Genetic Tracing Research Articles

Investigation of an outbreak of hepatitis E virus 4d reveals the importance of good kitchen hygiene

In July–August 2022, an outbreak of hepatitis E was reported following a funeral dinner in a village of Yantai, June 2022. The aim of this study was to characterise the outbreak, search for more cases, and investigate the risk factors to help prevent hepatitis E infection in the future. A 1:2 case-control study was used to investigate the suspected causative food, and HEV antibody testing and genetic tracing were performed on clinical and environmental samples, and HEV antibody of 69 healthy people who did not attend the dinner in the same village were tested. A total of 80 people were exposed to this outbreak and 18 (22.5 %, 18/80) had acute infections, which was much higher than the positivity rate of healthy people of the same age group in the village (2.9 %, 2/69). Multifactorial analysis showed that the infection was significantly associated with the consumption of cucumber in sauce (aOR = 4.44, 95%CI = 1.23–16.06). Further investigation revealed that there was a mixing of pots and pans for washing raw pork products with those for cold meals during dish preparation. A whole genome sequence of HEV was obtained from sera of cases and from an environmental sample from the pork supplier's refrigerator. All sequences were typed as HEV-4d. This foodborne outbreak was most likely caused due to a failure in kitchen hygiene to differentiate between raw and cooked pots and pans during dish preparation, resulting in cross-contamination from defrosted pork to cold dishes. Our findings emphasize the importance of education of food hygiene, especially in remote areas.

Prenatal diagnosis and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome.

The clinical phenotypes of 16p13.11 microduplication syndrome have been extensively reported in previous studies, mostly about adults and children, with limited information available on fetal cases. This study aims to explore the genotype-phenotype correlation of fetuses with 16p13.11 microduplication syndrome and analyze the characteristics of prenatal diagnosis indications and provide clinical information for prenatal and postnatal genetic counseling. We conducted a retrospective analysis of 3,451 pregnant women who underwent invasive prenatal diagnosis for SNP array between January 2018 and December 2022 at the Jinan Maternal and Child Health Hospital. Descriptive statistical analysis was performed on the prenatal diagnosis indications, pedigree analysis, pregnancy outcomes and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome. SNP array revealed that 15 fetuses had duplications in the 16p13.11 region with varying prenatal diagnosis indications. Among the cases, 6/15 exhibited ultrasound abnormalities, 5/15 had abnormal chromosomal copy number variations as indicated by non-invasive prenatal testing (NIPT), one case involved advanced maternal age, and 3/15 had other abnormalities. 16p13.11 microduplication syndrome was closely related to ultrasound abnormalities, especially structural abnormalities and soft marker anomalies (abnormal ultrasonic soft indicators), while the indication of NIPT could improve the detection rate of copy number variations (CNVs) in this region. Only 7/15 fetuses underwent pedigree verification, with one case of de novo 16p13.11 microduplication, and the others inherited from one parent. Pregnancy was terminated in 2/15 cases and the outcome of one case is unknown due to loss to follow-up. Among the remaining cases, only one case exhibited a ventricular septal defect, while another presented with omphalocele. No other obvious abnormalities were reported postnatally. The prenatal phenotypes of fetuses with 16p13.11 microduplication were highly associated with ultrasound abnormalities but lacked specificity. Comprehensive genetic tracing, outcome analysis, and follow-up are essential for providing accurate prenatal and postnatal genetic counseling.

Intercellular genetic tracing by alternative synthetic Notch signaling

Intercellular genetic tracing by alternative synthetic Notch signaling

Participation of ventricular trabeculae in neonatal cardiac regeneration leads to ectopic recruitment of Purkinje-like cells.

Unlike adult mammals, newborn mice can regenerate a functional heart after myocardial infarction; however, the precise origin of the newly formed cardiomyocytes and whether the distal part of the conduction system (the Purkinje fiber (PF) network) is properly formed in regenerated hearts remains unclear. PFs, as well as subendocardial contractile cardiomyocytes, are derived from trabeculae, transient myocardial ridges on the inner ventricular surface. Here, using connexin 40-driven genetic tracing, we uncover a substantial participation of the trabecular lineage in myocardial regeneration through dedifferentiation and proliferation. Concomitantly, regeneration disrupted PF network maturation, resulting in permanent PF hyperplasia and impaired ventricular conduction. Proliferation assays, genetic impairment of PF recruitment, lineage tracing and clonal analysis revealed that PF network hyperplasia results from excessive recruitment of PFs due to increased trabecular fate plasticity. These data indicate that PF network hyperplasia is a consequence of trabeculae participation in myocardial regeneration.

The cell fates of intermediate cell population in prostate development

Organ development, regeneration and cancer initiation are typically influenced by the proliferation and lineage plasticity of tissue-specific stem cells. Prostate intermediate cells, which exhibit characteristics of both basal and luminal cells, are prevalent in pathological states and during organ development. However, the identity, fate and function of these intermediate cells in prostate development are not well understood. Through single-cell RNA-seq analysis on neonatal urogenital sinus tissue, we identified intermediate cells exhibiting stem cell potential. A notable decline in the population of intermediate cells was observed during prostate development. Prostate intermediate cells were specifically labeled in early and late postnatal development by the enhanced dual-recombinase-mediated genetic tracing systems. Our findings revealed that these cells possess significant stem cell capabilities as demonstrated in organoid formation and cell fate mapping assays. These intermediate cells also exhibited intrinsic bipotential properties, enabling them to differentiate into both basal and luminal cells. Additionally, we discovered a novel transition from intermediate cell expressing neuroendocrine markers to neuroendocrine cell during prostate development. This study highlights intermediate cells as a crucial stem cell population and enhances our understanding of their role in prostate development and the plasticity of prostate cancer lineage.

Neural crest and sons: role of neural crest cells and Schwann cell precursors in development and gland embryogenesis.

In this review, we consider the multipotency of neural crest cells (NCCs), Schwann cell precursors (SCPs), and their role in embryogenesis base on genetic tracing and knock out model animals and single cell transcriptomic analysis. In particular, we summarize and analyze data on the contribution of NCCs and SCPs to the gland development and functions.

In situ reprogramming of cardiac fibroblasts into cardiomyocytes in mouse heart with chemicals.

Cardiomyocytes are terminal differentiated cells and have limited ability to proliferate or regenerate. Condition like myocardial infarction causes massive death of cardiomyocytes and is the leading cause of death. Previous studies have demonstrated that cardiac fibroblasts can be induced to transdifferentiate into cardiomyocytes in vitro and in vivo by forced expression of cardiac transcription factors and microRNAs. Our previous study have demonstrated that full chemical cocktails could also induce fibroblast to cardiomyocyte transdifferentiation both in vitro and in vivo. With the development of tissue clearing techniques, it is possible to visualize the reprogramming at the whole-organ level. In this study, we investigated the effect of the chemical cocktail CRFVPTM in inducing in situ fibroblast to cardiomyocyte transdifferentiation with two strains of genetic tracing mice, and the reprogramming was observed at whole-heart level with CUBIC tissue clearing technique and 3D imaging. In addition, single-cell RNA sequencing (scRNA-seq) confirmed the generation of cardiomyocytes from cardiac fibroblasts which carries the tracing marker. Our study confirms the use of small molecule cocktails in inducing in situ fibroblast to cardiomyocyte reprogramming at the whole-heart level and proof-of-conceptly providing a new source of naturally incorporated cardiomyocytes to help heart regeneration.

Clonal dynamics of surface-driven growing tissues.

The self-organization of cells into complex tissues relies on a tight coordination of cell behavior. Identifying the cellular processes driving tissue growth is key to understanding the emergence of tissue forms and devising targeted therapies for aberrant growth, such as in cancer. Inferring the mode of tissue growth, whether it is driven by cells on the surface or by cells in the bulk, is possible in cell culture experiments but difficult in most tissues in living organisms (in vivo). Genetic tracing experiments, where a subset of cells is labeled with inheritable markers, have become important experimental tools to study cell fate in vivo. Here we show that the mode of tissue growth is reflected in the size distribution of the progeny of marked cells. To this end, we derive the clone size distributions using analytical calculations in the limit of negligible cell migration and cell death, and we test our predictions with an agent-based stochastic sampling technique. We show that for surface-driven growth the clone size distribution takes a characteristic power-law form with an exponent determined by fluctuations of the tissue surface. Our results propose a possible way of determining the mode of tissue growth from genetic tracing experiments.

Genetic tracing of the illegal trade of the white-bellied pangolin (Phataginus tricuspis) in western Central Africa

The white-bellied pangolin is subject to intense trafficking, feeding both local and international trade networks. In order to assess its population genetics and trace its domestic trade, we genotyped 562 pangolins from local to large bushmeat markets in western central Africa. We show that the two lineages described from the study region (WCA and Gab) were overlapping in ranges, with limited introgression in southern Cameroon. There was a lack of genetic differentiation across WCA and a significant signature of isolation-by-distance possibly due to unsuspected dispersal capacities involving a Wahlund effect. We detected a c. 74.1–82.5% decline in the effective population size of WCA during the Middle Holocene. Private allele frequency tracing approach indicated up to 600 km sourcing distance by large urban markets from Cameroon, including Equatorial Guinea. The 20 species-specific microsatellite loci provided individual-level genotyping resolution and should be considered as valuable resources for future forensic applications. Because admixture was detected between lineages, we recommend a multi-locus approach for tracing the pangolin trade. The Yaoundé market was the main hub of the trade in the region, and thus should receive specific monitoring to mitigate pangolins’ domestic trafficking. Our study also highlighted the weak implementation of CITES regulations at European borders.

Dual recombination-mediated genetic tracing reveals the origin of alveolar stem cells during lung repair and regeneration

Dual recombination-mediated genetic tracing reveals the origin of alveolar stem cells during lung repair and regeneration

Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes.

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

Dual Genetic Tracing Reveals the Origin of Alveolar Stem Cells after Lung Injury

Dual Genetic Tracing Reveals the Origin of Alveolar Stem Cells after Lung Injury

The application of RAGA-PP model in environmental accounting information disclosure of enterprises in heavy pollution industries

The global concern for environmental protection has driven the demand for corporate environmental accounting information, especially in heavily polluting industries. Currently, there are deficiencies in China's research on environmental accounting information and its institutional development, and there is an urgent need for improved evaluation systems and models. The study proposes the RAGA-PP model, which is an environmental accounting information disclosure evaluation model that combines accelerated genetic algorithm and projection tracing algorithm. The model is based on 28 secondary evaluation indicators and aims to optimize the projection tracing through the accelerated genetic algorithm with real number coding to improve the stability and accuracy of data analysis. The experiment proves that the evaluation error value of the RAGA-PP model is 3.45 % on average, which is significantly lower compared to 9.55 % of the ant colony algorithm. The results show that the RAGA-PP model has significant advantages in accurately evaluating the quality of corporate environmental information disclosure, which confirms its application value in environmental accounting information disclosure in heavy pollution industries. The purpose of the study is to promote the improvement of the environmental accounting information disclosure system and to provide support for enhancing corporate environmental awareness and social responsibility.

Choroid plexus mast cells drive tumor-associated hydrocephalus

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH invivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

Abstract 14239: Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve

Background: Extracellular matrix (ECM) is involved in aortic valve development and aortic valve anomaly. ADAMTS gene mediated ECM dysfunction was reported to be related to aortic valve diseases. Hypothesis: The ADAMTS gene family members can contribute to aortic valve diseases. Methods: Morpholino based adamts gene-targeted phenotype-screening of outflow tract anomaly in zebrafish and the 19 ADAMTS family gene-targeted exon sequencing on a cohort BAV patients (304 BAV and 301 normal controls) were performed. Accordingly, both specific genetic tracing and different gene modified mouse models were generated to evaluate the role of specific genes in the aortic valve formation process. Movat’s pentachrome staining was used to describe the altered ECM maturation process. Bulk RNA-seq was performed on both mouse heart tissue and human iPSC-induced ECs to explore the potential molecular pathways, which was further confirmed in ex vivo embryonic outflow tract tissue culture studies. Results: We identified some novel adamts genes that resulted in cardiac outflow valve defects in zebrafish, while exon sequencing identified ADAMTS16 p.H357Q variant in an inherited BAV family. The generated Ad amts16-Cre; Rosa26-tdTomato mice showed Adamts16 -expressing cells were primarily restricted to valvular endothelial cells (VECs) at E12.5 and then expressed in valvular mesenchymal cells (VICs) at E13.5. Both Adamts16 +/- and Adamts16 +/H355Q mice exhibited an R-NC-type BAV phenotype, with progressive aortic valve thickening. Conditional Adamts16 mutant mouse models demonstrated that endothelial lineage cell deficiency in Adamts16 recapitulated the BAV phenotype, which was associated with worse ECM deposition without ECM trilaminar structure. Bulk RNA sequencing unveiled enhanced FAK signaling, which was accompanied by elevated fibronectin levels. Both in vitro iPSC-ECs culture and ex vivo embryonic tissue explant studies validated the altered FAK signaling resulted in exaggerated cell proliferation. Conclusions: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

Abstract 18042: Induction of Cardiac Reprogramming Following Injury Through Delivery of a Single Factor, PHF7

Direct reprogramming of resident cardiac fibroblasts into cardiomyocytes is an attractive strategy to restore cardiac function following myocardial infarction (MI). However, canonical and extensively studied reprogramming factors (e.g. Gata4, Mef2c, Tbx5) have shown to be ineffective in reprogramming adult human fibroblasts. Furthermore, current human cocktails require an excess of factors, limiting the clinical translation and safety of this technology. Recently, we undertook a large screen of gene regulatory factors and identified the epigenetic factor PHF7 as the most potent activating factor of adult human fibroblast reprogramming, and described its ability to bind and modify chromatin accessibility at cardiac super enhancers. In this manuscript, we describe the ability of PHF7 to activate adult reprogramming in vitro in absence of canonical reprogramming factors, with PHF7, MEF2C, and TBX5 (PMT) or PHF7 and TBX5 (PT) alone. These minimalist PHF7 cocktails induced broad and unique cardiac transcriptomes in comparison to canonical cocktails. Importantly, when overexpressed in isolation in fibroblasts, PHF7 strongly activated expression of several cardiac transcription factors, establishing its role as a cardiac master regulator. When delivered to the mouse heart following MI, minimalist PHF7 cocktails were found to significantly recover cardiac function and decrease fibrotic scar formation. Interestingly, delivery of PHF7 alone following MI was sufficient to improve cardiac function and induce remodeling through activation of bona fide reprogramming events in vivo as determined by genetic tracing of the Postn MCM lineage. Here, we report for the first time the ability of a single reprogramming factor, PHF7, to induce fibroblast to cardiomyocyte reprogramming and recover cardiac function following injury in vivo.

FRI272 Sex-Specific Role Of ProkR2 Neurons Of The Posterior Amygdala In The Integration Of Odors And Neuroendocrine Responses

Abstract Disclosure: B. Cisneros-Larios: None. C.F. Elias: None. Kallmann Syndrome (KS) is characterized by abnormal development of the reproductive axis and olfactory bulb dysgenesis. Genetic studies have revealed that Kallmann Syndrome is caused by loss-of-function mutations in several genes including the prokineticin receptor 2 (PROKR2) gene. Prokr2 null mice exhibit the same phenotype of KS patients. While the role of PROKR2 during development has been linked with GnRH neuron migration, limited data exists on PROKR2 neurons in adult reproduction. PROKR2 mRNA are highly expressed in reproductive control sites. Furthermore, our lab previously found sexually dimorphic expression of PROKR2 mRNA and Prokr2-Cre GFP+ cells in several reproductive control sites. The posterior amygdala had higher PROKR2 expression in males compared to female mice. More recently, we identified expression of sex steroid receptors in PROKR2 expressing cells of the PA. The amygdala is an important site of socio-sexual inputs and reproductive neuroendocrine responses in rodents and primates, including humans. We hypothesize Prokr2-Cre neurons in the PA have a role in male reproductive function. Using genetic tracing techniques, we mapped PA Prokr2-Cre neuronal projections in male mice and found dense innervation to reproductive control sites such as the medial preoptic area and the ventral premammillary nucleus. We found activation of these neurons, via fos expression, after opposite sex soiled bedding exposure (olfactory cues). Lastly, selective activation, using chemogenetic technology DREADDs, of PA Prokr2-Cre cells in males can induce a rise in circulating luteinizing hormone. These data suggest PA Prokr2-Cre cells have a role in pathways integrating olfactory cues and reproductive endocrine responses (e.g., aggression, mating behavior). We expect our studies will contribute to the understanding of the role of PROKR2 neurons in adult reproduction and reproductive deficits associated with PROKR2 mutations. Presentation: Friday, June 16, 2023

Spatial-temporal pattern evolution and geological influence factors analysis of ecological vulnerability in Western Sichuan mountain region

Compared to plains, hills and basins, mountainous areas with complex topography are more prone to ecological and environment vulnerability to climate change, human interference and geological disasters. This type of region is usually located in abrupt transitional zones, which marked by active neotectonic movements, high-incidence mountain disasters, abundant available resources, slower economic development, diverse eco-environment and insufficient cultivated land resources. Therefore, it is of great significance to determ the vulnerability status and spatial–temporal change process of the eco-environment in mountainous area with complex topography. In this study, data derived from vegetation, topography, soil, meteorological and human social factors were used to develop an evaluation index system that can reflect the characteristics of the eco-environment in the mountain area. Employing the Genetic Projection Tracing Model (RAGA-PPM), we developed a framework for assessing the vulnerability of mountainous areas with complex terrains and applied it to the western Sichuan mountain region, China. In addition, the relationship between the geological factors and the vulnerability of eco-environment was quantitatively analyzed by using the geographic detector. The results indicate that (1) The overall vulnerability was medium and the spatial distribution was higher in the central and eastern parts and lower in the surrounding areas. The area with high vulnerability accounted for 27.9%. (2) The overall degree of vulnerability increased from2010 to 2020, the eco-environment experienced a rapid deterioration from 2010 to 2015, followed by a slower improvement from 2015 to 2020. (3) Correlations were minimal between vulnerability results and fault density and earthquake dynamic peak acceleration, the influence of other geological factors ranged from 10% to 25.8%, indicating a nonlinear correlation. (4) This study reveals that the ecological vulnerability of mountain areas was the effect of multiple human and natural factors, and there was a certain correlation between geological environment and vulnerability distribution. These findings can assist decision-makers in providing a scientific basis for managing and developing the ecological environment in mountainous regions.

OS07.7.A GLIOBLASTOMA HYBRID CELL STATE CONVEYS RESISTANCE TO CONVENTIONAL THERAPIES

Abstract BACKGROUND Glioblastoma (GB) is a deadly disease and no therapeutic improvements have been made in the last 20 years. High phenotypic plasticity has been recognized as a major obstacle to the efficient treatment of GB. Single-cell sequencing revealed the coexistence of four different cell states within the same tumor. The transition from one subtype (or state) to another, such as from proneural (PN) to mesenchymal (MES), has been suggested as a mechanism for resistance to therapies. However, there is no prognostic value of the current GB states for patients, meaning that mechanisms of resistance may not lie in hardwire identities but in the transition from one subtype to another. Therefore, we investigated the role of the hybrid cell states in GB resistance to therapy. MATERIAL AND METHODS To dynamically follow spatiotemporal changes in GB plasticity in real time, we transduced patient-derived GB cells (patient-derived cell lines with different mutational and phenotypic landscapes) using two specific GB-subtype fluorescent synthetic genetic tracing cassettes. To follow the hybrid cell state dynamics and features, we functionally and transcriptionally investigated GB cells labeled with activated reporters for both the PN and MES subtypes. Using a combination of cytofluorimetry, live imaging, preclinical models, bulk and single-cell RNA-sequencing, we visualized the single-cell GB plasticity and studied the changes in the hybrid state subpopulation proportion over time and under therapeutic stress. RESULTS We discovered that the PN/MES hybrid state (GB-hybrid) has a definite molecular and phenotypic identity and that it is strongly induced after conventional therapies. The GB-hybrid cell state is highly resistant to therapy and more proliferative. Importantly, when implanted in mice the GB-hybrid cells were more aggressive than the rest of the cells, which is consistent with the marked prognostic power for the GB-hybrid geneset signature when tested in the TCGA database. Mechanistically, we showed that GB-hybrid cells were characterized by broad chromatin remodeling, an important increase in overall mRNA quantity per cell and intensification of the nuclear import/export machinery. CONCLUSION Here, we present a novel GB transition cell state between PN and MES subtypes/states. The GB-hybrid is at the same time highly proliferative and resistant to therapy, making it an interesting target to slow down GB recurrence after therapy.