In solid cancers, T cells typically function as cytotoxic effectors to limit tumor growth, prompting therapies that capitalize upon this antineoplastic property (immune checkpoint inhibition; ICI). Unfortunately, ICI treatments have been largely ineffective for high-grade brain tumors (gliomas; HGGs). Leveraging several single-cell RNA sequencing datasets, we report greater CD8+ exhausted T cells in human pediatric low-grade gliomas (LGGs) relative to adult and pediatric HGGs. Using several preclinical mouse LGG models (Nf1-OPG mice), we show that these PD1+/TIGIT+ CD8+ exhausted T cells are restricted to the tumor tissue, where they express paracrine factors necessary for OPG growth. Importantly, ICI treatments with alpha-PD1 and alpha-TIGIT antibodies attenuate Nf1-OPG tumor proliferation through suppression of two cytokine (Ccl4 and TGF beta)-mediated mechanisms, rather than by T cell-mediated cytotoxicity, as well as suppress monocyte-controlled T cell chemotaxis. Collectively, these findings establish a previously unrecognized function for CD8+ exhausted T cells as specialized regulators of LGG maintenance. With the emergence of immune checkpoint inhibitor therapies for cancer, the authors use single-cell sequencing to assess exhausted T cell content in human glioma samples and leverage these findings in mouse models to define the mechanisms by which exhausted T cells regulate low-grade glioma growth.

Abstract Brain tumors arise in close association with neurons, suggesting that these non-neoplastic cells may be critical stromal drivers of brain tumor initiation and growth. Previously, we have shown that murine low-grade optic glioma formation and progression in the setting of Neurofibromatosis type 1 (NF1) is dictated by neurons and neuronal activity. In these studies, these neuronal dependencies reflected neuronal activity-driven enzymatic cleavage of a growth factor (neuroligin-3) from oligodendrocyte precursors cells (tumor initiation) or neuronal production of a paracrine factor to stimulate T cell support of optic glioma growth (tumor progression). Since neurons typically communicate with other neurons through neurotransmitters, we sought to explore the possibility that neurotransmitters operate to modulate low-grade glioma growth using humanized mouse models of pilocytic astrocytoma (PA). Leveraging single cell RNA sequencing of three independent sets of pediatric PAs, we identified neurotransmitter pathway enrichment in the tumor cells. This neurotransmitter pathway enrichment reflected aberrant expression of specific neurotransmitter receptors, which we confirmed in three independently generated PA tissue microarrays and in five distinct primary PA cell lines grown in vitro. Moreover, this aberrant neurotransmitter receptor expression established differential neurotransmitter PA growth dependencies in vitro. Importantly, interruption of neurotransmitter signaling in human PA xenografts attenuated tumor growth and ERK activation in Rag1-/- mice in vivo. Finally, we discovered crosstalk between neurotransmitter receptor and receptor tyrosine kinase signaling that revealed another target for therapeutic inhibition. Taken together, these findings elucidate a previously unknown neurotransmitter PA growth dependency amenable to therapeutic targeting.

Abstract T cells are minor cellular constituents of the brain tumor microenvironment, but have recently been shown to play critical roles in glioma pathobiology and treatment. Previous studies from our laboratory have shown that T cells are required for the formation and growth of low-grade gliomas (LGGs) in murine models of the Neurofibromatosis type 1 (NF1) tumor predisposition syndrome. To better characterize the immune landscape of these pediatric low-grade tumors, we leveraged single cell RNA sequencing of human pediatric pilocytic astrocytomas and genetically engineered mouse LGG (Nf1-optic glioma) models. First, we showed that both human and murine LGGs contain a CD8+ T cell population expressing PD-1 and TIGIT, similar to conventional exhausted T cell populations. Second, these PD-1+/TIGIT+ CD8+ T cells are actively recruited to the tumor site by Ccl2 where they dictate tumorigenesis through the elaboration of Ccl4, a cytokine necessary for microglia Ccl5-mediated support of Nf1-OPG growth. As such, Ccl4 expression was enriched in PD-1+/TIGIT+ CD8+ T cells relative to all other T cell populations within these tumors. Third, this “exhausted” CD8+ T cell population was exclusively present in the optic nerves, but not in the blood, dura, or deep cervical nodes of optic glioma-bearing mice. Fourth, targeting this PD1+/TIGIT+ exhausted CD8+ T cell population with immune checkpoint monoclonal antibody inhibitors reduced CD8+ T cell tumoral recruitment, Ccl4 production, and optic glioma growth. Collectively, these findings establish an obligate role for PD-1+/TIGIT+ CD8+ T cells in Nf1-optic glioma control and suggest that immune checkpoint modulation may be useful for the treatment of LGGs in children.

ABSTRACT Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.

Introduction: Over the past decade, use of cannabidiol (CBD) to manage common symptoms such as anxiety, sleep disturbance, and pain has expanded rapidly. However, few clinical trials have investigated CBD's safety or efficacy. Furthermore, whether effects vary by characteristics of the product or individual characteristics is largely unknown. Materials and Methods: The safety and health effects of 13 orally ingested, commercially available CBD products were evaluated using an open-label, randomized, waitlist controlled design. Participants (n?=?2816; 94% female) residing in the United States and reporting symptoms of anxiety, sleep disturbance, or chronic pain were randomized to receive a 4-week supply of a CBD product or no product (waitlist control). CBD products varied by dose, form (e.g., capsules), and spectrum (isolate, broad, or full). Participants completed online surveys assessing well-being, anxiety, sleep disturbance, and pain using validated outcome measures weekly for 4 weeks. Linear mixed models were used to assess the effect of taking any product relative to waitlist control and the interaction effects of participant prior (prestudy) CBD use. Effects of CBD spectrum were investigated using post hoc analyses. Results: Well-being, anxiety, sleep disturbance, and pain significantly improved among those assigned to take a CBD product relative to waitlist control. Among those assigned to a CBD product, the percentage of individuals with a given condition who experienced an improvement which could be considered clinically meaningful or important was 46.6% for anxiety; 47.9% for sleep disturbance, and 35.2% for pain. No significant differences in effect were found as a function of spectrum or prior CBD use for any outcome. Approximately 9.5% of participants reported one or more side effects, the most common of which were gas/flatulence (1.6%), headache (1.4%), diarrhea (1.2%), and bloating (1.2%). No severe side effects were reported. Discussion: Our results suggest that the commercially available CBD products included in this study are safe and may serve as potentially effective complementary therapies for management of anxiety, sleep disturbance, and pain. These effects appear independent of the prior CBD use and product spectrum. Clinical Trial Registration number: NCT05003882.

Neuronal activity is emerging as a driver of central and peripheral nervous system cancers. Here, we examined neuronal physiology in mouse models of the tumor predisposition syndrome Neurofibromatosis-1 (NF1), with different propensities to develop nervous system cancers. We show that central and peripheral nervous system neurons from mice with tumor-causing Nf1 gene mutations exhibit hyperexcitability and increased secretion of activity-dependent tumor-promoting paracrine factors. We discovered a neurofibroma mitogen (COL1A2) produced by peripheral neurons in an activity-regulated manner, which increases NF1 -deficient Schwann cell proliferation, establishing that neurofibromas are regulated by neuronal activity. In contrast, mice with the Arg1809Cys Nf1 mutation, found in NF1 patients lacking neurofibromas or optic gliomas, do not exhibit neuronal hyperexcitability or develop these NF1-associated tumors. The hyperexcitability of tumor-prone Nf1 -mutant neurons results from reduced NF1 -regulated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function, such that neuronal excitability, activity-regulated paracrine factor production, and tumor progression are attenuated by HCN channel activation. Collectively, these findings reveal that NF1 mutations act at the level of neurons to modify tumor predisposition by increasing neuronal excitability and activity-regulated paracrine factor production.

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors.

Background Brain tumor formation and progression are dictated by cooperative interactions between neoplastic and non-neoplastic cells. This stromal dependence is nicely illustrated by tumors arising in the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, where children develop low-grade optic pathway gliomas (OPGs). Using several authenticated Nf1-OPG murine models, we previously demonstrated that murine Nf1-OPG growth is regulated by T cell function and microglia Ccl5 production, such that their inhibition reduces tumor proliferation in vivo. While these interactions are critical for established Nf1-OPG tumor growth, their importance in tumor formation has not been explored. Methods A combination of bulk and single-cell RNA mouse optic nerve sequencing, immunohistochemistry, T cell assays, and pharmacologic and antibody-mediated inhibition methods were used in these experiments. Results We show that T cells and microglia are the main non-neoplastic immune cell populations in both murine and human LGGs. Moreover, we demonstrate that CD8(+) T cells, the predominant LGG-infiltrating lymphocyte population, are selectively recruited through increased Ccl2 receptor (Ccr4) expression in CD8(+), but not CD4(+), T cells, in a NF1/RAS-dependent manner. Finally, we identify the times during gliomagenesis when microglia Ccl5 production (3-6 weeks of age) and Ccl2-mediated T cell infiltration (7-10 weeks of age) occur, such that temporally-restricted Ccl2 or Ccl5 inhibition abrogates tumor formation >3.5 months following the cessation of treatment. Conclusions Collectively, these findings provide proof-of-concept demonstrations that targeting stromal support during early gliomagenesis durably blocks murine LGG formation.

Objective To evaluate sociodemographic and medical predictors of patient return to a neurofibromatosis subspecialty clinic. Study design Data were collected from the Washington University Neurofibromatosis Clinical Program electronic medical records. A total of 713 subjects with initial visits to the Washington University Neurofibromatosis Clinical Program between July 1, 2005 and December 18, 2020 were included. Variables collected included sex, race, ethnicity, age, date of first visit, place of residence, diagnosis, insurance payer, physician recommendation for return, and subject return.Return rates for each demographic group were calculated. Bivariate analyses were performed to inform variable inclusion in the model, and a binary logistic regression model was calculated to predict subject return. Results The overall return rate was 76%. The binary logistic regression model was statistically significant (x(2) (9) = 131.094; P <.001) and showed that subjects who self-identified as Black and/or African American, presented with or received a diagnosis of caf similar to e-au-lait macules at their initial visit, were from a rural area, were older, or who lived farther from the Washington University Neurofibromatosis Clinical Program were less likely to return to clinic. Conclusions These findings support the implementation of tailored communication and monitoring interventions to improve the care for children with neurofibromatosis type 1. (J Pediatr 2022;248:94-9).

BACKGROUND:Emerging insights from numerous laboratories have revealed important roles for nonneoplastic cells in the development and progression of brain tumors. One of these nonneoplastic cellular constituents, glioma-associated microglia (GAM), represents a unique population of brain monocytes within the tumor microenvironment that have been reported to both promote and inhibit glioma proliferation. To elucidate the role of GAM in the setting of low-grade glioma (LGG), we leveraged RNA sequencing meta-analysis, genetically engineered mouse strains, and human biospecimens.METHODS:Publicly available disease-associated microglia (DAM) RNA-seq datasets were used, followed by immunohistochemistry and RNAScope validation. CD11a-deficient mouse microglia were used for in vitro functional studies, while LGG growth in mice was assessed using anti-CD11a neutralizing antibody treatment of Neurofibromatosis type 1 (Nf1) optic glioma mice in vivo.RESULTS:We identified Itgal/CD11a enrichment in GAM relative to other DAM populations, which was confirmed in several independently generated murine models of Nf1 optic glioma. Moreover, ITGAL/CD11A expression was similarly increased in human LGG (pilocytic astrocytoma) specimens from several different datasets, specifically in microglia from these tumors. Using CD11a-knockout mice, CD11a expression was shown to be critical for murine microglia CX3CL1 receptor (Cx3cr1) expression and CX3CL1-directed motility, as well as glioma mitogen (Ccl5) production. Consistent with an instructive role for CD11a+ microglia in stromal control of LGG growth, antibody-mediated CD11a inhibition reduced mouse Nf1 LGG growth in vivo.CONCLUSIONS:Collectively, these findings establish ITGAL/CD11A as a critical microglia regulator of LGG biology relevant to future stroma-targeted brain tumor treatment strategies.

Background Brain tumors are the most common solid tumors of childhood, but little is understood about the factors that influence their development. Pediatric low-grade gliomas in particular display unique temporal and spatial localization associated with different genetic mutations (eg, BRAF genomic alterations, mutations in the neurofibromatosis type 1 [NF1] gene) for reasons that remain unclear. NF1 low-grade gliomas typically arise in the optic pathway of young children as optic pathway gliomas (OPGs), likely from a cell of origin that resides within the third ventricular zone (TVZ). However, the factors that contribute to their distinct temporal patterning and penetrance have not been adequately explored. Methods TVZ neuroglial progenitor cells (NPCs) were analyzed over the course of mouse brain development. Progenitors isolated by fluorescence-activated cell sorting (FACS) were assessed for functional and molecular differences. The impact of different germline Nf1 mutations on TVZ NPC properties was analyzed using genetically engineered mice. Results We identify 3 individual factors that could each contribute to Nf1 optic glioma temporal patterning and penetrance. First, there are 3 functionally and molecularly distinct populations of mouse TVZ NPCs, one of which (“M” cells) exhibits the highest clonogenic incidence, proliferation, and abundance during embryogenesis. Second, TVZ NPC proliferation dramatically decreases after birth. Third, germline Nf1 mutations differentially increase TVZ NPC proliferation during embryogenesis. Conclusions The unique temporal patterning and penetrance of Nf1 optic glioma reflects the combined effects of TVZ NPC population composition, time-dependent changes in progenitor proliferation, and the differential impact of the germline Nf1 mutation on TVZ NPC expansion.

To elucidate the mechanisms underlying the reduced incidence of brain tumors in children with Neurofibromatosis type 1 (NF1) and asthma, we leverage Nf1 optic pathway glioma (Nf1OPG) mice, human and mouse RNAseq data, and two different experimental asthma models. Following ovalbumin or house dust mite asthma induction at 4-6 weeks of age (WOA), Nf1OPG mouse optic nerve volumes and proliferation are decreased at 12 and 24 WOA, indicating no tumor development. This inhibition is accompanied by reduced expression of the microglia-produced optic glioma mitogen, Ccl5. Human and murine T cell transcriptome analyses reveal that inhibition of microglia Ccl5 production results from increased T cell expression of decorin, which blocks Ccl4-mediated microglia Ccl5 expression through reduced microglia NFκB signaling. Decorin or NFκB inhibitor treatment of Nf1OPG mice at 4-6 WOA inhibits tumor formation at 12 WOA, thus establishing a potential mechanistic etiology for the attenuated glioma incidence observed in children with asthma.

Neurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.

Pilocytic astrocytoma (PA) is the most common glioma in children, with an excellent overall survival following surgical resection; however, a small subset of tumors recur, especially following incomplete resection. Since PAs contain a large number of non-neoplastic cells, immunomodulatory therapies have been proposed, particularly in light of results using experimental mouse models of low-grade glioma (LGG), where microglia and T cells were found to be critical non-neoplastic drivers of tumor formation and growth.(1)

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.

Abstract Neurons have recently emerged as essential cellular constituents of the tumor microenvironment, where their activity increases the growth of a diverse number of solid tumors. While the role of neurons in tumor progression has been previously demonstrated, the importance of neuronal activity to tumor initiation is less clear, particularly in the setting of cancer predisposition syndromes. In the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, in which tumors arise in close association with nerves, 15% of individuals develop low-grade neoplasms of the optic pathway (optic gliomas) during early childhood, raising the intriguing possibility that postnatal light-induced optic nerve activity drives tumor initiation. Here, we employ an authenticated murine model of Nf1 optic glioma to demonstrate that stimulation of optic nerve activity increases optic glioma growth, while decreasing optic nerve activity via light deprivation prevents tumor formation and maintenance. By manipulating environmental light to modulate optic pathway (retinal) neuron activity, we show that Nf1 optic glioma initiation depends on neuronal activity during a developmental period susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly high optic nerve neuroligin-3 (Nlgn3) shedding in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacologic inhibition of Nlgn3 shedding blocks murine Nf1 optic gliomagenesis and progression. Collectively, these studies establish an obligate role for neuronal activity in the development of certain brain tumors, elucidate a therapeutic strategy to reduce optic glioma incidence or mitigate tumor progression, and underscore the role of Nf1 mutation-mediated dysregulation of neuronal signaling pathways in the NF1 cancer predisposition syndrome.

Neurons have recently emerged as essential cellular constituents of the tumor microenvironment, where their activity increases the growth of a diverse number of solid tumors. While the role of neurons in tumor progression has been previously demonstrated, the importance of neuronal activity to tumor initiation is less clear, particularly in the setting of cancer predisposition syndromes. In the Neurofibromatosis-1 (NF1) cancer predisposition syndrome, in which tumors arise in close association with nerves, 15% of individuals develop low-grade neoplasms of the optic pathway (optic pathway gliomas [OPGs]), during early childhood, raising the intriguing possibility that postnatal light-induced optic nerve activity drives tumor initiation. Here, we employ an authenticated murine model of Nf1-OPG to demonstrate that stimulation of optic nerve activity increases optic glioma growth, while decreasing visual experience via light deprivation prevents tumor formation and maintenance. We show that Nf1-OPG initiation depends on visual experience during a developmental period susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly high optic nerve neuroligin-3 (Nlgn3) shedding in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of Nlgn3 shedding blocks murine Nf1 optic gliomagenesis and progression. Collectively, these studies establish an obligate role for neuronal activity in the development of certain brain tumors, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumor progression, and underscore the role of Nf1 mutation-mediated dysregulation of neuronal signaling pathways in the NF1 cancer predisposition syndrome.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.

Brain tumors (gliomas) are heterogeneous cellular ecosystems, where non-neoplastic monocytic cells have emerged as key regulators of tumor maintenance and progression. However, relative to macrophages/microglia, comparatively less is known about the roles of neurons and T cells in glioma pathobiology. Herein, we leverage genetically engineered mouse models and human biospecimens to define the axis in which neurons, T cells, and microglia interact to govern Neurofibromatosis-1 (NF1) low-grade glioma (LGG) growth. NF1 -mutant human and mouse brain neurons elaborate midkine to activate naïve CD8 + T cells to produce Ccl4, which induces microglia to produce a key LGG growth factor (Ccl5) critical for LGG stem cell survival. Importantly, increased CCL5 expression is associated with reduced survival in patients with LGG. The elucidation of the critical intercellular dependencies that constitute the LGG neuroimmune axis provides insights into the role of neurons and immune cells in controlling glioma growth, relevant to future therapeutic targeting.