certain immune cell populations, resulting in a change in the immune environment.94 Although these immune changes were not directly associated with a reduction in metastatic capacity, several other studies have identified age-related changes in the immune microenvironment that could influence priming of the pre-metastatic niche and metastatic progression.95 The link between age-associated inflammation, priming of the metastatic niche and activation of resident fibroblasts is likely to play an important role in metastatic outgrowth. For example, in a mouse model of PDAC, the aged liver provides a permissive inflammatory environment that supports the activation of HSCs to secrete factors that promote the outgrowth of DTCs.78Changes in the TME that occur during ageing are now being recognised to play an important role in tumour progression and can have profound effects on the development of metastases.29Important new advances in our understanding of the TME have unveiled potential new therapeutic options, and a number of clinical trials that target interactions between tumour cells and stromal cells are underway in the metastatic disease setting and have previously been reviewed.8 Similar approaches have been undertaken to target CAFs, by depleting specific subtypes or reprogramming them back to their resting state, or by targeting their activation or downstream effectors.29 For example, metastatic outgrowth following primary tumour resection was delayed following treatment with a vaccine that eliminates FAP+ CAFs.96 Targeting FAP and thereby reversing the CAF-induced pro-inflammatory tumour environment has been the focus of many studies, although the results from clinical studies in patients with advanced metastatic disease have been disappointing with limited evidence of efficacy. Further work on understanding the differences in the TME at the metastatic site is required to help optimise potential therapeutic benefit.Inhibiting the recruitment of immune cells that drive a pro-tumorigenic environment as well as inhibition of their effector signalling pathways, or their re-education to anti-tumour phenotypes, have all been shown to prevent metastases in preclinical models.+ T-effector cells in the peritumoural stroma (rather than the tumour parenchyma).97 In a model of metastatic colorectal cancer, inhibition of TGF-β initiated a cytotoxic T cell response, and, importantly, in both these studies, inhibition of TGF-β signalling enhanced the activity of immune checkpoint inhibitors.98 Although direct effects on metastatic outgrowth have not been reported, effective targeting of other CAF-derived factors, including CXCL12, have also been shown to enhance the effectiveness of checkpoint immunotherapy in mouse models, and work on optimising this approach for the treatment of metastatic disease will be important going forward.99 CAFs are also able to restrict immune cell infiltration through remodelling of the ECM, although studies in the metastatic setting are more limited.29Within the TME, CAFs are a major source of TGF-β, which plays a key role in controlling anti-tumour immunity. In metastatic urothelial cancer, lack of response to the checkpoint inhibitor anti-programmed death-ligand 1 in clinical trials was linked to TGF-β signalling in fibroblasts and the consequent presence of CD8100 Interestingly, fibroblast-directed deletion of FAK, in a genetically engineered mouse model of breast cancer, reduced metastasis but had no effect on primary tumour growth. Mechanistically, this result was attributed to the secretion from FAK-deficient CAFs of exosomes enriched with the tumour-suppressive microRNAs miR-16 and miR-148a, which suppressed an EMT and migratory phenotype in the cancer cells.101 These data unveiled an additional role for FAK signalling in the tumour stromal compartment, promoting a more migratory and metastasis-competent phenotype. In addition, in a model of PDAC, inactivation of the kinase activity of FAK in fibroblasts reduced fibrosis, immunosuppressive cell populations and tumour progression.102 However, by contrast, work from the Hodivala-Dilke group has shown that loss of FAK from CAFs can promote breast tumour growth through alterations in tumour metabolism.103 The use of different promoters to drive Cre recombinase-mediated fibroblast-specific deletion of FAK might represent targeting of distinct CAF subpopulations and explain the disparate results in these studies. However, the results highlight the need to consider the impact of treatments on both stromal and tumour-derived signals, and that subtle differences in the crosstalk between specific subpopulations of stromal cells will be important in determining the response. To this end, understanding the early interplay between immune cells, HSCs/CAFs and tumour cells during metastatic colonisation is important in developing therapeutic intervention strategies. It would not be necessary to target all of these components: a single aspect of this TME could be targeted, which would destabilise the formation of early metastases. For example, by targeting CCR5 on immune cells using a Food Drug Administration-approved antagonist (maraviroc), Adwan and colleagues104 were able to reduce the liver colonisation of colorectal cancer cells in an animal model. The TME should be seen as a well-balanced and evolving microenvironment that is sensitive to disruption with targeted therapies.It is also important to consider that many agents in development that have been designed with the intention of directly targeting tumour cells might also have an effect on the activity of stromal cells. For example, the importance of focal adhesion kinase (FAK) within the tumour cell compartment in driving tumour proliferation and metastasis is well documented and has led to the development of a number of small-molecule inhibitors, which are currently under clinical evaluation.105 Single-cell analysis of CAFs has also identified specific subsets of CAFs associated with immunosuppression and immunotherapy resistance in patients with breast cancer raising the possibility of targeting specific CAF populations to enhance the effects of immunotherapies.106 Such analysis also highlights the growing complexity governing the metastatic process, and future studies might benefit from incorporating a systems-level approach to help uncover key hubs that are responsible for driving disease progression.107The TME clearly plays a critical role in shaping the progression of highly aggressive metastatic disease, for which current treatment options are limited. Our increased understanding of the complex interplay between different stromal compartments and how they influence tumour cell phenotypes is key to developing more effective therapies. These endeavours are being aided by new technologies, such as single-cell RNA-sequencing. This approach has identified distinct transcriptomic profiles in micrometastatic lesions from breast cancer-patient-derived xenograft models and revealed mitochondrial oxidative phosphorylation to be the main pathway upregulated in micrometastases in these models. Notably, targeting oxidative phosphorylation could reduce metastatic seeding.A number of organs, including the liver, appear to be privileged sites for tumour metastasis. The liver has evolved to be highly regenerative and contains a number of cell lineages  that can exit mitotic quiescence and proliferate to repair damage or deposit scar tissue to maintain the integrity of the tissue. Given the central role the liver plays in detoxification and how it is subject to recurrent insult/injury throughout adult life, this is a necessary evolutionary adaptation. However, this regenerative capability has also allowed it to become a prime metastatic site for CTCs. While the local immune repertoire of the sinusoid can sense tumour cells and orchestrate their killing, once a cancer cell has overcome this surveillance it can enter the space of Dissé where it co-opts local HSCs and immune cells to build a metastatic microenvironment, changing the physical and signalling parameters around these early tumour cells to facilitate their survival and promote their growth. While some of the cellular and acellular players have been identified in liver, we should not overlook the fact that these components may differ between tissues. Indeed, the process of colonisation could be very different depending on the tissue of origin of the cancer cell(s), with different signals and cells required to initiate metastasis.109 Taken together, the complexity and heterogeneity of signalling within the stroma mean that a pan-treatment targeting individual stromal cells is unlikely to be effective, and that tumour-specific approaches might instead be required. A greater understanding of how the reciprocal communication between immune cells and CAFs sustains a pro-tumorigenic environment provides additional impetus for combining therapies that target CAFs and immune cell functions and opens up exciting opportunities for treating patients with metastatic disease, an important area of unmet clinical need.The TME is highly dynamic; however, most studies monitoring metastatic spread are only able to provide a snapshot of what is happening. Intravital imaging approaches that allow imaging in both the primary and metastatic sites are now providing key insights into the dynamic temporal and spatial regulation of different stromal cell populations and how they interact with each other and the tumour cells.


As a highly conserved signaling pathway in metazoans, the Notch pathway plays important roles in embryonic development and tissue regeneration. Recently, cardiac injury and regeneration have become an increasingly popular topic for biomedical research, and Notch signaling has been shown to exert crucial functions during heart regeneration as well. In this review, we briefly summarize the molecular functions of the endocardial Notch pathway in several cardiac injury and stress models. Although there is an increase in appreciating the importance of endocardial Notch signaling in heart regeneration, the mechanism of its activation is not fully understood. This review highlights recent findings on the activation of the endocardial Notch pathway by hemodynamic blood flow change in larval zebrafish ventricle after partial ablation, a process involving primary cilia, mechanosensitive ion channel Trpv4 and mechanosensitive transcription factor Klf2. Drosophila melanogaster)  Dexter . In the Drosophila melanogaster. The very first cleavage, which occurs at site 1 (S1) by Furin-like convertase, starts at the Golgi compartment before the receptor locates onto the cell membrane, rendering it divided into the Notch extracellular domain (NECD) as well as the Notch transmembrane and intracellular domain (NTM-ICD), which is held together in a noncovalent manner  to ADAM family proteins, which is followed by the cleavage of the transmembrane domain (TMD) at site 3 (S3) and site 4 (S4) by γ-secretase; These events set the Notch intracellular domain (NICD) free to activate the Notch pathway . Next, Notch-activated Hey1 and Hey2 can inhibit Bmp2 and downstream Tbx2 expression in the heart chambers, thus limiting their expression specifically in the atrioventricular canal (AVC), which is required for normal AVC development  . By analyzing anterograde and retrograde flow velocity and calculating the fundamental index, we revealed that there was increased oscillatory blood flow in ablated hearts  has been the focus of many studies because of its ability to respond to hemodynamic alterations, which often occur through the Notch pathway  proteins can regulate cilia assembly. Additionally, klf2a and klf2b expression is downregulated in ift88 morphant ablated hearts; Notch signaling activation is also downregulated, and cardiomyocyte proliferation is decreased, suggesting the essential role of primary cilia in bridging hemodynamic alterations and cardiac regeneration (Li et al. The next question to explore is what factors sense hemodynamic alterations and trigger + influx and respond to vasodilatory responses (Mendoza et al. klf2a expression during valve formation (Heckel et al. klf2a and Notch expression in ablated hearts is markedly inhibited in trpv4−/− mutants. The expression of early cardiac transcription factors is impaired, as is cardiomyocyte proliferation and reprogramming, leading to a reduction in heart regeneration (Galvez-Santisteban et al. Certain ion channels on the plasma membrane can also sense mechanical shear stress and can subsequently activate signaling (Kim et al. klf2 expression after ventricle ablation, subsequently leading to Notch signal activation, cardiomyocyte proliferation and reprogramming. Although these data shed light on the regulation of Notch signaling during regeneration, many questions remain unanswered. Does hemodynamic alteration play important roles in inducing the damage response and regenerative program in other heart injury models or in other species? What is the relationship between primary cilia and mechanosensitive ion channels, and what are the factors involved in signal transduction? What are the ligands for Notch receptor activation and where are they localized? These are all important questions that warrant further investigation.In this review, we briefly summarize the functions of the Notch signaling pathway in embryonic development and tissue regeneration, and we focus especially on several heart injury and stress models. Then, we reveal the essential role of hemodynamic alteration in endocardial Notch activation in larval cardiac regeneration. We demonstrate that primary cilia and ion channels can respond to mechanical force and activate


This review provides an up-to-date source of information on the primary auditory neurons or spiral ganglion neurons in the cochlea. These neurons transmit auditory information in the form of electric signals from sensory hair cells to the first auditory nuclei of the brain stem, the cochlear nuclei. Congenital and acquired neurosensory hearing loss affects millions of people worldwide. An increasing body of evidence suggest that the primary auditory neurons degenerate due to noise exposure and aging more readily than sensory cells, and thus, auditory neurons are a primary target for regenerative therapy. A better understanding of the development and function of these neurons is the ultimate goal for long-term maintenance, regeneration, and stem cell replacement therapy. In this review, we provide an overview of the key molecular factors responsible for the function and neurogenesis of the primary auditory neurons, as well as a brief introduction to stem cell research focused on the replacement and generation of auditory neurons.  According to the World Health Organization, hearing impairment, the partial or total inability to hear sounds, is among the top 10 disabilities of today’s society and affects approximately 6% of the world’s population. Neurosensory hearing loss is permanent and results from the death of neurons or sensory cells that have no ability to regenerate. Auditory neurons are a critical component of the auditory pathway, as they transmit auditory information from sensory hair cells to the cochlear nucleus in the brainstem. The current effective therapies for hearing impairment utilize either hearing aids to increase hair cell stimulation or cochlear implants as a substitute for hair cells. These medical devices require the presence of functional auditory neurons in the inner ear. Therefore, recent studies focus on possibilities for neuronal replacement, including exogenous stem cell transplantation and endogenous cell source replacement. Transcriptional networks are key in controlling the regeneration or replacement of auditory neurons from stem cells. Understanding and identifying individual transcription factors involved in the development and survival of auditory neurons are crucial for future more effective treatments for hearing loss ..Sox2 thrjections . In contjections , suggestelopment ,49,50,51Neurog1 is an essential gene for the induction of neurogenesis, as eliminating Neurog1 results in a complete absence of inner ear neurons [Neurog1 not only eliminates neuronal development but, also, affects the sensory development, resulting in a significantly shortened cochlea with multiple rows of inner and outer hair cells in the apex [Neurog1-expressing precursors within the proneurosensory domain of the otocyst also differentiate into sensory cells [+Neurog1 precursors. Thus, some precursors can differentiate either into sensory cells or into neurons. Both the bHLH factors NEUROG1 and NEUROD1 promote the neuronal fate in the inner ear.  neurons ,53. The the apex ,37. Neurry cells , suggestNeurod1 null mutants lose eventually most of the sensory neurons [Neurog1-null mice, Neurod1-null mutants have a shorter cochlea and disorganized sensory cells in the apex [Neurod1 from developing neurons after their delamination shows that NEUROD1 participates in the segregation of vestibular and auditory neurons and in the regulation of peripheral and central projections [Neurod1 conditional deletion mutant is comparable to the control, with three rows of outer hair cells and one row of inner hair cells  family of transcription factors, is more strongly expressed in type I neurons, with the highest levels in the Ic type [Pou4f1 is broadly expressed in all auditory neurons with the onset of neurogenesis, and its germline deletion results in significant pathfinding defects and a partial loss of neurons [Pou4f1 beyond the onset of hearing, play an instructive role in presynaptic properties [Recent single-cell RNA sequencing identified transcription factors specific for different types of auditory neurons ,14,15. F neurons . A subseoperties . Runx1 (runt-related transcription factor 1) is expressed mainly in Ia and Ib types of neurons, although its function is not clear [Runx1 controls neuronal differentiation and increases the expression of Neurod1 [In contrast, ot clear . In cert Neurod1 , as well Neurod1 . Etv4 and Prox1 and increased expressions of Mafb and Gata3 [Gata3 is first expressed in all inner ear neurons. The early elimination of Gata3 results in no neurosensory cell differentiation in the cochlea [Gata3 deletion severely disrupts cochlear wiring and causes disorganized and prematurely extended neurites in the cochlea [Gata3 is expressed in mature type II auditory neurons, its functional role in these neurons is still unclear.The type II auditory neurons, representing 5% of neurons in the spiral ganglion, are morphologically and also molecularly different from type I neurons. They are not essential for the transmission of acoustic information but seem to have a role in the damage response and pain signaling ,12. Theynd Gata3 ,14,15. D cochlea , whereas cochlea ,69. AlthAs sensory cells and auditory neurons have little ability to regenerate, it is only recent advances in stem cell engineering that are increasing the feasibility of cell-based therapies for hearing disorders. For future advances in the treatment of hearing disorders and cochlear implant technology, the development of methodologies for the replacement of auditory neurons represents tremendous clinical potential. This goal can be only achieved by understanding normal neuronal development, as well as the mechanisms involved in the dysfunction and degeneration of neurons in the inner ear.Neurog1 and Neurod1 [Neurog1 and Neurod1-transfected cells demonstrate neuronal phenotypes, this transcription factor-induced reprogramming is not sufficient on its own to generate mature neurons in cochlear explants [One direction for the replacement of neurons is to reprogram endogenous cells in the inner ear, which would be more physiological. Proliferative cells in the inner ear showing stem cell properties were identified by several groups ,71. Howe Neurod1 . Althougexplants . Additioexplants , which rA second direction for neuron replacement is to use exogenous pluripotent stem cells (PSCs). PSCs have the capacity to self-renew and are capable of differentiating into multiple cell types. There are two sources of PSCs: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) generated by the reprogramming of somatic cells. PSCs have been used to generate inner ear cells for cell-based therapy and for in vitro analyses (


Apoptotic cells are commonly observed in a broad range of tissues during mammalian embryonic and fetal development. Specific requirements and functions of programmed cell death were inferred from early observations. These inferences did not hold up to functional proof for a requirement of apoptosis for normal tissue development in all cases. In this review, we summarize how the appraisal of the importance of developmental apoptosis has changed over the years, in particular with detailed functional assessment, such as by using gene-targeted mice lacking essential initiators or mediators of apoptosis. In recent years, the essentials of developmental apoptosis have emerged. We hypothesize that apoptosis is predominantly required to balance cell proliferation. The two interdependent processes—cell proliferation and apoptosis—together more powerfully regulate tissue growth than does each process alone. We proposed that this ensures that tissues and cell populations attain the appropriate size that allows fusion in the body midline and retain the size of cavities once formed. In addition, a limited number of tissues, albeit not all previously proposed, rely on apoptosis for remodeling, chiefly aortic arch remodeling, elimination of supernumerary neurons, removal of vaginal septa, and removal of interdigital webs in the formation of hands and feet. The components of such dying cells are packaged into cytoplasmic membrane–enclosed vesicles awaiting phagocytosis by other cells. In contrast, during necrosis, which can occur during injury and inflammation, larger areas of tissue are usually affected, cells swell, the plasma membrane and nuclear envelope rupture, and cell contents spill out.The observation that cells can undergo cell death during development was first made in the 1920s  was coined –regulated pathway and the extrinsic or death receptor pathway  and in disease or experimental settings by DNA damage and diverse cytotoxic agents. The response to these stimuli is regulated by BCL-2 protein family members4. In response to death-inducing stimuli, pro-apoptotic members of the family  inhibit the anti-apoptotic BCL-2 family members , which under steady-state conditions keep the pro-apoptotic effectors, the multi-BH domain proteins BAX and BAK, in an inactive state. In addition, some BH3-only proteins  have been reported to activate BAX and BAK directly5. Once activated, BAX and BAK form pores within the mitochondrial outer membrane, which leads to mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome C and other apoptogenic factors from the mitochondria into the cytoplasm , RIPK3, and mixed lineage kinase domain-like pseudokinase (MLKL)  family to their cognate receptors that belong to the TNF receptor (TNFR) family . This can set in train two different series of events: if available, pro-caspase-8, which directly activates effector caspase-3, -6 and -7, is activated. The pro-apoptotic BH3-only protein BID, after proteolytic conversion to tBID, can also activate the BAX/BAK-dependent mitochondrial apoptotic pathway to thereby amplify the apoptotic cascade . If casp (MLKL) . These a (MLKL) . The forIn the following section, we summarize originally proposed roles of apoptotic cell death during development and more recent detailed functional studies that have identified the much more restricted gambit of processes that critically depend on developmental apoptosis. In the conclusion, we propose why developmental apoptosis occurs in many tissues, seemingly without being essential.Caenorhabditis elegans toMus musculus, were used. InC. elegans, specific individual cells destined to undergo cell death were identified10. In contrast, clusters of cells undergoing cell death in mouse embryos were noted at specific stages in specific regions, but death did not appear to be pre-programmed to target specific individual cells  cells were present in certain locations at given stages of development. Diverse animal models, froml cells . In mice11, and during the development of the epiblasts at E6 to E712, neural crest cells13, lens vesicle14, and the pro-amniotic cavity15, for digit separation by removing interdigital tissue at E13.516, shaping the limbs17, neural tube closure18, and elimination of supernumerary neurons during early postnatal development of the brain26.Subsequent reports proposed that apoptotic cell death was essential for normal development in regions where pyknotic cells were observed , includiMice lacking individual apoptotic regulators provided evidence for the requirements for specific regulators and suggested that developmental apoptosis was essential for mammalian development .28. However, it appears that the lack or restriction of neurotrophic support may be the cause of neuronal apoptosis on both occasions29.Indeed, from the original large number of developmental processes proposed to require apoptosis to proceed normally , knockou31. Male mice lacking a functionalTrp53 gene are born in the expected numbers, but female–/–Trp53 mice are under-represented because of a partially penetrant failure of neural tube closure33. Originally, electron microscopy32 and terminal deoxynucleotidyl transferase dUTP nick end labeling of DNA fragments (TUNEL) (a technique used to identify cells undergoing apoptosis) on sections33 were employed to determine whether loss of p53 affected developmental apoptosis, but neither method detected a difference between wild-type and–/–Trp53 mouse embryos. It was hypothesized that the methods employed may not have been sensitive enough to detect differences33. More recently, developmental apoptosis in–/–Trp53 embryos was assessed by TUNEL flow cytometry on single cell suspensions and this method was sufficiently sensitive to detect a difference between–/–Trp53 and wild-type embryos34. Flow cytometry proved to be a sensitive and precise method that also easily detected differences between wild-type and–/–;Bak–/–Bax double knockout (DKO) embryos 35 or+/–;Bclx+/–Mcl1 double heterozygous mice 36. However, the low percentage of apoptotic cells in developing wild-type embryos (only about 1.5%36) does not provide a large dynamic range for detecting a gradual reduction in apoptosis. Indeed, although the more severe developmental abnormalities in mice with combined loss of pro-apoptotic BAX, BAK, and BOK compared with loss of only BAX and BAK suggested that the–/–;Bak–/–;Bok–/–Bax triple knockout mice (TKO) should have a greater reduction in developmental apoptosis than–/–;Bak–/–Bax DKO mice, flow cytometric analysis was unable to detect significant differences between these two genotypes35. Therefore, a claim of zero apoptosis is hard to support with the current methods. A more sensitive approach may be the use of mice carrying theCAG-sA5-YFP transgene, which encodes a fluorescently labeled, secreted form of annexin V. The fluorescently labeled annexin V accumulates to detectable levels when binding to phosphatidylserine exposed on the surface of apoptotic cells37. A caveat here may be that necroptotic cells also stain positive for annexin V38.As a corollary of identifying developmental processes that require apoptosis, those processes that occur normally without apoptosis might also have been defined. However, to make such a claim with certainty, the possibility of residual apoptosis occurring would have to be excluded. The difficulties in assessing absolute numbers of apoptotic cells during development can be illustrated by using the example of embryos lacking the tumor suppressor p53, which in response to a number of stress stimuli can induce either cell cycle arrest or apoptosisC. elegans development8 and from time to time it was speculated that apoptosis in mammalian development, too, might be less critical than its prevalence might suggest39. Throughout the decades of intense research on the role of apoptosis in a number of developmental processes, information surfaced indicating that, despite previous inferences, apoptosis was not required for specific processes. An example is the role of apoptosis in the formation of the pro-amniotic cavity, which was viewed as a model of cavity formation/tube formation by apoptosis and supported by work inin vivo and culture models41 but was later called into question by the development of embryos past this point in the absence of detectable apoptosis35. Similarly, apoptosis had been observed in the ridge of the closing neural folds and therefore it had been proposed that neural tube closure required apoptotic cell death at the point where the two neural folds met. This was supported by the observation that the application of caspase inhibitors at a critical time point blocked neural tube closure18. On the surface, this conclusion appears to be amply supported by the many knockout mouse mutants of apoptotic regulators that display exencephaly or spina bifida or both . However, one study presented evidence that apoptosis is not actually required for the specific time of neural tube closure and that lack of apoptosis did not affect tissue remodeling and separation of the neural tube from the surface ectoderm at the fusion site47. Similarly, it is not clear whether apoptosis is required at the specific time of palate fusion51.It was surprising that apoptosis, given its stereotypic occurrence, was not essential forThe studies showing that neural tube closure and palate fusion can occur without developmental apoptosis raise the question of why so many mouse mutants of critical constituents of the mitochondrial apoptotic pathway display exencephaly, spina bifida, and cleft palate . A possi78 and mice lacking RIPK1 die shortly after birth79 whereas RIPK3-deficient mice develop normally80. However, if both death receptor–activated processes (apoptosis and necroptosis) are inactivated by simultaneously deleting genes necessary for each process, the resulting mice are viable . The studies using these compound mutant mice provided evidence that death receptor–mediated apoptosis is not required for normal mouse development; rather, a balance between caspase-8 and RIPK1/RIPK3 activity needs to be maintained to prevent MLKL activation and consequent necroptosis. The observation that mice lacking MLKL85, the essential mediator of necroptosis, develop normally reveals that this programmed cell death process is not required for embryogenesis. In agreement with these findings, early histological studies did not detect substantial involvement of necrotic processes in mammalian development.Activation of the death receptor pathway can lead to apoptosis via caspase-8 activation or, when caspase-8 is inhibited, to necroptosis via RIPK1/RIPK3/MLKL activation . Death rBax,Bak, andBok did not appear to cause a compensatory increase in necroptosis, pyroptosis, or autophagy35, suggesting that these other forms of cell death may not be poised to compensate for a deficiency of the mitochondrial apoptotic pathway.The most abundant form of developmental cell death appears to be apoptosis, and more specifically mitochondrial apoptosis, as the death receptor pathway does not appear to be essential for normal development (discussed above). Furthermore, reduction of mitochondrial apoptosis to below current detection levels by simultaneous deletion of86, autophagy88, cell extrusion from epithelia90, and cell senescence92. However, their prominence and roles during mammalian development remain to be determined. Autophagy is morphologically distinct from apoptosis93 and so could be assessed and its frequency could be compared to apoptosis. An overlap between apoptosis and entosis exists in so far as apoptotic cells are eventually engulfed by other cells. Whether mammalian cells are extruded from epithelia  before or after they have initiated cell death appears contentious95, and the ultimate fate of senescent cells seems to be cell death, possibly mostly through apoptosis92.A number of alternative cell removal processes have been proposed to play a role during development, including entosisOverall, the mitochondrial apoptotic pathway appears to be the major form of developmental cell death. However, it should be noted that low prevalence of a given process does not necessarily imply little importance. Accordingly, it is possible that in specific cases these alternative forms of cell death play an essential role during mammalian development, which remains to be functionally tested. This will probably require the generation and analysis of mice deficient in multiple cell death pathways and possibly even mice lacking several cell death pathways plus other processes  to uncover redundancy.The following open questions exist in the field of developmental apoptosis.(1) The field might benefit from the results of a comprehensive, sensitive, and quantitative analysis of apoptosis throughout the sequence of mammalian development that either does not rely on tissue sections or uses sophisticated methods of three-dimensional reconstruction and numerical assessment.(2) Once a sensitive technical approach to (1) has been developed, the question of whether developmental apoptosis is dynamically regulated during development in response to disruptions  could be answered.(3) If similarly sensitive and quantitative detection methods were developed for other forms of cell death, their relative importance compared with apoptosis could be assessed.(4) The role of a number of BCL-2 family members in development remains to be determined, while single knockout of the genes encoding these does not result in overt phenotypic anomalies, which is possibly due to functional redundancies.(5) In addition, based on the theory of evolution, one would expect that even these BCL-2 family members have essential functions (that provide a selective advantage), which remain to be discovered.The available data identify developmental processes that are clearly dependent on functional apoptosis, whereas other processes previously thought to depend on programmed cell death do not appear to be as reliant on apoptosis.et al., 201835.) However, the caveat that residual apoptosis not detectable by current methods (discussed in the preceding sections) needs to be kept in mind.Processes apparently independent of apoptosis based on normal embryonic and fetal development in the combined absence of BAX, BAK, and BOK include, for example, the formation of the pro-amniotic cavity, epiblast, intestine, and lens development as well as enchondral ossification. .Another possible explanation is that a twofold control of developing tissue size and cell population homeostasis by maintaining a balance between proliferation and cell death affords not just one but two cellular mechanisms of fine-tuning cell number. This would provide a more robust regulation, less prone to errors and variation in response to negative impact.97 but perhaps also by actively downregulating developmental apoptosis. It can be assumed, on the basis of its evolutionary conservation, that apoptotic cell death, even in regions where it is not absolutely required, provides a competitive advantage.Indeed, mammalian and other embryos appear to have an astounding capacity to compensate for the loss of cells, certainly by upregulating cell proliferation


Plant growth, development, and productivity are adversely affected by environmental stresses such as drought (osmotic stress), soil salinity, cold, oxidative stress, irradiation, and diverse diseases. These impacts are of increasing concern in light of climate change. Noticeably, plants have developed their adaptive mechanism to respond to environmental stresses by transcriptional activation of stress-responsive genes. Among the known transcription factors, DoF, WRKY, MYB, NAC, bZIP, ERF, ARF and HSF are those widely associated with abiotic and biotic stress response in plants. Genome-wide identification and characterization analyses of these transcription factors have been almost completed in major solanaceous food crops, emphasizing these transcription factor families which have much potential for the improvement of yield, stress tolerance, reducing marginal land and increase the water use efficiency of solanaceous crops in arid and semi-arid areas where plant demand more water. Most importantly, transcription factors are proteins that play a key role in improving crop yield under water-deficient areas and a place where the severity of pathogen is very high to withstand the ongoing climate change. Therefore, this review highlights the role of major transcription factors in solanaceous crops, current and future perspectives in improving the crop traits towards abiotic and biotic stress tolerance and beyond. We have tried to accentuate the importance of using genome editing molecular technologies like CRISPR/Cas9, Virus-induced gene silencing and some other methods to improve the plant potential in giving yield under unfavorable environmental conditions. In their natural environments, plants are persistently and simultaneously confronted to diverse biotic and abiotic stresses, whose impacts are enhanced by climate change resulting from anthropogenic activities because of dramatic population increases which resulted in restraining the water availability and upsurge the temperature . The incSolanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena) and hot pepper (Capsicum annuum) are in high demand worldwide for their nutritional and medicinal values, high vitamin content and psychotropic effects i.e., solanaceous crop can be used as a source of drugs and energy [Phytophthora capsici, Fusarium species and Collectricum species [The major Solanaceous food crops such as tomato . Transcription factors (TFs), which is also known as trans-acting factors, are proteins that identify and bind to specific DNA sequences (often in the promoter region) and control cellular processes by sustaining and regulating the expression of target genes during the transcription processes . This meMoreover, up-to-date more than 60 transcription factors have been identified in plants using bioinformatics tools, next generation sequencing and other methods in plant genome-wide association and other molecular studies . But in Research in tomato and potato have underscored that plants use different methods to hold out the environmental stresses such as drought, salinity, oxidative stress, cold, heat, and diverse diseases that affect the plant growth and development in different ways. Given the sessile nature of plants, environmental stresses may lead to sub-optimal growth conditions, imposing plasticity in different metabolic pathways that allows plants to function while resisting, tolerating or recovering from the stress conditions. As shown in The complexity of the abiotic stress response arises from its polygenic nature and the coupling of signal transduction events with the production of stress response factors when the plant gets exposed to stresses. Noticeably, abiotic stress factors such as drought(water stress), water logging( excessive water), salinity, mineral toxicity, variation in temperature are unfavorably impact the physiological components of plants by altering their metabolism, growth and development status as plants are sessile (immobile) in nature and cannot escape from the environmental cues . As it cEnvironmental stresses (primary and secondary) which have been mentioned in the In plant breeding and improvement, creating variation in the gene pool is an important way of improving plant traits and ensuring genetic diversity at prominent level. The conventional method of crop improvement has many bottlenecks such as mutations, gene duplications/deletions and chromosomal rearrangements and etc. However, nowadays, there is a conspicuous promise for addressing the evolving challenges in plant production because of the ongoing climate change. To challenge the climate change which is the big issue for the world right now, the currently emerged molecular technology, CRISPR/Cas9 may play pivotal role in improving the traits of plant against the climate change. It is sequence-specific genome editing technology which is becoming a powerful tool in improving agronomic trait of crops and play role in ward off pathogens and minimizing the risk of different stresses even at multiplex genome editing techniques either by adding or removing the gene of interest . Most imMLO1, DMR6, MAPK3, BZR1, CBF1, AGL6, IAA9, MPK20, GAD , BOP  [Basically, the CRISPR/Cas9 can cut or add the target regions of genes directly in a DNA sequence in very specific way. This means that, it is easy to generate stable and heritable mutations without affecting the existing valuable traits in plants or other organism. In this way, it is possible to develop the homozygous modified transgene-free plants in only one generation and its stable transmission to successive generations which can save time immensely ,23. Take1, 2, 3) . As a reSlCBF1 protein and chilling stress tolerance have shown that it was positive, meaning that it helps in tolerating the chilling stress. On the other hand, the highly conserved CBFs are cold-response system components found in many plant species. Therefore, it was concluded that the generated SlCBF1 mutants using the CRISPR/Cas9 system has shown that SlCBF1 protein profoundly helped tomato in chilling stress tolerance [SlMAPK3 gene is responsible for drought in tomato, the knockout mutants produced by CRISPR/Cas9 has shown the same thing on the drought stress [MLO1 gene is also responsible for powdery mildew vulnerability just that the mutant generated by CRISPR Cas9 has shown tolerance to the disease comparing with the untreated tomato plant.For instance, in tomato, chilling stress is the main restraint because it is tropical in origin and very-sensitive to chilling stress, which can restrict the flowering and fruiting stage. But, currently, it has been revealed that the mutant that has been made using the CRISPR/Cas9 gene system to understand the functional relationship between olerance . Alternat stress ,27. Accot stress , MLO1 geSlGRX1 is responsible for drought stress in tomato as the satellite-virus-based vector, DNAmβ was used for confirmation at physiological level by measuring chlorophyll and relative water content (RWC) which both of them have shown low content compared to the control plant in addition to phenotypic differences [CaPO2) gene in Capsicum annum is responsible for osmotic stress in which bleaching was displayed more than the control plant. The research that has been done on the silencing of the ABI3/VP1 transcription factor (CaRAV1) alone or together with OXIDOREDUCTASE (CaOXR1) was shown the same thing in the plant [The other genome editing technology is Virus-induced gene silencing (VIGS) which is also the most valuable tool to understand the functional confirmation of stress-responsive genes which shows high transcript profile (high expression level) when the plant gets exposed to different kind of stresses. Most importantly, the TRV-VIGS-mediated silencing method is very imperative in solanaceous crops for studying the gene function at the transcription level. For instance, it has been pinpointed that a GLUTAREDOXIN gene, ferences . On the he plant ,31. MostThe plant-specific multigene DOF (DNA-binding one finger) transcription factor family is highly involved in many growth and developmental processes such as phytohormone regulation, light signaling, and seed germination. It plays major role in assisting protein-protein interaction in addition to aiding in DNA-binding activities . ImportaDOF proteins can physically cooperate with each other or modulate transcription in the genome at defined sites  and can The Zinc finger motif has 29 amino acid residues and another 21 amino acid residues at its C-terminal region just that the residues can participate between Dof domain and DNA interactions. Because of the strong similarity among the Dof DNA binding domain, it is characterized by a C2C2-type zinc finger motif that distinguishes the specific regulatory elements of four nucleotide bases of “AAAG” or “CTTT” in the promoters of the specific genes as it has been studied in vivo and in vitro experiments ,43,44. OTFs belonging to the same family may exhibit various activities owing to the occurrence of a transcriptional regulatory domain that may act as a repressor or activator during the gene expression . The repSmeDof25 and SmeDof4), and others were extremely low  and high  which may be promising for the development of transgenic lines [StDof genes were expressed in all potato tissues, although the expression levels of individual genes varied in tomato, eggplant, and hot pepper. Seventeen of 33 genes have shown relatively high expression levels in the root. In the same way, 22 SlDofs in tomato and 22 GmDofs in soybean were expressed at relatively high levels in the root [Heat maps showing the global transcription patterns of hot pepper Dof genes were generated based on publicly available RNA-seq data for the plant tissues in different developmental stages and varieties  Table 1Table 1. ic lines . Similarthe root . Therefothe root ,56,57,58SlWRKY30, SlWRKY83 and SlWRKY75 genes in tomato have been upregulated in drought [SlWRKY72 gene has a significant role in basal immunity and makes the plant withstand the pathogen attack when the pathogen gets conducive environment and has shown a positive role in abiotic stress tolerance. The WRKY genes studied so far in major solanaceous crop have mostly shown upregulation under different extremities like drought, salt, heat, cold and other different stresses which obstruct the plant from finishing their life cycle and producing the genetically expected yield as shown in This transcription factor is widely studied in different plants including the solanaceous crops and it is considered as one of the largest and important transcription factors in connection with the environmental stresses both biotic and abiotic stresses. The WRKY protein has one WRKY domain which includes 60 amino acid residues like most the other transcription factors. Structurally, its N terminus has the WRKYGQK motif and the C-terminal has metal-chelating zinc finger motif, either C2H2 or C2HC as carbon and hydrogen play an important role in defining its structure. Therefore, the WRKY family has three groups depending on the number of WRKY domains and number of zinc finger it has in its structure. Among the three groups, Group I and II WRKY domains placed in the C- and N-terminus, respectively ,60. Alte drought . SimilarCaWRKY27 gene from Capsicum annum, has shown positive role in regulating the stress resistance to Ralstonia solanacearum infection by modulating and regulating SA, JA and ET mediated signaling pathways in tobacco (Nicotiana tabacum) after the plant has been exposed to the mentioned pathogen [StWRKY72 gene in potato has been also highly up-regulated under stress treatment that sounds its importance better in developing a resistant transgenic line in solanaceous crop to make sure that the developed plant can grow under a water shortage and help in marginalized land utilization utmost [Most importantly, signaling molecules which are endogenous to plants such as SA, JA, ET and ABA play a tremendous role in regulating the signaling pathways which subsequently has capable of changing the transcription level of stress related genes and protein post-processing which as a result help as a mediators in basal defense systems ,75. Thispathogen ,75. Thusn utmost .https://solgenomics.net/), NCBI (https://www.ncbi.nlm.nih.gov/) and PLANTTFDB (http://planttfdb.cbi.pku.edu.cn) which are major databases for many plant transcription factors. By and large, WRKY protein has many functions in regulation of plant growth and development in response to different stresses such as salinity, drought, heat and cold ,77,78. Ihttp://solanaceae.plantbiology.msu.edu/pgsc_download.shtml). In Solgenome database is very common for major solanaceous crops and provides all the information for genomes, genes and protein sequences whereas PLANTTFDB database has all 60 plant transcription factors. Most prominently, the WRKY protein and gene sequences can be downloaded from the PGSC database  has been also used to understand the function of CaMYB31 gene by suppressing it. After the CaMYB31 gene was silenced, the plant has shown a substantial decrease in capsaicinoid accumulation compared to the control chilli pepper plant [The expression of miRNAs has been regulated most of the time during the pathogen infection or when the plant gets attacked by the pathogen. Most importantly, miRNA58 regulate the expression of numerous genes taking part in a specific pathway including the phenylpropanoid pathway which plays major roles in the production of antifungal compounds to increase the plant’s ability to stand against pathogen infection massively .The moster plant . The infThis transcription factor was discovered 30 years ago and participates in transcriptional activation of the genes controlled by thermal stress (heat stress), which has capable of encoding heat shock proteins i.e., it shows a high level of gene expression under thermal stress. On the other hand, the expression of heat-shock genes increases when the plant gets exposed to thermal stress which results in the rapid accumulation of heat-shock proteins (HSPs). Understanding heat transcription factor started when it was first isolated from tomato  and has 2+ and salicylate which is highly toxic to human health [Therefore, when the plant gets exposed to high temperature , heat transcription factor which is also known as the central regulator of heat shock stress response, regulate the expression of many heat-stress-inducible genes at transcription level by recognizing the conserved binding motifs  that exist in the promoter region so that plant can withstand thermal stress. Additionally, HSPs guard the cells against thermal and other environmental stresses which at the same time participate in protein folding that helps in protein function, cell differentiation, dimensional structure and conformation ,107. Notn health ,109.HSFA1a) has shown a unique function in master regulator for acquired thermotolerance which cannot be replaced by any other HSFs families [CS-SlHsfA1a), in which the expression levels of HSPs were decreased under normal conditions and this plant has shown more sensitivity to thermal or heat stress. Hence, it is helpful in developing solanaceous transgenic plant to improve heat stress resistance so that it is possible to use the marginal land where thermal stress is problematic. HSF gene sequences of each major solanaceous food crops can be obtained from the Plant Transcription Factor Database [http://pfam.xfam.org/search). Although the solanaceous plant could survive in a different range of agro-ecological conditions, both vegetative and reproductive growth is severely affected by heat  stress which resulted in low yield and/or poor quality fruits ,111. Expfamilies . This roDatabase . The comhttp://blast.ncbi.nlm.nih.gov/blast.cgi) and Spud DB for major solanaceous food crops  and Genomics Resources database (http://solanaceae.plantbiology.msu.edu/) with E-value of 0.001 to screen candidate Hsfs with homologous amino acids sequences. These candidate genes can be analyzed using the domain identification function of the Pfam database (E = 1.0) to remove the Hsfs without the conserved domain sequences. This helps in the multiple protein sequence alignment using Clustal W and MEGA 4.0 to remove repetitive sequences. Depending on the genome wide identification and characterization of heat shock transcription factor in solanaceous crops, the total lists of HSTs were listed in The protein sequences of HSF conserved domains are aligned by BLAST in the NCBI , eggplant (SmNAC), potato  and hot pepper (CaNAC1) are take part in different signaling pathways to induce local and systemic disease resistance in which most of them are discussed in When the plant gets attacked by the pathogen (biotic stresses), there are at least three signaling mediators such as SA, JA) and ET that can be harmonized to generate the plant defense system ,130. MosAtNAC1 gene was induced by lateral root development and it is also regulated by auxin which at the same time enhances the cell division and regulates the leaf senescence [SlNAC1 gene play an important role in chilling stress resistance in tomato (4 °C), heat stress (40 °C), and different osmotic (drought) stresses. This gene also aids the plant against the mechanical wounding and other kinds of similar stresses which directly or indirectly help plant’s tolerance against these kinds of stresses [The research that has been done on Arabidopsis has shown that the expression of the nescence ,155,156.nescence . Most ststresses ,157,158.It is also one of the largest transcription factors found in all eukaryotes including plants. It has a bZIP domain that regulates gene expression in response to different abiotic and biotic stresses. As the name suggests, the basic region/leucine zipper (bZIP) domain consists of persistent α-helix involving a basic region (BR) which is very important in binding the DNA followed by a C-terminal leucine zipper (LZ) motif accountable for the dimerization . In planEthylene is a recognized gaseous plant hormone that plays crucial and regulatory roles in plant growth and development. It is also functioned as a stress-related hormone required in biotic and abiotic stress responses in different ways. Hence, the Ethylene Responsive Factor family which was first isolated from tobacco two decades ago is one of the largest transcription factors that involved in the ethylene signaling pathways and lead to the expression of various defense-related genes such as pathogenesis-related (PR) genes and abiotic stress-responsive genes . ERFs alBy the same token, it was reported that the ERF transcription factor family members are responsive to drought, ABA, and saline conditions and can serve as activators or repressors of ABA signaling under salt stress . JasmonaAuxin is one of the most important plant hormone that plays significant roles in plant growth and developmental processes such as cell division, expansion, and differentiation. It regulates the expression of early auxin-responsive genes under different developmental processes and stress conditions. Thus, the ARF proteins are a plant-specific transcription factor, which structurally consists of three components: a conserved amino-terminal DNA binding domain (DBD), a highly conserved carboxyl-terminal domain (CTD) and a variable middle region (MR) . The ARFThe genome wide identification and characterization of a major transcription factors in solanaceous food crops have been almost completed and the lists have been shown in the following The effects of abiotic stresses such as drought and salinity on photosynthesis are multifaceted and are directly interrelated with stomatal closure and mesophyll limitations for the diffusion of gases, which ultimately changes the net photosynthesis process. It is therefore very significant to measure the chlorophyll content of plants using spectrophotometric analysis to know how much the plant is affected physiologically. Acetone and DMSO are the two most commonly used in the extraction of Chlorophyll a, chlorophyll b and carotenoid in plants and are measured at 665, 643 and 470 nm respectively . Most ofOn the other hand, measuring the relative water content  is very Genome-wide studies of transcription factors such as WRKY, MYB, DOF, HSF, bZIP and NAC in solanaceous plants play a vital role in understanding the genes responsible for different stresses i.e., transcription factors might help in boosting up the yield and developing a tolerant plant to different environmental stress conditions. A major future challenge is the mitigation of climate change effects on crop production despite reduced water availability in which the systems of drought resistance may vary depends on the climate change and soil condition with variation in temperature. The advantage of focusing on plant TFs lies in the potential production of the genetically modified cultivar in which plant stress response pathways and factors can be fine-tuned to make the plant tolerate the environmental stresses. Increasing the water use efficiency of solanaceous crops is very important particularly in arid and semi-arid areas in most developing countries like in Africa because of the fact that solanaceous crop demand more water. There are no many transgenic plants developed using TF candidate genes in solanaceous crops to the wanted extent, but it may represent a good strategy in improving the production and productivity in the area of water shortage and where the disease is very severe. The currently available genome editing technologies like CRISPR-Cas9, VIGS and other gene editing and silencing technologies are invaluable in improving the traits of not only solanaceous plant but also other very important food crops. This has at least two major functions: firstly, it is worth emphasizing that a single TF can regulate the expression of multiple genes to help in withstanding different stresses and secondly it may also use for developing a transgenic plant of desirable characteristics. This regulatory capacity might, therefore, be useful for improving water use efficiency and yield. The expression analyses of the mentioned eight transcription factors in different solanaceous crops by testing the upregulation in different tissues  or under different abiotic or biotic treatments (stress regulation). This could promise in understating the role of each gene as the potential of each specific gene is unique in terms of environmental stresses and the conditions facing dry land agriculture. Therefore, the development of transgenic plants harboring TF genes that improve stress tolerance in field conditions represents a gap that will need to be filled by future research which would be environmentally friendly. There are still a lot of transcription factors that need to be characterized and the variation in each gene with its function in solanaceous crop sounds better in developing better traits. Another challenge is the difficulty of ‘pyramiding’ drought-tolerance related genes in highly heterozygous tetraploids plants like potato cultivars because most abiotic stress like drought and salinity are controlled by many genes. Therefore, TF genes need to be further studied to identify suitable candidate genes to improve different stress tolerance and water use efficiency through reverse genetics analyses, gene expression studies, transgenics and using different available molecular assays technologies to keep the food security. By and large, genome editing technologies should get better attention as climate change is ongoing and number of populations is also dramatically increasing which as a result giving attention to improve the yield in quality and quantity is so much important.


Hyperventilation is a commonly used therapy to treat intracranial hypertension (ICTH) in traumatic brain injury patients (TBI). Hyperventilation promotes hypocapnia, which causes vasoconstriction in the cerebral arterioles and thus reduces cerebral blood flow and, to a lesser extent, cerebral blood volume effectively, decreasing temporarily intracranial pressure. However, hyperventilation can have serious systemic and cerebral deleterious effects, such as ventilator-induced lung injury or cerebral ischemia. The routine use of this therapy is therefore not recommended. Conversely, in specific conditions, such as refractory ICHT and imminent brain herniation, it can be an effective life-saving rescue therapy. The aim of this review is to describe the impact of hyperventilation on extra-cerebral organs and cerebral hemodynamics or metabolism, as well as to discuss the side effects and how to implement it to manage TBI patients. Intracranial hypertension (ICHT) is the most critical and potentially devastating complication in traumatic brain injury (TBI) patients . Since t2) has been used since decades in neuro-anesthesia and in neuro-intensive care, because lowering PaCO2  through increased minute volume ventilation  can rapidly contribute to reduce the volume of the swollen brain and help control ICP  have been evaluated, while pre-hospital hyperventilation or spontaneous hyperventilation will not be discussed.2 <38 mmHg) in order to manipulate cerebral hemodynamics and compliance , forced (PaCO2 26–30 mmHg), or intensified forced (PaCO2 <26 mmHg), according to PaCO2 levels  or dilating  , and is proportionally more relevant with hypercapnia than with hypocapnia  could improve indices of cerebral autoregulation function in TBI patients with disturbed pressure-reactivity at baseline, whereas those with intact pressure-reactivity at baseline would have no effect of such intervention , although these results were not consistent in all studies  is a regional technique, is well-correlated with local CBF and can directly monitor the areas at higher-risk of secondary ischemia    , 74. Rec30 mmHg) , 40.n = 9) showed that moderate and intense hyperventilation resulted in reduced CBF and increased oxygen extraction, but a constant oxygen metabolism  (2 <30 mmHg) increased the volume of hypoperfused cerebral areas within the injured brain, which, in the absence of increased oxygen extraction, would result in tissue hypoxia.Cerebral metabolic function can be assessed at bedside using the microdialysis technique, or performing PET and magnetic resonance imaging (MRI) spectroscopy studies. In one study, early hyperventilation  was associated with a significant increase in tissue lactate and lactate/pyruvate ratio, suggesting anaerobic metabolism and tissue hypoxia ; these mfailure) . In two failure) , 75, hypn = 251), Gordon et al. (2 between 25 and 30 mmHg for 6 to 41 days); however, more severe neurological sequelae were observed among survivors in the hyperventilation group when compared to the other. Only one prospective randomized clinical trial has investigated the effects of hyperventilation in this setting; Muizelaar et al. , there was a higher proportion of patients with long-term favorable neurological outcome when compared to the others.With all these potential side effects, which is the effect of controlled hyperventilation on the outcome of TBI patients? In one large retrospective study  monitoring, is necessary in this phase, as hyperventilation in the absence of elevated ICP is frequently observed in adult TBI patients   and therefore be less accurately predicted, using PaCO2 levels and quantifying changes in cerebral hemodynamics and physiology to PaCO2 changes at bedside is more feasible for physicians. If ICP remains within acceptable values after 48 h from the injury , lower PaCO2 values  could result in improved cerebral autoregulation  could be tolerated, although a more comprehensive neuromonitoring would be the only effective solution to detect the potential occurrence of tissue hypoxia.The initial PaCOagement [, internaagement [, 50. Proagement [ as it wopatients . Importa output) . Whethergulation , howevergulation ; as such2 values of 30 mmHg because: (a) the most important effects of PaCO2 on ICP are observed between 30 and 50 mmHg and (b) most of the relevant cerebral side effects were reported for forced hyperventilation. If possible, the use of hyperventilation should be minimized for these patients during the first 24 h after injury, when CBF often is the most reduced  are present  and coul reduced , unless 2 around 33–36 mmHg and avoid values <30 mmHg , international guidelines recommended the use of controlled hyperventilation only as “tiers 2” therapy, after the failure of increased sedation and osmotics infusion ; indeed,<30 mmHg . In the 2 values on cerebral oxygenation and metabolism. As such, PaCO2 values and hyperventilation could be adjusted to the brain tolerance and therapeutic targets individualized on the patients' need. In case of the need for low PaCO2 values and concomitant lung injury, other interventions aiming at reducing CO2 production, such as increasing sedation or hypothermia, could be considered to induce hypocapnia and avoid lung stress and VILI. However, this strategy should be further evaluated in clinical studies.In our opinion, invasive neuromonitoring should be also considered in severe TBI patients and ICHT to optimize overall management and, in particular, to assess the effects of low PaCO2 values are mandatory in severe TBI patients because of the significant effects on brain hemodynamics and compliance, high doses of sedatives, often in association with neuromuscular blocking agents (NMBAs), are required to adjust ventilatory parameters to obtain desired PaCO2 targets. As such, in the absence of aggressive therapies for ICHT, it remains unknown when it would be safe to discontinue sedatives and eventually tolerate spontaneous hyperventilation in these patients. If this occurs after several  days from injury, the risk of oligemia would be probably limited. Also, spontaneous hyperventilation after acute brain injury is often triggered by local acidosis , which result in vascular dilation and would probably compensate from vasoconstriction induced by hyperventilation. In these cases, non-invasive  and invasive  monitoring would again be helpful to individualize therapeutic decisions.If controlled PaCO2 values should be maintained in the early phase after TBI if ICP remains within acceptable values. Controlled hyperventilation  should be used as a temporary life-saving intervention in case of severe intracranial hypertension; PaCO2 levels should be also adjusted and individualized in each patient using CTP and cerebral ultrasound of, whenever possible, advanced multimodal neuromonitoring.In conclusions, controlled hyperventilation is effective in reducing ICP but it also reduces CBF and might have both cerebral and systemic serious side effects. As such, normal PaCOEG, LP, JC, and FT contribute equally to the elaboration of this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.