The pelvic organs receive no parasympathetic innervation.

The pelvic organs (bladder, rectum, and sex organs) have been represented for a century as receiving autonomic innervation from two pathways - lumbar sympathetic and sacral parasympathetic - by way of a shared relay, the pelvic ganglion, conceived as an assemblage of sympathetic and parasympathetic neurons. Using single-cell RNA sequencing, we find that the mouse pelvic ganglion is made of four classes of neurons, distinct from both sympathetic and parasympathetic ones, albeit with a kinship to the former, but not the latter, through a complex genetic signature. We also show that spinal lumbar preganglionic neurons synapse in the pelvic ganglion onto equal numbers of noradrenergic and cholinergic cells, both of which therefore serve as sympathetic relays. Thus, the pelvic viscera receive no innervation from parasympathetic or typical sympathetic neurons, but instead from a divergent tail end of the sympathetic chains, in charge of its idiosyncratic functions.

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of ETS transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS::FLI1 and EWS::ERG fusions in Ewing sarcoma (EwS) and TMPRSS2::ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS::FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs) and are highly elevated in plasma of EwS patients with metastatic disease. High HSAT2,3 levels in EWS::FLI1 or ERG expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs which transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Evaluation of the role of AXL in fusion-positive pediatric rhabdomyosarcoma identifies the small-molecule inhibitor bemcentinib (BGB324) as potent chemosensitizer.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. Over 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response - and subsequent tumor recurrence - is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration and chemotherapy response, and whether targeting of AXL with small molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDXs), primary cultures and cell line models of RMS, consistent with its stem-cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacological blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner, and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong anti-tumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor bruden compared to single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in RMS patients.

Imaging characterization of paediatric tumours with the neurotrophic tyrosine receptor kinase fusion transcript.

OBJECTIVES: The neurotrophic tyrosine receptor kinase (NTRK) fusion transcript (FT) is a major genetic landmark of infantile fibrosarcoma (IFS) and cellular congenital mesoblastic nephroma (cCMN) but is also described in other tumours. The recent availability of NTRK-targeted drugs enhances the need for better identification. We aimed to describe the anatomic locations and imaging features of tumours with NTRK-FT in children. CASE SERIES: Imaging characteristics of NTRK-FT tumours of 41 children (median age: 4 months; 63% <1 year old; range: 0-188) managed between 2001 and 2019 were retrospectively analysed. The tumours were located in the soft tissues (n = 24, including 19 IFS), kidneys (n = 9, including 8 cCMN), central nervous system (CNS) (n = 5), lung (n = 2), and bone (n = 1). The tumours were frequently deep-located (93%) and heterogeneous (71%) with necrotic (53%) or haemorrhagic components (29%). Although inconstant, enlarged intratumoural vessels were a recurrent finding (70%) with an irregular distribution (63%) in the most frequent anatomical locations. CONCLUSION: Paediatric NTRK-FT tumours mainly occur in infants with very variable histotypes and locations. Rich and irregular intra-tumoural vascularization are recurrent findings. ADVANCES IN KNOWLEDGE: Apart from IFS of soft tissues and cCMN of the kidneys, others NTRK-FT tumours locations have to be known, as CNS tumours. Better knowledge of the imaging characteristics may help guide the pathological and biological identification.

PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution.

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

Fusion-negative rhabdomyosarcoma 3D organoids to predict effective drug combinations: A proof-of-concept on cell death inducers.

Rhabdomyosarcoma (RMS) is the main form of pediatric soft-tissue sarcoma. Its cure rate has not notably improved in the last 20 years following relapse, and the lack of reliable preclinical models has hampered the design of new therapies. This is particularly true for highly heterogeneous fusion-negative RMS (FNRMS). Although methods have been proposed to establish FNRMS organoids, their efficiency remains limited to date, both in terms of derivation rate and ability to accurately mimic the original tumor. Here, we present the development of a next-generation 3D organoid model derived from relapsed adult and pediatric FNRMS. This model preserves the molecular features of the patients' tumors and is expandable for several months in 3D, reinforcing its interest to drug combination screening with longitudinal efficacy monitoring. As a proof-of-concept, we demonstrate its preclinical relevance by reevaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on transcriptomic data exploitation.

Simple droplet microfluidics platform for drug screening on cancer spheroids.

3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipment for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplet microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorogenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from a patient-derived-xenografts (PDX). As compared to microtiter plates methods, our system reduces the initial number of cells up to 10 times and opens new avenues towards primary tumors drug screening approaches.

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups.

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.

A biobank of pediatric patient-derived-xenograft models in cancer precision medicine trial MAPPYACTS for relapsed and refractory tumors.

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient's tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies.

Sequential genomic analysis using a multisample/multiplatform approach to better define rhabdomyosarcoma progression and relapse.

The genomic spectrum of rhabdomyosarcoma (RMS) progression from primary to relapse is not fully understood. In this pilot study, we explore the sensitivity of various targeted and whole-genome NGS platforms in order to assess the best genomic approach of using liquid biopsy in future prospective clinical trials. Moreover, we investigate 35 paired primary/relapsed RMS from two contributing institutions, 18 fusion-positive (FP-RMS) and 17 fusion-negative RMS (FN-RMS) by either targeted DNA or whole exome sequencing (WES). In 10 cases, circulating tumor DNA (ctDNA) from multiple timepoints through clinical care and progression was analyzed for feasibility of liquid biopsy in monitoring treatment response/relapse. ctDNA alterations were evaluated using a targeted 36-gene custom RMS panel at high coverage for single-nucleotide variation and fusion detection, and a shallow whole-genome sequencing for copy number variation. FP-RMS have a stable genome with relapse, with common secondary alterations CDKN2A/B, MYCN, and CDK4 present at diagnosis and impacting survival. FP-RMS lacking major secondary events at baseline acquire recurrent MYCN and AKT1 alterations. FN-RMS acquire a higher number of new alterations, most commonly SMARCA2 missense mutations. ctDNA analyses detect pathognomonic variants in all RMS patients within our collection at diagnosis, regardless of type of alterations, and confirmed at relapse in 86% of FP-RMS and 100% FN-RMS. Moreover, a higher number of fusion reads is detected with increased disease burden and at relapse in patients following a fatal outcome. These results underscore patterns of tumor progression and provide rationale for using liquid biopsy to monitor treatment response.

Transmorph: a unifying computational framework for modular single-cell RNA-seq data integration.

Data integration of single-cell RNA-seq (scRNA-seq) data describes the task of embedding datasets gathered from different sources or experiments into a common representation so that cells with similar types or states are embedded close to one another independently from their dataset of origin. Data integration is a crucial step in most scRNA-seq data analysis pipelines involving multiple batches. It improves data visualization, batch effect reduction, clustering, label transfer, and cell type inference. Many data integration tools have been proposed during the last decade, but a surge in the number of these methods has made it difficult to pick one for a given use case. Furthermore, these tools are provided as rigid pieces of software, making it hard to adapt them to various specific scenarios. In order to address both of these issues at once, we introduce the transmorph framework. It allows the user to engineer powerful data integration pipelines and is supported by a rich software ecosystem. We demonstrate transmorph usefulness by solving a variety of practical challenges on scRNA-seq datasets including joint datasets embedding, gene space integration, and transfer of cycle phase annotations. transmorph is provided as an open source python package.

Neurotrophic tropomyosin receptor kinase (NTRK) fusion positive tumors: a historical cohort analysis.

BACKGROUND: NTRK gene fusions have been identified in various tumors; some requiring aggressive therapy and sometimes new TRK inhibitors (TRKi). We aimed to describe a national, unselected, retrospective, multicenter cohort. RESEARCH DESIGN AND METHODS: Patients were identified through the French sarcoma diagnostic laboratory at Institut Curie through samples analyzed by RT-qPCR or whole-transcriptome sequencing. RESULTS: From 2001 to 2019, 65 NTRK fusion tumors were identified within 2120 analyses (3.1%): 58 by RNA sequencing (including 20 after RT-qPCR analysis) and 7 exclusively by RT-qPCR. Of the 61 patients identified, 37 patients had infantile soft tissue or kidney fibrosarcomas (IFS), 15 other mesenchymal (Other-MT) and nine central nervous system (CNS) tumors. They encompassed 14 different tumor types with variable behaviors. Overall, 53 patients had surgery (3 mutilating), 38 chemotherapy (20 alkylating agents/anthracycline), 11 radiotherapy, two 'observation strategy' and 13 received TRKi. After a median follow-up of 61.0 months [range, 2.5-226.0], 10 patients died. Five-year overall survival is, respectively, 91.9% [95%CI, 83.5-100.0], 61.1% [95%CI, 34.2-100.0] and 64.8% [95%CI, 39.3-100.0] for IFS, Other-MT, and CNS groups. CONCLUSIONS: NTRK-fusion positive tumors are rare but detection is improved through RNA sequencing. TRKi could be considered at diagnosis for CNS NTRK-fusion positive tumors, some IFS, and Other-MT. TRIAL REGISTRATION: Not adapted.

Cell-Free DNA Extracted from CSF for the Molecular Diagnosis of Pediatric Embryonal Brain Tumors.

BACKGROUND: Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis. METHODS: The CSF cfDNA of pediatric patients with medulloblastoma (n = 18), ATRT (n = 3), ETMR (n = 1), CNS NB FOXR2 (n = 2) and pediatric EBT NOS (n = 1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA. RESULTS: 15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES (n = 15), a mean of 83 (range 1-160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility. CONCLUSION: CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.

Impact of age on survival according to molecular tumor findings in children and adolescents with soft-tissue and bone sarcoma: The BIOSCA project.

BACKGROUND: Adolescents (15-19 years) with sarcoma are known to have significantly worse survival than children (0-14 years). One possible reason may be that the adolescent sarcomas exhibit specific biological characteristics resulting in differences in clinical presentation and treatment resistance behaviors. The BIOSCA project aims to further explore these age-related differences in survival accounting for molecular tumor characteristic in children and adolescents with sarcoma. METHODS: A retrospective national population-based observational study with documented somatic genetic analyses was conducted between 2011 and 2016 of all patients aged from 0 to 17 years with a diagnosis of sarcoma using the National Registry of Childhood Cancers Database. RESULTS: A total of 1637 children (0-9years: 40%), preadolescents (10-14years: 35%) and adolescents (15-17 years: 25%) with a diagnosis of bone (N = 845) or soft-tissue (N = 792) sarcoma were included. Adolescents had significantly worse outcome for undifferentiated small round cell sarcoma (USRCS), alveolar rhabdomyosarcoma (ARMS), and epithelioid sarcoma. Five-year overall survivals were worse among CIC-rearranged USRCS cases (47% [95%CI:21-69]) as compared to other USRCS, and PAX3::FOXO1 ARMS patients (44% [95%CI:32-55]) as compared to other ARMS. Adjusting for stage and genomic-profiling status, adolescents with USRCS were 1.6-fold more likely to die than children (P = 0.05), while the difference in survival between age of ARMS patients was weaken. Indeed, the prevalence of PAX3::FOXO1 increased significantly with age. CONCLUSION: Age was an independent prognostic factor of outcome only in patients with USRCS, while the association between age and survival of patients with ARMS could be partly explained by differences in prevalence of PAX3::FOXO1.

Adaptive nanopore sequencing to determine pathogenicity of BRCA1 exonic duplication.

BRCA1 and BRCA2 are tumour suppressor genes that have been characterised as predisposition genes for the development of hereditary breast and ovarian cancers among other malignancies. The molecular diagnosis of this predisposition syndrome is based on the detection of inactivating variants of any type in those genes. But in the case of structural variants, functional consequences can be difficult to assess using standard molecular methods, as the precise resolution of their sequence is often impossible with short-read next generation sequencing techniques. It has been recently demonstrated that Oxford Nanopore long-read sequencing technology can accurately and rapidly provide genetic diagnoses of Mendelian diseases, including those linked to pathogenic structural variants. Here, we report the accurate resolution of a germline duplication event of exons 18-20 of BRCA1 using Nanopore sequencing with adaptive sampling target enrichment. This allowed us to classify this variant as pathogenic within a short timeframe of 10 days. This study provides a proof-of-concept that nanopore adaptive sampling is a highly efficient technique for the investigation of structural variants of tumour suppressor genes in a clinical context.

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma.

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

PAX-FOXO1 fusion status in children and adolescents with alveolar rhabdomyosarcoma: Impact on clinical, pathological, and survival features.

BACKGROUND: Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer and cases with fusion PAX3-FOXO1 and PAX7-FOXO1 seem to have a poor prognosis. The aim is to evaluate whether PAX-FOXO1 alterations influence clinical outcome in childhood and adolescence population with ARMS. PROCEDURE: A population-based study was conducted between 2011 and 2016 in patients less than 17 years with a diagnosis of ARMS. Overall survival (OS) depending on fusion status with clinical factors was analyzed. RESULTS: Out of 111 ARMS patients recorded in the French National Childhood Cancer Registry during the 2011-2016 period, 61% expressed PAX3-FOXO1, 15% expressed PAX7-FOXO1, 13% were FOXO1 fusion-positive without PAX specification, and 7% were PAX-FOXO1 negative (n = 4 missing data). Compared to patients with PAX7-FOXO1 positive ARMS, those with PAX3-FOXO1 positive tumor were significantly older (10-17 years: 57.4% vs. 29.4%), and had more often a metastatic disease (54.4% vs. 23.5%). Poorer 5-year OS for patients with PAX3-FOXO1 and PAX not specified FOXO1-positive tumor were observed (44.0% [32.0-55.4] and 35.7% [13.1-59.4], respectively). After adjustment for stage at diagnosis, patients with positive tumor for PAX3-FOXO1 were 3.6-fold more likely to die than those with positive tumor for PAX7-FOXO1. CONCLUSION: At the population level, PAX3-FOXO1 was associated with a significant higher risk of death compared to PAX7-FOXO1-positive and PAX-FOXO1-negative tumors, and could explain poorer 5-year OS observed in adolescence population diagnosed with ARMS. A continuous risk score derived from the combination of clinical parameters with PAX3-FOXO1 fusion status represents a robust approach to improving current risk-adapted therapy for ARMS.

Alarm communication predates eusociality in termites.

Termites (Blattodea: Isoptera) have evolved specialized defensive strategies for colony protection. Alarm communication enables workers to escape threats while soldiers are recruited to the source of disturbance. Here, we study the vibroacoustic and chemical alarm communication in the wood roach Cryptocercus and in 20 termite species including seven of the nine termite families, all life-types, and all feeding and nesting habits. Our multidisciplinary approach shows that vibratory alarm signals represent an ethological synapomorphy of termites and Cryptocercus. In contrast, chemical alarms have evolved independently in several cockroach groups and at least twice in termites. Vibroacoustic alarm signaling patterns are the most complex in Neoisoptera, in which they are often combined with chemical signals. The alarm characters correlate to phylogenetic position, food type and hardness, foraging area size, and nesting habits. Overall, species of Neoisoptera have developed the most sophisticated communication system amongst termites, potentially contributing to their ecological success.