Glioblastoma (GBM) tumor microenvironment (TME) is a highly heterogeneous and complex system, which in addition to cancer cells, consists of various resident brain and immune cells as well as cells in transit through the tumor such as marrow-derived immune cells. The TME is a dynamic environment which is heavily influenced by alterations in cellular composition, cell-to-cell contact and cellular metabolic products as well as other chemical factors, such as pH and oxygen levels. Emerging evidence suggests that GBM cells appear to reprogram their the TME, and hijack microenvironmental elements to facilitate rapid proliferation, invasion, migration, and survival thus generating treatment resistance. GBM cells interact with their microenvironment directly through cell-to-cell by interaction mediated by cell-surface molecules, or indirectly through apocrine or paracrine signaling via cytokines, growth factors, and extracellular vehicles. The recent discovery of neuron–glioma interfaces and neurotransmitter-based interactions has uncovered novel mechanisms that favor tumor cell survival and growth. Here, we review the known and emerging evidence related to the communication between GBM cells and various components of its TME, discuss models for studying the TME and outline current studies targeting components of the TME for therapeutic purposes. In addition to these mechanisms, tumor cells are known to employ exosomes, gap junctions, circulating tumor cells, tunneling nanotubes, cell-free DNA, and horizontal DNA transfer to interact with other tumor or normal cells.7–9 In addition, the recognition of the essential nature of the TME and the complex communication network between tumors and normal cells for GBM development and progression has yielded a new focus for therapeutic targeting of GBM.9–12Glioma cells take a central role in regulating the functions of cellular and noncellular components of the TME via complex signaling networks which enables them to regulate processes such as biomass synthesis, maintenance of cellular processes and resistance to therapies that facilitates their survival.14The focus on the GBM development of novel therapies targeting the TME has also raised the need for reliable models that recapitulate the TME in patients. In addition to conventional rodent intracranial xenograft models, newer genetically engineered and humanized rodent models have been developed to address specific questions related to the TME; additionally, more recent development of advanced 3-dimensional tumor models have helped further bridge the gap between basic discoveries and their translation through therapeutic modeling of GBM.In this review, we discuss the components of GBM TME and their interactions with the tumor cells and among one another . We alsoEmerging knowledge of the tumor-related immune system has led to novel therapeutic approaches and immunotherapies including immune checkpoint inhibition (ICI) that has revolutionized the management of a growing list of cancer types. Newer methods of immune therapy including chimeric antigen receptor (CAR) T cells, vaccine-based approaches and oncolytic virus-based therapies are under rigorous investigation. While the development of such approaches has seen a significant impact on survival of patients in other cancers, the same has not been seen in GBM patients due to multiple limitations that are unique to brain cancer including its characteristic heterogeneity, low immunogenicity, a profound immune suppressive microenvironment, and the blood–brain barrier (BBB) that restricts immune infiltration. In this section, we review recent findings regarding the mechanisms underpinning anti-glioma immunology with a focus on the major immune components of the glioma microenvironment and an emphasis on data from human studies.16 This significant progress has provoked speculation of a role for the meningeal lymphatic system in glioma immune response and has been demonstrated subsequently in mouse models of brain tumors.18 However, the meningeal lymphatic compartment is not directly connected to the brain parenchyma, and the relevance of its role in glioma immunity in humans awaits further study. Indeed, extracranial metastases of primary gliomas are rare and lymphatic spread is extremely rare.19 In addition to the meningeal lymphatic system, tertiary lymphatic structures (TLSs) have been found to exist in brain tumors.20 TLSs are alternative sites for T-cell priming and the only known routes of lymphocyte infiltration, which may permit glioma immune responses and warrant further studies. The CNS relies heavily on innate mechanisms that are mediated by neurons, astrocytes, and resident immune cells, mainly microglia, for immune defense against pathogens. This distinct version of immune defense has a clear impact on the glioma microenvironment where immune cells, which can constitute up to 50% of the tumor cellularity, consist of microglia, glioma-associated macrophages (GAMs), and, less abundantly, monocytes, neutrophils, and TILs.21–23 These innate and adaptive immune cells and inflammatory infiltrates co-exist with cancer cells, neurons, multiple lineages of glial cells, and vascular endothelial cells, which shape the glioma immune microenvironment through direct and indirect interactions. Recent studies, as facilitated by single-cell approaches, among others, have begun to paint a complex picture of an interplay of tumor, immune, and host cells, which is conducive to a profound immunosuppressive microenvironment. This scenario can be further exacerbated by metabolic stress, hypoxia, and damage-associated molecular patterns that are common in high-grade gliomas and GBMs. While the immune suppressive microenvironment plays an important role in thwarting the launch of anti-glioma immunity and therapeutic immune responses, most gliomas are intrinsically poorly immunogenic across the cancer continuum, adding an additional hurdle for immunotherapy. As such, therapeutic interventions may benefit from a dual strategy exploiting glioma immunogenicity and circumventing the immunosuppressive glioma microenvironment.The central nervous system (CNS) is historically considered as an immune-privileged organ guarded by the BBB where adaptive immunity and inflammatory response are tightly controlled. However, this concept has been recently challenged with the discovery of the existence of a glymphatic system  and lymphatic CNS drainage to cervical lymph nodes, forming a foundation for adaptive immunity associated with CNS inflammation and certain neurodegenerative diseases.24 It remains unclear to what extent the same route of infiltration operates in brain tumors.Microglia and GAMs are the most abundant immune cells in the microenvironment of most primary gliomas, pertaining to the CNS milieu. As such, these myeloid cell lineages have been the focus of extensive investigations, which have demonstrated a remarkable origin, phenotype, and functional diversity and plasticity. Microglia and some of the macrophage populations are derived from embryonic progenitors in yolk sac, whereas bone marrow-derived macrophages are believed to originate from peripheral monocytes that infiltrate the TME as in other pathological conditions. Additionally, new findings in mice showed a meningeal pool of monocytes and neutrophils that were supplied directly from the adjacent bone marrow of the skull, which could reach the brain parenchyma under pathological conditions of CNS injuries and inflammation.23 which constitute highly dynamic cell populations. As demonstrated extensively in cancer as well as noncancer contexts, different cytokine and chemokine conditions can induce macrophages to undergo either M1 or M2 polarization with M1 macrophages exhibiting immune-supportive and cancer-inhibitory functions, as opposite to M2 macrophages. Although this conventional phenotyping may not adequately cover the functional complexity of GAMs,25 these myeloid lineages are generally immunosuppressive in the glioma microenvironment. Although the underlying mechanisms are not fully understood, GAMs can produce anti-inflammatory cytokines  and tumor-promoting factors , promote angiogenesis (VEGF and IL8), disrupt metabolism (ARG1- and IDO-mediated amino acid depletion), and activate immune checkpoints by expressing PD-L1 and CD39.26–28 Given the overwhelming abundance of GAMs, which are professional cells that perform phagocytosis , it is tempting to develop therapeutic strategies based on harnessing some of these cell populations. One such approach targets the colony-stimulating factor 1 receptor (CSF1R) because glioma cells produce the ligand cytokines CSF1 and IL34 abundantly, which act on GAMs through CSF1R. Several CSF1R inhibitors have been developed that are effective in reprogramming GAMs toward an antitumor phenotype, reducing M2 polarization, inhibiting tumor growth, and increasing survival in a murine GBM model in multiple preclinical settings.30 However, a phase II study of PLX3397, a CSF1R inhibitor, conducted to treat patients with recurrent GBM failed to show therapeutic efficacy despite the excellent tolerance and blood–tumor barrier penetration capacity and on-target drug action.31 Additional preclinical studies and clinical trials have also been conducted to test other CSF1R inhibitors, such as BLZ945, pexidartinib, and blocking antibodies, either as single agents or in combination with radiotherapy or ICI.32–35 In brain cancer models, however, as demonstrated in mice, following initial therapeutic benefit, CSF1R inhibitor-treated tumors recur frequently driven by elevated macrophage-derived insulin-like growth factor 1 (IGF-1) and high IGF-1 receptor (IGF-1R) levels on tumor cells and other mechanisms.37 Further, unlike in other tumors, inhibition of CSF1R had minimal impact on CD8+ T-cell infiltration in a syngeneic mouse GBM model, highlighting the immunosuppressive microenvironment of brain tumors, and that CSF1R inhibition alone appears to be insufficient to elicit strong anti-glioma immunity.38–40The glioma microenvironment can have a significant impact on microglia and GAMs,42 although they are distinguished from GAMs and microglia based on the expression of different cell-surface markers. However, single-cell mRNA profiling23 failed to distinguish MDSCs from other myeloid cell lineages. Additional investigation of brain tumor-associated MDSCs at single-cell resolution with increased depth may reveal heterogeneous cell states and provide improved clarity. On the other hand, these single-cell studies showed the presence of abundant neutrophils in the glioma TME. Although neutrophils also appear to play a generally immunosuppressive role in GBM tumors, the role of this cell population is not well studied. Given their phagocytic and secretory activities and their unique ability to interact with tumor cells, neutrophils are likely to have a more important role in the glioma TME than has been demonstrated to date.As found in many other cancer types, myeloid-derived suppressive cells (MDSCs), which are derivatives of monocytic or granulocytic cells, are also detected in the glioma TME. These cells suppress tumor-specific effector T cells in a manner similar to that of GAMs,+CD3− cells) are potent effector lymphoid cells that mediate antigen-independent immune surveillance against pathogens and stressed cells through “missing-self” recognition, which can also perform innate antitumor immunity. In GBM, however, NK cells were found to be one of the least numerous immune cell populations of all tumor-infiltrating immune cells and were predominantly the CD56dimCD16− subtype.43 Newer single-cell studies have confirmed the scarcity of NK cells in primary gliomas with identification of CD16− immature NK cells in IDHwt tumors, whereas CD16+ cytotoxic NK cells are present in IDH1mut tumor and brain metastases.22 Further, NK cells are inhibited by classic (antigen-presenting) and nonclassic HLA-I molecules through binding to the inhibitory killer immunoglobulin-like receptors. In gliomas, although HLA-I genes are subject to loss of heterozygosity, complete deletion of all of these genes is uncommon. In addition, the glioma TME plays an important role in suppressing NK cell function, since TGFβ, which is secreted by cancer and noncancer cells, downregulates the expression of NKG2D-activating receptor on NK cells isolated from GBM patients.44 Thus, the role of NK cells in anti-glioma surveillance remains to be demonstrated. Nonetheless, adoptive NK cells have been demonstrated to exhibit cytotoxic activities against glioma cells, including glioma stem cells, in preclinical settings, and targeting some of the inhibitory mechanisms was recently shown to increase NK cell function.45–47Natural killer  and hence a lower availability of immunogenic neoantigens and antigen-specific T-cell pools.57 In addition to TMB and the resultant nonsynonymous single (or double) nucleotide variants that give rise to potential neoantigens, several other mechanisms can produce immunogenic peptides and cancer-associated antigens, which have been the subject of emerging new studies, including genetic and epigenetic alterations, transcriptional and pre-mRNA splicing events, noncanonical translation, and post-translational modifications.58–63 Pan-cancer studies thus far have demonstrated that gliomas are less affected by these various tumor antigenic mechanisms as compared with most other cancers, consistent with an immunologically “cold” phenotype. Tumor-specific antigens undergo MHC-I or -II restriction for T-cell priming and recognition. Most neoantigen discovery and vaccine development studies focus on HLA-I restricted neoepitopes, which can be recognized by CD8+ effector and memory T cells enabling T-cell-mediated cytotoxicity. However, naive T cells must first be primed by dendritic cells (DCs), which access and process tumor antigens and then mediate HLA-I antigen presentation and co-stimulation normally observed in lymphoid organs. Somatic HLA-I loss of heterozygosity, a pan-cancer genetic aberration underlying the immune escape of cancer cells, affects ~10% of gliomas, which is less frequent than most other cancers.64 However, the brain parenchyma is largely devoid of appropriate lymphatic vessels limiting direct access to potential tumor antigens by DCs. Non-DC antigen-presenting cells, such as macrophages, process tumor antigens through the endocytic pathway and HLA-II complex and the effectiveness of this mechanism in eliciting anti-glioma immune response remains unclear despite the abundance of these cells in the glioma TME. Further, most GAMs are tumor promoting, and their antigen-presenting function is inhibited due to STAT3 activation.65 Instead, it is conceivable that the phagocytic phenotype of GAM due to M2 polarization may play a role in scavenging damaged tumor cells further reducing tumor antigen accessibility to professional antigen-presenting DCs. Indeed, although necrosis is a common phenotype of high-grade GBMs associated with reduced patient survival, these tumors remain poorly inflamed.Unlike many other cancer lineages, gliomas are poorly responsive to ICI, an immunotherapy modality that marks one of the most significant advances in cancer management. ICI counteracts the exhaustion phenotype of cytotoxic T cells expressing the immune checkpoint receptor PD1 and, therefore, it requires preexisting tumor antigen-specific T cells. While gliomas are in general resistant to ICI despite the remarkable genetic and epigenetic heterogeneity of the tumor lineages, brain metastases are more sensitive to immune checkpoint blockade with complete responses being achieved in some patients.67 Immunogenicity defects can also be bypassed by CAR T cells, which are engineered to express artificial tumor antigen ligands eliminating the priming step and the dependence on DC-mediated antigen presentation. The CAR T-cell approach has been demonstrated to be highly effective in B-cell malignancies and can be used in drug-resistant diseases, where these cells can remain therapeutically active in patients for an extended period of time.68 CAR T cells specific for IL13RA2, CD276, CD133, αvβ3 integrin, EGFR, and the oncogenic EGFRvIII variant that target GBM cells have been developed and tested in mouse models69–71 However, CAR T cells have demonstrated limited therapeutic efficacy in patients in clinical trials except for a recent report of prolonged survival achieved with EGFRvIII CAR in a patient with recurrent GBM.72 Effectiveness of CAR T-cell therapies in solid cancers, including gliomas, is confounded by the limitations of adequate distribution of these cells in the TME, low antigen coverage due to inter- and intratumoral heterogeneity, antigen escape, suppression by the TME, and failure to attain long-term persistence in recipients because of exhaustion, senescence, and cell death. The mechanisms of CAR T-cell dysfunction may also operate in other antigen-specific effector T cells, and some of the mechanisms are only beginning to be elucidated. For example, compelling evidence shows that CAR T cells can extract tumor antigen from target cells through a mechanism known as trogocytosis, which underlies multiple aspects of CAR T-cell function, including tumor antigen escape, fratricidal CAR T-cell death, and the triple exhaustion phenotype.73 Further, epigenetic reprogramming underpins the exhaustion phenotype in CAR as well as non-CAR T cells.74–79 In addition, cancer cell-intrinsic IFNγR pathway has been shown to be essential for CAR T-cell-mediated killing of GBM and other solid tumor cells but not for hematologic malignancies,68 suggesting that impaired IFNγR signaling may represent a cancer cell-intrinsic mechanism of resistance to CAR T cells. Therapeutic approaches targeting these mechanisms are currently being integrated into immunotherapy modalities based on ICI and CAR T cells.Given that insufficient immunogenicity and the immunosuppressive TME are the major hurdles limiting anti-glioma immunity, several immunotherapeutic strategies have been devised to increase glioma immunogenicity, including DC vaccines, STING agonists, oncolytic virus, and epigenetic modulators. The therapeutic effects of these approaches rely on elicitation of tumor antigen-specific immune responses and the cytotoxicity of effector T cells, providing a clear rationale for combination with ICI.80 The unique characteristics of both glioma cells and the native cells of the CNS facilitate even more complex means of interaction  and mature forms, both of which play crucial roles in both early development of the nervous system and modulation of synaptic plasticity in adulthood. BDNF and TrkB expression are increased in cancers of various types, including gliomas.85 Intrinsic expression of BDNF and TrkB has been noted in C6 glioma cells.86 and exogenous exposure of these cells to recombinant murine mature BDNF increased proliferation, where antibody-mediated blocking of the BDNF abrogated these effects in a dose-dependent fashion. Additionally, glioma cells had increased motility and invasion in the presence of BDNF, as well as decreased rates of apoptosis. The balance in proportions of proBDNF and mature BDNF, which appear to exert opposing functions in cell growth, appears to correlate with tumor grade in glioma.87 These studies suggest that targeting mature BDNF signaling may represent a potential therapeutic strategy against gliomas. Of note, given the delicate homeostatic balance between BDNF and proBDNF expression, as well as the important roles these proteins play in the function of the nervous system and development of neurologic disease , agents to block BDNF signaling, such as NTRK inhibitors, could potentially carry the risk of neurological toxicity and additional studies are needed to balance the risks and benefits of targeting the BDNF pathway.The study of paracrine interactions between the nervous system and glioma cells has centered on the roles of brain-derived neurotrophic factor (BDNF) and neuroligin 3 (NLGN3). BDNF , is a molecule that has been implicated in neuronal growth, differentiation, and apoptosis via signaling through the transmembrane tyrosine kinase B (TrkB) receptor (NTRK2).93 Based on the hypothesis that neuronal activity has a mitogenic effect on glioma cells, 1 study revealed that soluble NLGN3 activated the PI3K–mTOR pathway in human glioma cell lines.94 Beyond promoting cell proliferation and growth through the previously characterized mechanisms of the PI3K–mTOR pathway, NGLN3-mediated PI3K–mTOR activation also resulted in the unexpected feed-forward secretion of additional NLGN3. Optogenetic studies utilizing light-mediated modulation of neuronal activity in vivo specifically within deep cortical neurons expressing channelrhodopsin-2 in the premotor cortex in a mouse orthotopic xenograft model of pediatric GBM have demonstrated a correlation between neuronal activity and glioma proliferation which was mediated by NGLN3. Conversely, NLGN3 depletion had an adverse effect on proliferation, although this effect was only partial suggesting a role for other mitogens such as BDNF.NLGN3 is a member of the neuroligin family of cell adhesion molecules which regulate synaptic structure, plasticity, and function; it is expressed in both excitatory and inhibitory neurons as a postsynaptic transmembrane protein and NLGN3 alterations can alter synaptic function.95 In addition, Nlgn3 knockout mice are unable to harbor patient-derived orthotopic xenografts of several molecular subtypes of high-grade glioma and show durable inhibition of glioma growth despite minimal impact on neurologic function . The important role of the PI3K–mTOR pathway was confirmed, with the additional discovery of focal adhesion kinase-mediated downstream effects.Subsequent studies identified that NLGN3 is cleaved by ADAM10 and secreted into the TME 0and ADAM10 inhibitors were found to inhibit the release of NLGN3 and consequently prevent growth of patient-derived orthotopic xenograft.96 Light deprivation, achieved by rearing mice in the dark during weeks when tumors are normally expected to develop, decreased the formation and growth of optic pathway gliomas. Optogenetic promotion of optic pathway glioma growth was mediated in these low-grade gliomas by BDNF and NLGN3 secretion, which in turn was promoted by retinal activity. A direct link between neuronal activity and gliomagenesis was reported through stimulation of a unilateral optic nerve in mouse models of optic pathway glioma, also expressing channelrhodopsin-2 in retinal ganglion cells. The optic nerves in blue-light stimulated mice were larger and exhibited a higher proportion of glial or glioma cells. It should be highlighted that the effects of NLGN3 appeared to be similar across different histologic and molecular subtypes of glioma, at least in these models. The NF1 mutation, synergistically with optogenetic stimulation of the retina, has been shown to contribute to NLGN3 secretion and optic glioma growth and further drives neuronal hyperexcitability and tumor progression through altered hyperpolarization-activated cyclic nucleotide-gated channels.97 In a similar context, sensory input of other types impacts glioma formation as well, particularly in the olfactory bulb wherein olfactory receptor neurons were shown to excite mitral and tufted cells into releasing IGF-1, thereby promoting gliomagenesis.98Additional evidence of BDNF and NLGN3 involvement in glioma is reflected in the discovery that photic stimulation can modulate the growth of glioma cells, specifically in mouse models of neurofibromatosis type 1 with optic pathway glioma.99 The gap junctions between glioma cells themselves also were noted to facilitate broad propagation of these impulses throughout the tumor, demonstrated through experiments including the administration of gap junction blocking agents which reduced the amplitude of glioma currents.Beyond the effects of soluble synaptic proteins, the synapse itself has a unique structure that may affect glioma cells via direct electrochemical signaling from neurons. Some glioma cells robustly express synaptic genes and form functional connections with neurons. Signaling from neurons was shown in work by Venkatesh et al. to induce AMPA (glutamate) receptor-mediated depolarization and excitatory postsynaptic potentials in glioma cells, which could be abolished with the sodium channel blocker tetrodotoxin.100 These studies build on the work by Osswald et al. and who characterized “tumor microtubes” or protrusions emanating from the membranes of human astrocytoma cells, and first demonstrated the ability of these structures to propagate intracellular calcium waves via gap junctions.101 More recently, Venkataramani et al. found that these currents represented a functional connection both among glioma cells and between them and local astrocytes, and further demonstrated that neuronal activity increased tumor microtubule turnover and accelerated invasive behavior.102 Knockdown studies of the gene encoding connexin 43, a gap junction protein, in both in vitro and in vivo models further emphasize the importance of such junctions on intratumoral cell communication and glioma progression.103Connectivity via gap junctions was also reflected in studies revealing that spontaneous slow inward currents in neuron–glioma co-cultures increased input resistance and similarly could be blocked by gap junction blocking agents.105 The mechanism through which they may do so is thought to largely depend on increased expression of a transporter that exchanges extracellular cysteine for glutamate.106 The resulting increase in neuronal activity may contribute to seizure risk and invasiveness.107 Despite this, clinical trials of agents targeting glutamatergic neurotransmission have not shown significant benefit to date.108Glutamate-mediated signaling may contribute to feedback loops resulting in glioma–neuron network hyperexcitability, as glioma cells also release glutamate into the surrounding tissue.110 There is conflicting evidence regarding the potential impact on gliomas of antidepressants that modulate serotonergic tone; glioma incidence is apparently decreased in patients who have taken tricyclic antidepressants on a long-term basis,112 but not in those taking selective serotonin reuptake inhibitors, and there is no association between selective serotonin reuptake inhibitor use and survival in GBM.113 Tricyclic antidepressants may act via other broad neurotransmitter effects or separate mechanisms. Blocking dopamine receptor D4 has been shown to inhibit glioma cell proliferation,114 and ONC201, a small molecule used to treat diffuse midline glioma, works to block the dopamine receptors DRD2 and DRD3, in addition to inhibiting the phosphorylation of Akt and ERK. There may also be an emerging role of GABAergic neurotransmission in the electrical microenvironment. Glioma cells have been noted to upregulate expression of GABA receptors when in proximity to neurons,115 and GABAergic currents have been described in glioma cell lines.116 One study linked increase in endogenous GABA levels impede tumor growth, and further work in this field is ongoing.117The way in which synaptic transmission affects glioma cells is an area of active investigation; beyond glutamatergic transmission, glioma cells also express functional serotonin, GABA, and dopamine receptors and are therefore potentially vulnerable to the effects of these neurotransmitters.119 The release of neurotransmitters and neuropeptides may also influence neo-angiogenesis. Neuronal activity is also known to affect immune function,120 although how such interactions might shape the glioma immune microenvironment specifically is less certain.121Neurons may be able to interact with cancer via indirect effects on the TME. It has been demonstrated that adrenergic nerves in the periphery are capable of modulating endothelial cell metabolism, with a demonstrable impact on angiogenesis in models of prostate cancer.122 These microglia are described further as components of the immune microenvironment in the immunology section of this review. On histologic examination, gliomas are also commonly seen in association with reactive, large astrocytes.123 These astrocytes modulate glioma invasiveness, likely through the release of neurotrophic factors and increased production of matrix metalloproteases.124 Astrocytes may additionally link with glioma cells via gap junctions which, as in physiologic astrocyte–astrocyte gap junctions, mediate the exchange and distribution of toxins and small molecules across a wide network to promote cell health and homeostasis.125Glial cells are also able to interact with tumor cells. Ameboid-appearing microglia, which were first described in association with glioma cells by Wilder Penfield in 1925, appear to promote glioma cell migration and proliferation.127 Tumor cells adopt several aberrant metabolic processes to meet the energy demands associated with rapid proliferation and survival. For instance, glioma cells increase the uptake of glucose and other nutrients to support the aggressive biomass production in GBM. However, nutrient supply to the tumor can significantly vary due to the heterogeneity of the neoangiogenesis and the vascular network in GBM; such intratumoral variances in vascular supply can also alter the availability of oxygen within tumor, which in turn can influence the metabolic properties and energy utilization of cancer cells.129 Therefore, tumor cells within the same tumor can have differential metabolic signatures  which allows the cells to accumulate high levels of metabolic intermediates, resulting in lower extracellular TME glucose concentration, higher lactic acid and H+ production, and secretion, and changes in tumor energy utilization. Given that the lowering of intracellular pH may result in programmed cell death, cancer cells enhance their ability to secrete these metabolites into extracellular space through secretory mechanisms such as upregulated MCT-4 (monocarboxylated transporter) and Na+-H+ transporters131–135 and lowering the pH of the TME.136 The acidic environment in the TME can facilitate several other pro-tumorigenic processes including proliferation, migration, and angiogenesis. Also, given that such an acidic environment is a potential threat to cancer cells, to counter this, malignant cells activate protective mechanisms such as increasing the expression of proteins such as LAMP2 which can protect lysosomal and plasmalemmal membranes from acid proteolysis and increase expression of autophagy-related proteins such as ATG5, and anti-apoptotic proteins such as BCL-2 upon chronic exposure to low pH conditions; such emergent of defense mechanisms related to tumor-associated acidosis can further enhance the ability of the cells to survive harsh environmental conditions.127A major characteristic of developing malignancies is the emergence of tumor acidosis which progressively affects both intrinsic cellular processes as well as the TME which can help establish an immunosuppressive TME and confer treatment resistance.138 Beyond its impact on immunity, acidosis in general is known to provide a protective microenvironment for dormant tumor cells that supports the survival and invasion of circulating tumor cells and promotes resistance to chemotherapy and radiotherapy. In addition, the acidic environment has been found to promote anoikis resistance via mTOR/NF-κB signaling.139 Whether similar mechanisms also apply to glioma cells in the brain microenvironment to support cell survival, treatment resistance, and infiltrative phenotype remains under study.Acidosis can also strongly contribute to the GBM TME becoming highly immunosuppressive; such immunosuppression is in part achieved by the acidosis and hypoxia induced upregulation of the synthesis of hypoxanthine and transmembrane CD44 receptors given that adhesion between tumor cells can be reduced as a result of hypoxanthine and CD44 binding; this has profound effects on multiple components of immune cell function which could potentially be targetable for therapeutic strategies.140 This plasticity has been demonstrated in a subset of the GBM cell population which were found to have both high glycolytic and oxidative phosphorylation phenotypes, a phenomenon that was leveraged to use metabolic inhibitors leading to a change in their bioenergetics state. Thus, targeting of acidosis with the use of inhibitors that target pH balance in the TME along with standard-of-care therapy is emerging as another therapeutic strategy against GBMs,141 a concept that is being tested in ongoing clinical trials (NCT03011671).Cancer cells use the Warburg effect (aerobic glycolysis) to meet their high energy demands. However, the results of recent studies suggest that cancer cells under aerobic glycolysis can switch to oxidative phosphorylation by downregulating hypoxia-inducible factors (HIF)-1α, resulting in a more aggressive phenotype. The metabolic plasticity of tumor cells, which allows them to adjust according to the changing microenvironment, gives them a selective advantage.143 Tumor cells respond to hypoxic conditions by upregulating HIFs which in turn activate gene expression of downstream targets needed for cell survival.144 A change in oxygen tension (pO2) is sensed by prolyl-4-hydroxylase 2 (PHD2), which regulates the expression of HIFs.145 During normoxic conditions, PHD2 is activated in the presence of oxygen and 2-oxoglutarate and downregulates HIF-1α by hydroxylation at its Pro402 and Pro564 residues at C terminus.146–148 The hydroxylated HIF-1α binds to pVHL and is degraded by proteasome polyubiquitination.147 During hypoxic conditions, low oxygen tension leads to inhibition of pHD2, causing HIF-1α accumulation. The dimerization of HIF-1α and HIF-1β leads to the upregulation of proangiogenic genes, such as VEGF, fibroblast growth factor, and several others, which leads to an increase in angiogenesis, invasiveness, and tumor migration.149 Depending on the cellular context, PHD2 exerts both pro- and antitumor properties. In breast cancer, PHD2 has been reported to promote tumor cell migration via cancer-associated fibroblast activation. On the other hand, downregulation of PHD2 showed an antitumor effect in GBM,150 head and neck squamous cell carcinoma,151 and bone marrow-derived cells.152 Considering the significance of PHD2 in tumor progression and HIF regulation, PHD2 could be a potential therapeutic target in the treatment of GBM and other cancer types. There are 2 ongoing clinical trials targeting hypoxia using MBM-02 (Tempol) and belzutifan to inhibit HIFs in GBM patients (NCT04874506 and NCT02974738).Another chemical environmental factor that helps cancer cells acquire resistance is hypoxia which is a fundamental driver of oncogenesis which develops when oxygen consumption demands from cell proliferation outstrips the ability of neoangiogenesis or diffusion to restore oxygenation. The hypoxic environment has been well established as a key trigger for angiogenesis, invasion, survival, and resistance to therapies in GBMs; additionally, tumor hypoxia also directly correlate with disease progression and poor prognosis in GBM patients.101 The ideal model should be similar to human glioma genetically and should adequately represent intratumoral heterogeneity along with relevant host factors.To enhance knowledge of the GBM TME and the development of specific treatment strategies related to TME, a variety of in vitro and in vivo preclinical models have been developed. In this section, we discuss the features and limitation of TME models . An idea157 Simpler 3D models using cellular aggregates or neurospheres provide an improved representation of glioma and the TME that allow studies of simple cell–cell interactions and spatial relationships and in the case of patient-derived glioma stem-like cell spheres, also represent human tumors more faithfully genetically than attached cells. Tumor cell secretions, responses to changes in nutrients, pH and oxygen, and tumor–normal cell interactions can be preliminarily studied using such models but they still do not represent the native TME and do not have aBBB.162 More recent developments have to led to matrix-assisted 3D tumor models which can be used to study drug delivery systems and soluble factor signaling. They can also be used to prescreen drugs before in vivo experiments, reducing the time and effort required and minimizes use of animals (162Based on cellular composition, these models are categorized as cell-based, tissue engineering-based and animal models. In vitro 2D models are the most used ones because of the ease of use and maintenance. However, they lack the 3D representation of the various TME cellular components and do not have a BBB. animals .158,162172 Recent findings have shown that neurons and glial cells are preserved in organotypic hippocampal slice cultures.173 Similar models have also been developed from glioma tissue in which organotypic slice cultures generated from freshly resected human or animal tumors have been successfully used to study tumor biology and therapeutic responses. Such slice cultures contain both tumor and nontumor microenvironmental cells in their native spatial distribution which provides a window to examine the intercellular relationships and the opportunity to modulate the same therapeutically in an ex vivo setting. Because of the true representation of tissue heterogeneity and microenvironmental components in these ex vivo cultures, they have become an integral part of preclinical drug testing.174–176 Although organotypic slice cultures have several advantages over conventional 2D and 3D models, they also have limitations including the effects of the specific culture conditions used and the lack of a functional BBB; however, this model provides the most direct data related to biology and treatment response of human tumor and TME outside of clinical studies. Another relevant model uses tissue engineering methods including microfluidic systems and tissue matrices in an effort to reconstruct the complexity of human gliomas through the so-called tumor-on-a-chip model.; these are also increasingly used to study the impact of TME components on tumor development and progression.177A more relevant 3D model uses organotypic slice cultures which are derived directly from the brain tissue and hence closely mimics the in vivo system making them suitable for various brain and spine studies. Delbridge et al. found that microglia isolated from organotypic slice cultures were similar to acutely isolated adult microglia; they used this model successfully to study neuroinflammation.168–171Development of preclinical models, while useful for studying specific questions related to the GBM TME, do not fully represent the highly complex nature of TME and cell-to-cell interactions. Therefore, in vivo tumor models may be more relevant to study the TME and related cellular interactions. Most studies involve using patient-derived xenograft cells implanted in immune-compromised mice, limiting the representation of the immune component of TME. This shortcoming can partly be resolved using a syngeneic brain tumor model. However, this may lead to differences between species and not completely represent a native TME. The development of humanized mouse models, which have components of the human immune system interacting with tumor xenografts, allows a better representation of the immune TME; in addition, these models can be genetically modified to address specific biological questions. Continued optimization of such preclinical models by overcoming their various limitations is ongoing with the goal of generating a more relevant model with true fidelity to the human glioma.Recent advances in science have enhanced the understanding of the TME in GBM; however, translation of these findings to therapeutic strategies against the disease has not yet been optimally achieved and the disease prognosis remains poor. The bidirectional interaction of tumor cells with the neighboring microenvironment results in a complex multicellular system that enables tumor cell proliferation and resistance to treatments including chemotherapy, radiotherapy, and other related treatments. Recent in-depth studies have helped better understand the composition of the TME and the interaction of the TME with glioma cells. Additionally, development of robust tumor models derived from patient specimens with immune, stromal, and cancer cells have further improved preclinical target assessment and help to identify key elements for therapeutic targeting and develop novel approaches to anti-glioma therapy targeting the points of interaction between gliomas and their TME.


Dedicated to the 130th anniversary of Dmitry Ivanovsky’s discoveryof the virus kingdom as a new form of biological life.The genome of some RNA-containing viruses comprises ambipolar genes that arearranged in stacks (one above the other) encoding proteins in oppositedirections. Ambipolar genes provide a new approach for developing viraldiversity when virions possessing an identical genome may differ in itsexpression scheme (strategy) and have distinct types of progeny virions varyingin the genomic RNA polarity and the composition of proteins expressed bypositive- or negative-sense genes, the so-called ambipolar virions. So far,this pathway of viral genome expression remains hypothetical and hidden fromus, like the dark side of the Moon, and deserves a detailed study. Ivanovskyreported having discovered a new form of biological life, the so-called“contagium vivum fixum” [m fixum” , which wm fixum” . Accordim fixum” .According to the well-known classification by D. Baltimore , which iDifferences in the viral genome structure and variations in the patterns of itsexpression in an infected cell underlie virus diversity, pantropic adaptation of viruses to various organismssuch as bacteria, fungi, plants, fish, and animals, in particular humans, andensure the global spread of viruses on Earth, and possibly in space and otherplanets .The genetic diversity of viruses, which underlies the Baltimore classification,was considered as follows: one unique viral genome develops one genomestrategy; i.e., one genome has one replication scheme and directs the formationof one structural and functional class of virions . This implies a uniform and unified process for the synthesis ofviral particles (virions) within one viral genus (or family) , 8.HowePB1,PB2, PA, HA,NP, NA, M,NS) with negative coding polarity in the genomic RNA in the3’ → 5’ direction, had additional positive coding polarity(in the 5’ → 3’ direction of the genomic molecule).The peculiarity of these ambipolar genes was theirlocalization in genome regions overlapping the corresponding classicalnegative-sense genes; the so-called stacking arrangement [Fig. 1B).The ambipolar genes identified in the genomes oforthomyxo- and coronaviruses were found to be characterized by the presence ofall the functional elements necessary for expression of these geneticframeworks as translational genes [In 2007, we analyzed the negative-sense genome of influenza A viruses(orthomyxovirus family) and found extended open reading frames (ORFs) that,unlike the canonical influenza virus genes , and thal genes  possessial genes , 14, 18.Today, the genome of one virus species (genus) is believed to have one strategythat determines the formation of viral particles of a certain structure and a characteristic range of hosts. The discovery of ambipolarstacking genes in the genomes of RNA viruses suggests the existence ofalternative strategies in the genome of one virus species (genus) whoseexpression pathways may (1) provide the synthesis of several structural andfunctional classes of virions that differ in both their protein composition andthe structural form (polarity) of genomic RNA and/or (2) develop severaldifferent strategies for virus replication and its pathogenesis in an infectedmacroorganism. The presence of several strategies in one viral genome providesa reserve of viral adaptive properties, which may be considered as a pathway(or modification) of genetic bet-hedging .Fig. 2 forthe influenza virus and coronavirus models. Theinfluenza virus comprising genomic (–)-RNA is characterized by thepossibility of both a classical pathway of genome implementation and alternative strategies. Implementation of alternative genomestrategies may lead to the formation of ambipolar virions that may contain bothclassical proteins  and additionalproteins–products of the ambipolar genesNSP1–NSP8 (NSP –Negative Strand Protein) of appropriate genomic RNA segments.Expression of the classic coronavirus strategy also leads to theformation of virions containing the canonical (+)-RNA genome and classicalstructural proteins: N (nucleocapsid protein), S (surface glycoprotein), E(membrane protein), and M  and a number of auxiliarynon-structural regulatory proteins (nsp1-nsp16) that support viral replicationin target cells and suppression of the host’s immune response. However,the products of the main ambipolar genesNGP1–NGP5 , escape the attention of researchers. So far, these proteinsencoded by open ambipolar genes have not been found in infected cells. Apossible reason lies in either the minor level of their synthesis or theirstrictly selective expression only in specialized body cells containing theunique factors necessary for the expression of these viral stacking genes undercertain conditions of the intracellular and/or surrounding extracellularenvironment. At the same time, there are indirect approaches to observeambipolar gene expression in an infected macroorganism. Animals infected withthe influenza A virus were found to develop clones of cytotoxic lymphocytesthat recognize specific peptide domains of the influenza virus ambipolarproteins, in particular the NSP8 protein encoded by the ambipolar NSP8gene of the influenza A virus NS segment [The multiple strategies of the genome in one virus species (genus) and theexpression schemes of its classical and alternative strategies are shown inproteins), which  segment , 25,26.Fig. 2).Thus, ambipolar viral particles may containboth ambipolar proteins and ambipolar genomic RNA replicas, providing analternative pathway for the viral genome strategy. As a result, one uniqueviral genome may be implemented in several, alternative strategies – withor without involvement of ambipolar genes – and viruses may possessseveral possible life pathways, depending on the context of the surroundingcellular processes. This idea is illustratedin Fig. 2. Thismultivariant mechanism of a unique viral genome strategy may be considered as away of bet-hedging by viruses, which promotes the establishment of alternativeways of virus replication and the creation of reserve adaptive potentials forviruses of various families. In this aspect, RNA viruses may be similar to DNAviruses and RNA-containing retroviral (virus-like) transposons that have adual-track lifestyle: as a DNA provirus and a mature virus, respectively, whichdetermines the vertical  and horizontal (mature virions) ways of their existence in the host,depending on the propagation environment and the range of hosts [We may posit that these non-canonical proteins are able to decorate the viralgenome from a new class of viral particles performing unique regulatoryfunctions, and altering the virus behavior in an infected organism; e.g.,switching from productive virus infection to a latent persistent (lowreproductive) viral infection process. Furthermore, there may be an alternativewhen a genome molecule becomes an RNA chain complementary (ambipolar replica)to the canonical virus genome: the coronavirus (–)RNA or influenza virus(+)RNA . So far, these pathwaysof multiple expression strategy of the viral genome remain as hypothetical andenigmatic as the “dark side of the Moon.” Experimental verificationof this crystal-ball reading exercise will enable us to evaluate the possibleexistence of ambipolar classes of stealth virions hidden from the eye ofresearchers. To date, mature protein products encoded by identified ambipolarviral genes in an infected organism have not yet been detected. But this doesnot mean that expression of these viral stacking genes is not implemented innature. Identification of the expression of these genes requires a targetedsearch using original approaches and highly sensitive methods for identifyingproteins in various organs and the specific cells of an infected hostmacroorganism. It is possible that the unraveling of alternative strategies ofviral genomes may be important for understanding virus evolution and thepathogenesis of viral infections, as was the case in covid-2019 when long-termand severe complications of the viral infection could develop due to theformation of ambipolar virions hidden from the attention of researchers andmedical practitioners.The ambipolar genes of viruses are endowed with high evolution stability. Inparticular, in the natural population of highly variable influenza viruses,these genes have been observed in the genome with all the necessary regulatoryelements for more than 100 years, despite a noticeable population variabilityin both canonical and identified ambipolar genes with a characteristic highdN/dS coefficient that indicates pronounced immunological pressure from thehost macroorganism in nature . TheevoFig. 3 usingan arenavirus model . In this regard, the difference in stacking allows us toconsider two major groups of ambipolar viruses. To date, the following divisionseems logical: in the first group of viruses with gene stacking in the viral genome, pathways of ambipolar genome strategiesmay have an alternative  character, while in viruses lacking genestacking , the implementation of theambisense genome strategy should be considered as an obligatory (mandatory)reality for virus replication. Further targeted search for the expressionpathways of alternative genome strategies in one viral species andidentification of a hypothetical class of ambipolar virions will answer thequestion of the existence of this type of viral life diversity and its role inthe evolution of viruses of various genera. This knowledge will come handy inthe development of new vaccines and antiviral drugs and add to ourunderstanding of the molecular basis of viral disease pathogenesis.Obviously, the ambipolar stacking of genes found in RNA viruses provides thevirus with, first, an enhanced information capacity of the genome. Second, itunderlies the linked  evolution of viral genes when mutations inone gene generate changes in a stacking gene and, thus, represent a kind ofgenetic synteny. Third, the protein products of stacked genes may befunctionally linked and have a predetermined structural correspondence to eachother, which remains a hypothetical and requires experimental evidence, 17.The


Studies suggest that increasing body O2 stores supports breath-hold diving, but less is known about metabolic specializations that underlie underwater locomotion. We measured maximal activities of 10 key enzymes in locomotory muscles  to identify biochemical changes associated with diving in pathways of oxidative and substrate-level phosphorylation and compared them across three groups of ducks—the longest diving sea ducks (eight spp.), the mid-tier diving pochards (three spp.) and the non-diving dabblers (five spp.). Relative to dabblers, both diving groups had increased activities of succinate dehydrogenase and cytochrome c oxidase, and sea ducks further showed increases in citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HOAD). Both diving groups had relative decreases in capacity for anaerobic metabolism (lower ratio of lactate dehydrogenase to CS), with sea ducks also showing a greater capacity for oxidative phosphorylation and lipid oxidation . These data suggest that the locomotory muscles of diving ducks are specialized for sustaining high rates of aerobic metabolism, emphasizing the importance of body O2 stores for dive performance in these species.Diving animals must sustain high activity with limited O The best-fitting model (lowest AIC) to estimate dive time included all variables of interest: [Hb], [Mb] in the gastrocnemius, mass, the three enzymes in the gastrocnemius  or pectoralis (SDH) that were significantly different between the sea ducks and pochards versus dabblers, and the two almost significantly different enzymes  in the gastrocnemius . In this model, none of the variables was a significant predictor of dive time. Removal of SDH activity in the pectoralis to focus only on changes in the gastrocnemius only decreased the AIC by 0.3 (104.16 versus 104.46). When only gastrocnemius enzyme activity was considered, the increase in oxidative capacity seen in the sea ducks and pochards, as indicated by increased CS activity, was found to be the most significant predictor of dive time overall (p = 0.0379).Select enzyme activities in the pectoralis or gastrocnemius, O. 4Our results show significant remodelling of metabolic pathways in the locomotory muscle of the sea ducks, with a generally intermediate phenotype seen in the pochards. CS and COX are known to be biochemical indicators of mitochondrial abundance, and CS activity in the gastrocnemius was the best predictor of dive time –60. Beca (a)2 supply remains critical to maintaining aerobic metabolism while diving. Indeed, in the closely related tufted duck (Aythya fuligula), blood flow to the leg muscles was five times higher during a dive compared with resting levels, which probably helps meet the high demands for transporting circulatory O2 stores to the working muscle ) is generally higher in swimming versus non-swimming muscles [2 availability during the dive, further extending aerobic dive times. Since we did not see consistent changes in the activities of either CK or AK, increased capacities for cytoplasmic ATP buffering and substrate-level phosphorylation do not appear to contribute to increases in dive capacity.Myoglobin is important in muscles as both an intracellular O muscles ,62,63. [ muscles , which w2 to the electron transport chain, this increase in CS could function to maximize electrons funnelled into oxidative phosphorylation and creation of the protonmotive force for ATP synthesis. The lower ratio of ATP synthase to CS could also be indicative of an increase in the proportion of subsarcolemmal mitochondria, the subfraction of mitochondria located next to the cell membrane, which have a lower overall activity of ATP synthase compared with intermyofibrillar mitochondria [2 stores during diving.The increases in both CS and COX activity were not matched by an equivalent increase in ATP synthase activity, as evidenced by the decrease in the ATPSyn:CS ratio in the sea ducks and pochards. Interestingly, this trend was seen in both the gastrocnemius and pectoralis and could represent a specific diving phenotype in the muscle generally. However, ATP synthase activity remained relatively high across all three clades, and differences seen in the ATPSyn:CS ratio reflect the higher CS activity in the sea ducks and pochards. As a key enzyme involved in the first step of the TCA cycle, which provides energy-rich NADH and FADHchondria . Because (b)Sea ducks and pochards had significantly elevated HOAD activity in the gastrocnemius in comparison with dabblers, probably reflecting a higher capacity for fuelling aerobic metabolism with fatty acids. Both sea ducks and pochards prey mostly on molluscs, crustaceans and fish, which generally have a higher lipid content than the vegetation preferred by the dabblers ,66. An i2. Fatty acid oxidation requires more O2 per mole ATP compared with carbohydrates, and a preference for carbohydrates is a strategy used by some taxa to improve exercise performance under low O2 conditions [If the increased capacity for fatty acid oxidation is reflected by increases in fatty acid use during dives, it could suggest that muscle metabolism is not restructured to maximally conserve Onditions ,69,70. Mnditions ,71,72. T (c)b). Compared with the other sea ducks, the long-tailed duck had the greatest CS and SDH activities and the lowest ATP synthase activity in the gastrocnemius . The long-tailed duck was also an outlier for the activity of several enzymes in the pectoralis . This suggests that the consistent remodeling of metabolic pathways that we observed in sea ducks is pushed to the extreme in the deepest diving duck among the group.Within the sea ducks, the long-tailed duck has the greatest dive time and dive depth , making figure 5. 52 stores for extending dive capacity in diving ducks, because we found no evidence that diving ducks had consistently greater capacities for anaerobic metabolism or substrate-level phosphorylation. The present study highlights some of the key metabolic specializations that support increased dive performance across many species of specialized diving waterfowl.The locomotory muscles of sea ducks and pochards, but especially the sea ducks, appear to be specialized to sustain high rates of aerobic metabolism during diving locomotion. The combined increases in CS and COX activity probably reflect high overall capacities for oxidative phosphorylation, and high HOAD activities may enable high rates of lipid oxidation. Our findings also emphasize the importance of body O


Ccn1, are also essential genes in humans. However, a deficit in loss of function mutants in humans indicated that THBS1 is also highly loss-intolerant. In addition to roles in embryonic development or adult reproduction, genes may be loss-intolerant in humans because their function is needed to survive environmental stresses that are encountered between birth and reproduction. Laboratory mice live in a protected environment that lacks the exposures to pathogens and injury that humans routinely face. However, subjecting Thbs1−/− mice to defined stresses has provided valuable insights into functions of thrombospondin-1 that could account for the loss-intolerance of THBS1 in humans.Matricellular proteins comprise several families of secreted proteins that function in higher animals at the interface between cells and their surrounding extracellular matrix. Targeted gene disruptions that result in loss of viability in mice have revealed critical roles for several matricellular proteins in murine embryonic development, including two members of the cellular communication network (CCN) gene family. In contrast, mice lacking single or multiple members of the thrombospondin (THBS) gene family remain viable and fertile. The frequency of loss of function mutants, identified using human deep exome sequencing data, provided evidence that some of the essential genes in mice, including  Several families of secreted proteins have been identified in higher animals that regulate cell behavior by interacting with structural components of the extracellular matrix, secreted cytokines and proteases, and specific cell surface receptors. These are collectively known as matricellular proteins  . Compared to the 56 expected LoF mutants, only 7 were observed, and the pLI for THBS1 was 1.00, indicating this gene to be highly loss-intolerant   , loss of which is perinatal lethal in mice , pLI = 1.00) (Karczewski et al. Loss of Recently, thrombospondin-1 was identified as an essential component of supramolecular attack particles produced by CD8 T cells and natural killer cells (Ambrose et al. Thbs1 gene disruption on the ability of mice to survive exposure to specific pathogens or respond to a variety of physiological stresses (McMaken et al. THBS1 in humans, it is premature to rank their relevance. To date, the evidence is most compelling for protective functions of thrombospondin-1 in hemostasis to account for its loss-intolerance. Future genome wide association studies may identify additional missense mutations in THBS1 that contribute to essential functions in humans and provide clues to the specific thrombospondin-1 interaction partners involved. The insights gained from stress models that revealed protective functions of Thbs1 and Cd47 in mice may also be applicable for identifying molecular mechanisms underlying the deficits in LoF and missense mutants observed for other thrombospondins and members of other matricellular gene families in humans.Studies have identified beneficial as well as detrimental effects of


Is it feasible to collect prospective clinical safety and activity data for the use of compassionate or off-label innovative anticancer medicines at a multicenter, national level?This cohort study included information collected from 366 children, adolescents, and young adults treated with 55 compassionate or off-label innovative drugs as part of the SACHA -France study. Pharmacovigilance monitoring confirmed significant adverse drug reactions in one-third of them; 25% of the patients achieved at least a partial response.These results confirm the feasibility of this prospective real-word study. This cohort study of children, adolescents, and young adults treated at pediatric oncology centers in France examines outcomes and safety measures for the use of off-label treatments for cancer. Innovative anticancer therapies for children, adolescents, and young adults are regularly prescribed outside their marketing authorization or through compassionate use programs. However, no clinical data of these prescriptions is systematically collected.To measure the feasibility of the collection of clinical safety and efficacy data of compassionate and off-label innovative anticancer therapies, with adequate pharmacovigilance declaration to inform further use and development of these medicines.This cohort study included patients treated at French pediatric oncology centers from March 2020 to June 2022. Eligible patients were aged 25 years or younger with pediatric malignant neoplasms  or related conditions who received compassionate use or off-label innovative anticancer therapies. Follow up was conducted through August 10, 2022.All patients treated in a French Society of Pediatric Oncology (SFCE) center.Collection of adverse drug reactions and anticancer activity attributable to the treatment.MEK/BRAF inhibitors followed by multi-targeted tyrosine kinase inhibitors. In 34% of patients at least a grade 2 clinical and/or grade 3 laboratory adverse drug reaction was reported, leading to delayed therapy and permanent discontinuation of the innovative therapy in 13% and 5% of patients, respectively. Objective responses were reported in 57 of 230 patients (25%) with solid tumors, brain tumors, and lymphomas. Early identification of exceptional responses supported the development of specific clinical trials for this population.A total of 366 patients were included, with a median age of 11.1 years ; 203 of 351 patients (58%) in the final analysis were male. Fifty-five different drugs were prescribed, half of patients (179 of 351 [51%]) were prescribed these drugs within a compassionate use program, mainly as single agents (74%) and based on a molecular alteration (65%). Main therapies were This cohort study of the SACHA-France (Secured Access to Innovative Medicines for Children with Cancer) suggested the feasibility of prospective multicenter clinical safety and activity data collection for compassionate and off-label new anticancer medicines. This study allowed adequate pharmacovigilance reporting and early identification of exceptional responses allowing further pediatric drug development within clinical trials; based on this experience, this study will be enlarged to the international level. Childhood and adolescent cancers comprise a heterogeneous group of rare diseases, mostly distinct to those diagnosed in adults. Survival of children and adolescents with cancer has considerably improved in recent decades, but cancer remains the leading cause of disease-related mortality in high-income countries.1Although better understanding of cancer biology has led to increased development of novel targeted therapies and immunotherapies in the last 2 decades, only few have been approved for the treatment of childhood cancers over the last 15 years. There are more anticancer medicines approved for the treatment of childhood cancers in the US than in the EU, with a median time of 1.9 years between pediatric anticancer drug authorization by the US Food and Drug Administration (FDA) and the authorization in the EU.2 and, since 2020, within the French National Program, the 2025 France Genomic Medicine Initiative (PFMG2025) as part of health care. In order to increase the access to matched innovative therapies within the framework of early phase clinical trials the Innovative Therapies for Children with Cancer (ITCC) AcSé-ESMART platform trial (NCT02813135) was open in parallel in France and Europe in 2016 in addition to early phase trials running in the ITCC centers. However, the current clinical trials portfolio still remains insufficient and pediatric hematologist-oncologists regularly prescribe unauthorized drugs on compassionate use and/or innovative medicines outside their marketing authorization and clinical trials. Before 2020, no information on those treatments was collected in France. In order to collect clinical data of the administration of compassionate and off-label used innovative therapies, the French Society of Pediatric Oncology (SFCE) developed in 2020 the Secured Access to Innovative Medicines for Children with Cancer (SACHA)-France observational study (NCT04477681).In France, since January 2016 all pediatric and young adult patients with recurrent or refractory malignant neoplasms have access to tumor molecular profiling at the time of disease recurrence, first within the MAPPYACTS trial3 Before inclusion in the SACHA-France study, interregional multidisciplinary discussion is mandatory to ensure that all patients receive the most adapted available treatment, preferably within clinical trials. This study is a Research Involving Human Subjects of category 3  according to the French Jardé law, that request patient or their parents or legal representative’s nonopposition. This study has been reviewed and approved by the French ethics committee in compliance with the EU General Data Protection Regulation, which confirmed that informed consent was not needed. SACHA-France is open in all SFCE centers. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The SACHA-France study is a prospective, multicenter, national observational registry that collects clinical data of innovative therapies  administered to patients aged 25 years or younger at the time of prescription with pediatric malignant neoplasms (solid tumor or leukemia) or related conditions, provided through the French early access authorization and compassionate use programs or as an off-label anticancer medicine that has been first approved in adults in Europe after the implementation in 2017 of the EU Pediatric Regulation.Adverse drug reactions (ADRs) attributable to the treatment under study are described according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5. Only ADRs grade 2 or higher for clinical and/or 3 or above for laboratory are reported. All reported ADRs that lead to drug dose reduction, delay, or end of treatment as well as all unexpected ADRs and all serious ADRs (SADRs) are remotely monitored by the Pharmacovigilance Unit of Gustave Roussy.Progression-free survival (PFS) and best response from the start of treatment to tumor relapse or progression is assessed by the patient’s treating physician following the standard response criteria for each tumor type. In case of reported objective tumor response (ORR), radiological reports at baseline or time of response are reviewed by the SACHA-France coordinating investigator to confirm the coherence of the reported response.All cohorts of 10 patients or more with measurable or evaluable disease treated with the same drug for the same indication and reported at least 6-month follow-up as well as patients with observed antitumor activity previously not reported are reviewed by the SACHA France Steering Committee. In cases where no activity is observed or if there is a major safety concern (severe toxic effects or death), the steering committee recommends to all SFCE centers to no longer prescribe the medicine in that specific indication. If outstanding not previously reported activity results are observed, the steering committee recommends the development of an early phase clinical trial in that specific indication.Categorical variables were described with the frequency and percentage of each category; continuous variables as median and range value.NF1-related plexiform neurofibroma treated by trametinib [10 patients]; BRAF-altered low grade glioma [LGG] treated by dabrafenib plus trametinib [4 patients]; NTRK fusion–positive sarcomas treated by larotrectinib [3 patients]). Main tumor types of 342 reported patients were central nervous system (CNS) tumors (164 patients [48%]), non-CNS solid tumors (122 patients [36%]), leukemias (36 patients [11%]), and lymphomas (20 patients [6%]) , NF1 (30 patients), H3K27M (21 patients), ALK (20 patients), SMARCA/B12 (10 patients), and NTRK (10 patients).Main innovative therapies prescribed in 10 patients or more were trametinib (74 patients), dabrafenib plus trametinib (44 patients), ONC201 (25 patients), regorafenib (23 patients), cabozantinib (19 patients), pazopanib (18 patients), tazemetostat (14 patients), venetoclax (12 patients), larotrectinib (10 patients), and crizotinib (10 patients). Main reported genetic alterations that led to a matched targeted therapy were alterations in 4 None of the study participants were lost to follow-up.Information on follow-up was provided for 322 of 351 patients (91%). Treatment was stopped for 194 of 322 patients (60%). Main reasons for end of treatment were disease progression (124 patients [64%]), physician decision , adverse drug reaction (17 patients [9%]), disease progression and adverse drug reaction (4 patients [2%]), and patient’s decision.With a date cut-off of the August 10, 2022, 284 CTCAE grade 2 to 4 ADRs related to 26 out of 55 prescribed innovative therapies were reported in 121 of 351 patients eligible for analysis (34%). More than a third of them (122 of 284 patients [43%]) were related to trametinib as a single agent.Overall, 72 of 284 ADRs (25%) led to a delayed therapy in 45 patients, 38 of 284 (13%) to a drug reduced dose in 23 patients, and 29 of 284 (10%) to a discontinued treatment in 22 patients (related to 12 different therapies), including 5 patients with a temporarily interruption only. All nonserious ADRs reported are described in eTable 3 in A total 24 SADRs (8%) related to 12 therapies were reported in 17 patients . Of themMET fusion–positive malignant supratentorial tumor treated with the MET inhibitor capmatinib; a patient younger than 5 years with a FGFR1 fusion brain stem high-grade glioma treated with the multitargeted tyrosine kinase inhibitor (MTKI) covering FGFR1 lenvatinib; a patient aged between 5 and 10 years with Burkitt lymphoma treated with an anti-CD20/CD3 antibody.Of the 242 patients with solid tumors, brain tumors, or lymphomas with treatment started before December 31, 2021, 230 (95%) had measurable or evaluable disease at study entry and were evaluable for response assessment. Of them, 57 of 230 (25%) achieved a partial response or complete response as best response , confirmed by remote monitoring of radiological reports at baseline or time of response. Most responses were related to drugs already approved by the European Medicines Agency or the FDA for adults or other indications (54 of 57 drugs) (eTable 4 in MEK inhibitor trametinib: cohort 1 (BRAF fusion–positive LGG), cohort 2 (NF1-related optic pathway glioma), and cohort 3 (NF1-related plexiform neurofibroma). Patients in cohort 4  were treated by the combination of BRAF/MEK inhibitors (dabrafenib and trametinib), and patients in cohort 5 (osteosarcoma) were treated with the MTKI regorafenib. Patient characteristics and reported outcomes are detailed in According to the predefined criteria for activity assessment, there were 5 cohorts with 10 or more patients treated by the same medicine for the same indication. Three cohorts of patients were treated with the The SACHA-France study is a unique innovative initiative that collects clinical data on safety and activity of compassionate or off-label new anticancer medicines prescribed outside clinical trials for children, adolescents, and young adults. It is worth nothing that all French pediatric hematology-oncology centers are actively involved, with more than 150 patients included annually and 55 different drugs prescribed.The goal of SACHA is not to encourage treating patients with innovative medicines outside clinical trials. For this reason, multidisciplinary discussion has been set up and is mandatory to ensure that all relevant options, starting with enrollment in a clinical trial, are identified and discussed for each patient at the time of disease recurrence. Nevertheless, compassionate and off-label prescriptions do happen and SACHA is collecting this information in a real-life setting, to our knowledge for the first time, to assure patient security and to inform antitumoral activity and toxicity.5 This increasing prevalence was confirmed in a retrospective single-institution cohort with 374 patients who received an off-label targeted anticancer drug from 2007 to 2017, with encouraging activity data, but with clinically significant ADRs (38%) and discontinuation due to toxic effects (13%).6 In a 5-year period retrospective review of all genomically targeted therapeutic single patient use in 4 US large pediatric cancer centers, ORRs were seen in 39.5% cases, treatment discontinuation due to toxicity was reported in 13% patients, but no information on other ADRs were reported, strongly reinforcing the high need of systematically and adequate collection of this data at the multi-center level.7Off-label and compassionate use prescription is common in pediatric oncology and its prevalence has increased over the last decade; however, only limited data are published and safety information remains largely underreported.BRAF V600E variant LGG.8 In March 2023, this combination granted FDA approval for patients ages 1 year and older with LGG with a BRAF V600E variation who require systemic therapy, but is not yet approved by the EMA. Therefore, the only access in Europe to this proven effective combination for children and adolescents continues to be off-label (tablets) or compassionate  use. Similarly, MEK inhibitors have been extensively explored in pediatric patients with BRAF fusion positive or NF1-related tumors. Selumetinib has shown promising single-agent activity in this group of patients9 and has been approved since October 2020 by the FDA and June 2021 by the EMA for the treatment of inoperable symptomatic plexiform neurofibromas (PNs) in children with NF1. Additionally, trametinib has shown single-agent activity in this population,10 but has not been approved for this last indication by the FDA or the EMA. Due to initial lack of access to selumetinib until EMA approval in June 2021, trametinib was prescribed for this indication in Europe until that date.In SACHA-France, there was a 25% reported ORR in patients with solid tumors, brain tumors, and lymphomas mostly related to innovative drugs already explored within pediatric clinical trials for either the same disease or biomarker match but that are not yet approved in children and adolescents. This is for example the case of the combination of dabrafenib with trametinib in pediatric patients with BRAF-variant LGG to dabrafenib plus trametinib was higher that previously reported,4 while similar for patients with BRAF fusion–positive LGG and NF1-related optic pathway gliomas treated with trametinib.11 These differences have to be interpreted with caution due to the lack of independent radiological review, which has been shown to significantly modify the final response assessment for these tumors.12 In our study, none of the 10 patients with NF1-related PNs treated by trametinib had an ORR, compared with 24% ORR reported,11 but most had a prolonged clinical benefit. This difference may reflect a more selected population in clinical trials, compared with a real-life prescription population. Based on these results and current selumetinib EMA approval for this indication, the SACHA steering committee recommended to no longer prescribe trametinib, and instead prescribe selumetinib for patients with NF1-related inoperable PNs. MTKIs with antiangiogenic activities have also shown activity in patients with bone sarcomas. Regorafenib is a MTKI with proven efficacy in adults with osteosarcoma in 2 randomized double-masked trials14 but with limited experience in pediatric patients.15 In our study, regorafenib 4-month PFS in patients with osteosarcoma was similar to other single-agent MTKI reported.16 Currently MTKIs are being included in upfront trials in combination with chemotherapy  and as maintenance therapy .In our study, we confirmed previously reported ORR within early phase clinical trials, although in our study ORR of MET and FGFR1 fusion–positive CNS tumors. The first case supported the development of the capmatinib plus everolimus arm in the ESMART trial (NCT02813135). This basket trial already has an arm with a FGFR inhibitor (futibatinib), which opened to enrollment after that patient had already been treated by lenvatinib. This underscores the role of the SACHA-France study in supporting the formal development of drugs in the pediatric population when antitumor activity is observed and not as a substitute for them. Although quality of data derived from off-label and compassionate use is not equivalent to clinical trial data, clinical data can support clinical trial data in the regulatory review of new drug applications or indications, and they increasingly are used in regulatory decision making.18 Nevertheless, in very rare populations with strong predictive biomarkers this may even lead directly to regulatory approval, as was the case of alpelisib, which obtained FDA accelerated approval for adult and pediatric patients with severe manifestations of PIK3CA-related overgrowth spectrum.19The SACHA-France study allowed the early identification of exceptional responders to inform further pediatric development of those drugs within clinical trials. This is the case of the antitumor activity not previously reported in patients with 20 Since March 2020, almost 300 ADRs have been reviewed and 24 SADRs reported to the national competent authorities. This includes, for example, a secondary malignant neoplasm in a patient younger than 5 years with ATRT and a long-lasting response to tazemetostat, which is the second case reported to our knowledge.21 Interestingly, some of the reported unexpected SADRs are well-known toxic effects already reported to a specific drug or drug class but not included in the summary of product characteristics , further stressing the importance of adequate reporting of these toxic effects.The SACHA-France study has an important pharmacovigilance objective with remote monitoring of all reported ADRs and special focus on unexpected and SADRs. This allows improving the knowledge of clinical toxic effects to innovative therapies that are otherwise usually underreported.24 Since 2021, ANSM actively recommends the inclusion of pediatric patients treated on a compassionate use program in the SACHA-France study in order to ensure adequate monitoring of these prescriptions. In 2017, the EU Commission Expert Group on Safe and Timely Access to Medicines for Patients (STAMP) released the results of a study of the public health aspects related to the off-label use of medicinal products, investigating the balance between the benefits and risks for patients, and the regulatory framework for the off-label use of medicines.25 Following the proposal by the STAMP Group, a medicines repurposing framework is being implemented in the EU.26 In this context the SACHA initiative could provide, beyond pediatric oncology, relevant information from real patient populations while assuring safety of patients receiving off-label treatments. Based on the SACHA-France experience, the ITCC has developed the SACHA International Project (ITCC-105) that will be launched in 2023.It is important to highlight the strong support to this study by ANSM, the French competent authority responsible for approving the compassionate access of unauthorized drugs that represents half of the drugs prescribed in our study. In France, early drug access before marketing authorization is granted by ANSM through the Temporary Authorization for Use program  that allows patients with unmet medical needs to be treated with an innovative drug before marketing authorization, if the assessment of preliminary data presents a positive risk-benefit balance and the patient has no therapeutic alternatives among the existing therapies and clinical trials.The SACHA-France study is limited by the absence of information on off-label prescriptions in France to calculate the proportion of patients enrolled. This information will be available in the near future for patients within compassionate use programs thanks to the collaboration with the ANSM.In this cohort study, we confirmed the feasibility of clinical safety and activity data collection of compassionate and off-label new anticancer medicines prescribed to children, adolescents, and young adults. Our study allowed the adequate pharmacovigilance reporting of unexpected and serious ADRs, confirmation of antitumor responses in real-life situation and the early identification of exceptional responses allowing further pediatric drug development within clinical trials.


Bifidobacterium, Faecalibacterium, Ruminococcus, and Roseburia) are associated with childhood respiratory diseases. However, results were inconsistent and studies had important limitations, including insufficient characterisation of bacterial taxa to species level, heterogeneous outcome definitions, residual confounding, and small sample sizes. Large longitudinal studies with stool sampling during the first month of life and shotgun metagenomic approaches to improve bacterial and fungal taxa resolution are needed. Standardising follow-up times and respiratory disease definitions and optimising causal statistical approaches might identify targets for primary prevention of childhood respiratory diseases.Data from animal models suggest a role of early-life gut microbiota in lung immune development, and in establishing susceptibility to respiratory infections and asthma in humans. This systematic review summarises the association between infant (ages 0–12 months) gut microbiota composition measured by genomic sequencing, and childhood (ages 0–18 years) respiratory diseases . Overall, there was evidence that low α-diversity and relative abundance of particular gut-commensal bacteria genera ( Childhood respiratory diseases, including respiratory infection, recurrent wheezing, and asthma, are important causes of morbidity and mortality in children and at ages thereafter. Up to 30% of children worldwide will develop at least one viral lower respiratory tract infection (vLRTI) during their first 2 years of life,Animal models have provided evidence that early-life gut microbiota composition might influence respiratory immunity and susceptibility to both asthma and respiratory infections.The gut microbiota is the largest and most diverse microbiota in the body, harbouring billions of bacteria (the predominant organisms), archaea, eukaryotes, and viruses.In the past decade, genomic sequencing technologies and bioinformatic analytical tools have advanced considerably. Available platforms now allow the simultaneous sequencing of most or all genetic material present in stool samples,We systematically reviewed the existing literature to examine the association between gut microbiota composition during infancy (measured by genomic sequencing) and the subsequent development of respiratory disease during childhood.A systematic literature review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.We comprehensively searched five electronic databases  for articles published with the full text in English between Jan 1, 2010, and April 27, 2021. A start year of 2010 was selected to ensure all studies using genomic sequencing for gut microbiota characterisation were captured.The first reviewer (CG-MA) screened all titles, abstracts, and full texts of shortlisted articles. A second reviewer (VMP) screened 10% of studies at each stage of the screening process and results were compared between reviewers to check agreement. Any disagreement was resolved with a third reviewer (CO). Data were extracted by the first reviewer. The second reviewer independently extracted information from 80% of included studies to ensure accuracy in reporting and to minimise reviewer bias.Differences in microbiota diversity, relative abundance of bacteria or fungi taxa , and any measure of association with 95% CIs relative to either diversity or abundance were extracted and reported as the main result. Other gut microbiota parameters analysed and findings related to gut microbiota composition and respiratory disease were also extracted .Critical appraisal was performed independently by two reviewers (CG-MA and VMP) to assess the quality of included studies and provide context for the interpretation of the findings. Studies were assessed with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) metagenomics framework for reporting of metagenomic studies.4759 titles and abstracts were screened and 4648 of these studies were excluded. Reasons for exclusion included non-primary research, animal studies, in-vitro studies, or studies in which exposure or outcome were not relevant to the research question. After a full-text review of the remaining 111 studies, 11 were included . AgreemeOf the 11 included studies, eight were cohort studiesFour of the 11 included studies were classified as poor quality,Regarding the STROBE metagenomics checklist , all incThe statistical analyses used to establish the association between diversity and relative abundance of taxa and respiratory disease included univariate statistical tests,28Study results have been summarised and stratified by type of respiratory disease and age at stool sample collection35Of the seven studies37BifidobacteriumFaecalibacterium,Roseburia,RuminococcusLachnospiraLachnospira at age 1 yearVeillonellaVeillonella at ages 3 months and 1 year was associated with asthma and atopic wheeze at age 5 years.Although results varied between studies, overall there was evidence of a low relative abundance of the genera 18S rRNA gene,ITS1 and ITS2).Candida and Rhodotorula and low abundance of Malassezia taxa measured at age 1 month in one study,Pichia kudriavzevii at age 3 months measured in another,Three studiesBifidobacterium and higher abundance of Klebsiella and Enterococcus at age 1 week in children with higher cumulative incidence of respiratory infections at age 1 year than for children with lower cumulative incidence of respiratory infections.Four studies were included in the respiratory infections and wheezing group, in which respiratory disease definitions were highly heterogeneous. One study evaluated respiratory infections,BifidobacteriumFaecalibacterium,Roseburia,Ruminococcus,To our knowledge, this systematic review is the first to consider the association between early-life gut microbiota and childhood respiratory diseases, including respiratory infections, with a focus on genomic sequencing to measure the gut microbiota. Large studies (>700 participants) reported high α-diversity as being protective of asthma and wheezing.A previous systematic review by Zimmerman and colleaguesKlebsiella and Enterococcus at age 1 weekStreptococcus at age 3 months was associated with atopic wheeze at age 5 years, and that high relative abundance of RothiaDialisterBifidobacterium,Faecalibacterium,Ruminococcus,Roseburia,All studies included in this systematic review used 16S rRNA gene amplicon sequencing, allowing a relatively untargeted starting point for exploring the gut microbiome and permitting us to summarise and compare study results at a bacterial genera level. Some studies reported that high relative abundance of non-commensal gut bacteria such as Bifidobacterium constitutes one of the most abundant bacteria in the gut of children during the first 4 months of lifeBifidobacterium spp have been shown to affect respiratory disease susceptibility in mouse models of asthma and respiratory infection.Bifidobacterium infantis regulates the equilibrium between Th1 and Th2 responses, reducing symptoms of atopic asthma in an induced mouse model.Bifidobacterium and Bacteroides showed increased influenza survival through an enhanced CD8 T-cell and well regulated macrophage response than mice with lower gut abundance, preventing excessive airway neutrophil influx.The genus Faecalibacterium, Ruminococcus, Lachnospira, Roseburia, and Veillonella correspond to the clostridia class, which has been described in the guts of children from around 4–6 months of age, coinciding with weaning off breastmilk.Ruminococcus  and Roseburia  have been described as signature taxa in infants aged 12 months.Roseburia and Faecalibacterium, which produce butyrate, a bacterial metabolite with anti-inflammatory properties in animal and in-vitro models.Lachnospira, Veillonella, Faecalibacterium, and Rothia improved airway inflammation in the adult progeny of these mice,The bacteria genera Comparison of studies exploring respiratory infections and wheezing episodes in children younger than 5 years is challenging because of the inconsistent definitions for upper and lower viral respiratory infections and recurrent wheezing in the literature and clinical guidelines, and the difficulty in diagnosing asthma at ages 0–6 years.Bifidobacterium and high relative abundance of Klebsiella and Enterococcus in stool samples collected at age 1 week were associated with a higher number of respiratory infections evaluated at age 1 year compared with those with low numbers of respiratory infections.Only two studies included in this systematic review contained respiratory infections per se in their outcome definition.Neonatal gut microbiota composition is known to influence subsequent colonisation patterns, which might also affect subsequent microbiota and immune crosstalk and development in the long term.Bacteroides spp are selectively depleted at –80°C dependent on time in storage),Despite relatively good reporting of laboratory methods and bioinformatic pipelines, these methods and pipelines were heterogeneous, important information was sometimes missing, and potential procedure-specific bias was barely discussed. For example, six studies did not clearly report time between sample collection and freezing and no studies mentioned freeze –thaw cycles, although both can affect microbial profiles.A more informative genomic sequencing approach than the targeted amplicon sequencing method is shotgun metagenomic sequencing, which enables completely untargeted sequencing of all genetic material present in a stool sample; robust bacterial resolution down to a species or strain level; provides information about functionality; and detects viruses, archaea, fungi, and other microeukaryotes that do not possess the 16S rRNA gene.This systematic review has additional limitations. The search was restricted to studies published after 2010, and it is possible that some smaller studies were missed. One included study had important issues with reverse causality, and one study was a post-hoc analysis of a randomised controlled trial (RCT).Bifidobacterium spp have been shown to influence respiratory disease pathogenesis in animal studies,Bifidobacterium spp and Lactobacillus spp) for subsequently informing intervention studies,Several frameworks have been developed to aid decisions in establishing causation in microbiome studies.Another important consideration is that the entire process of microbiota ecosystem development could be the cause of health or disease,Another outstanding question when evaluating causality between gut microbiota and lung disease is the role of the respiratory microbiota.Overall, there is observational evidence that low α-diversity and relative abundance of particular gut-commensal bacterial genera in the first year of life are associated with subsequent respiratory disease, especially asthma. There is less evidence for the association between low α-diversity and relative abundance of particular bacterial taxa in the first year of life and respiratory infections. However, the available evidence showed important limitations, and gut microbiota composition might not have a causal role in subsequent respiratory disease (despite the observed associations). Large longitudinal studies with stool and respiratory sampling during the neonatal period that use shotgun metagenomic sequencing to improve measurement resolution of the microbiome to a species or strain level are needed. Optimising statistical approaches for causal inference, standardising outcome definitions, and validating findings with experimental models will help move knowledge forward in the area of early-life microbiota and in the development of potential preventive and therapeutic interventions for childhood respiratory diseases.We declare no competing interests.