An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.

Mitochondrially-associated actin waves maintain organelle homeostasis and equitable inheritance.

First identified in dividing cells as revolving clusters of actin filaments, these are now understood as mitochondrially-associated actin waves that are active throughout the cell cycle. These waves are formed from the polymerization of actin onto a subset of mitochondria. Within minutes, this F-actin depolymerizes while newly formed actin filaments assemble onto neighboring mitochondria. In interphase, actin waves locally fragment the mitochondrial network, enhancing mitochondrial content mixing to maintain organelle homeostasis. In dividing cells actin waves spatially mix mitochondria in the mother cell to ensure equitable partitioning of these organelles between daughter cells. Progress has been made in understanding the consequences of actin cycling as well as the underlying molecular mechanisms, but many questions remain, and here we review these elements. Also, we draw parallels between mitochondrially-associated actin cycling and cortical actin waves. These dynamic systems highlight the remarkable plasticity of the actin cytoskeleton.

Invasive fungal disease in children with solid tumors: An Australian multicenter 10-year review.

Invasive fungal disease (IFD) occurs less frequently during treatment for solid compared to hematological malignancies in children, and risk groups are poorly defined. Retrospective national multicenter cohort data (2004-2013) were analyzed to document prevalence, clinical characteristics, and microbiology of IFD. Amongst 2067 children treated for solid malignancy, IFD prevalence was 1.9% overall and 1.4% for proven/probable IFD. Of all IFD episodes, 42.5% occurred in patients with neuroblastoma (prevalence 7.0%). Candida species comprised 54.8% of implicated pathogens in proven/probable IFD. In children with solid tumors, IFD is rare, and predominantly caused by yeasts.Routine prophylaxis may not be warranted.

Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis.

Objectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples. Methods: In this study, we conducted a high-dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non-age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias. Results: We observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57- NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A-HLA-E interaction may play a role in modifying NK cell responses to leukaemia cells. Conclusion: We provide an in-depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.

Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Incidence and survival for childhood cancer by endorsed non-stage prognostic indicators in Australia.

BACKGROUND: An international expert panel recently recommended 15 'non-stage prognostic indicators' (NSPIs) across eight childhood cancers, classified as essential or additional, for collection in population-based cancer registries. We aimed to describe the incidence distribution and survival of each of these NSPIs. PROCEDURES: Cases were extracted from the Australian Childhood Cancer Registry. The study cohort (n = 4187) comprised all children aged under 15 years diagnosed with an eligible cancer between 2010 and 2018, with follow-up until 31 December 2020. NSPI data were collected directly from each patient's medical records. Differences in 5-year relative survival were assessed using multivariable flexible parametric models, adjusted for sex and age group at diagnosis. RESULTS: The availability of data varied, exceeding 85% for all essential NSPIs apart from histologic subtype for Wilms tumours (69%) and lineage for acute lymphoblastic leukaemia (78%). Information on additional NSPIs tended to be recorded less often, particularly cytogenetic subtype for non-alveolar rhabdomyosarcoma (28%) and astrocytoma (4%). Eight NSPIs exhibited a significant difference in survival, with the largest disparity occurring among children with astrocytoma according to tumour grade (5-year relative survival of 18% for grade IV disease compared with 99% for grade I disease; p < .001). CONCLUSIONS: Our findings demonstrate that most of the recommended NSPIs can be retrieved from medical records in Australia in recent years, allowing the capability of assessing survival within patient subgroups of clinical interest. Reporting of NSPI data has the capability to inform local and global understanding of population-level disparities in childhood cancer survival.

Biologic and clinical features of childhood gamma delta T-ALL: identification of STAG2/LMO2 γδ T-ALL as an extremely high risk leukemia in the very young.

PURPOSE: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease. METHODS: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts. RESULTS: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping resulting in deregulation of gene expression associated with T-cell differentiation. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine. CONCLUSION: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL. SUPPORT: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

Reproducible Bioinformatics Analysis Workflows for Detecting IGH Gene Fusions in B-Cell Acute Lymphoblastic Leukaemia Patients.

B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by diverse genomic alterations, the most frequent being gene fusions detected via transcriptomic analysis (mRNA-seq). Due to its hypervariable nature, gene fusions involving the Immunoglobulin Heavy Chain (IGH) locus can be difficult to detect with standard gene fusion calling algorithms and significant computational resources and analysis times are required. We aimed to optimize a gene fusion calling workflow to achieve best-case sensitivity for IGH gene fusion detection. Using Nextflow, we developed a simplified workflow containing the algorithms FusionCatcher, Arriba, and STAR-Fusion. We analysed samples from 35 patients harbouring IGH fusions (IGH::CRLF2 n = 17, IGH::DUX4 n = 15, IGH::EPOR n = 3) and assessed the detection rates for each caller, before optimizing the parameters to enhance sensitivity for IGH fusions. Initial results showed that FusionCatcher and Arriba outperformed STAR-Fusion (85-89% vs. 29% of IGH fusions reported). We found that extensive filtering in STAR-Fusion hindered IGH reporting. By adjusting specific filtering steps (e.g., read support, fusion fragments per million total reads), we achieved a 94% reporting rate for IGH fusions with STAR-Fusion. This analysis highlights the importance of filtering optimization for IGH gene fusion events, offering alternative workflows for difficult-to-detect high-risk B-ALL subtypes.

Paediatric B lymphoblastic leukaemia with hyperdiploidy and a false-positive KMT2A fluorescence in situ hybridization result.

The dramatic improvement in the event-free survival of paediatric B-lymphoblastic leukaemia (B-ALL) has led to risk-stratified treatment. Through a combination of clinical features, cytogenetic abnormalities and assessment of treatment response, patients are stratified to receive different intensities of therapy. The presence of high hyperdiploidy (>50 chromosomes) is considered a favourable genetic feature. Conversely, KMT2A fusion genes in B-ALL are associated with a poor prognosis, resulting in intensification of treatment. We present a seven-year-old female with B-ALL, a high hyperdiploid karyotype (56 chromosomes) and KMT2A rearrangement detected on FISH, but with no productive fusion identified. Single nucleotide polymorphism (SNP) array suggested the KMT2A rearrangement was due to chromosome 11 chromothripsis. Subsequent targeted RNA fusion panel and whole transcriptomic sequencing (mRNA-seq) did not detect an expressed KMT2A fusion. Differential expression analyses of the mRNA-seq data led to clustering of this case with other hyperdiploid cases, consistent with the hyperdiploid cytogenetic results. Given the additional intensity and potential toxicity of high-risk treatment, unusual findings by chromosome analysis, FISH and/or chromosomal microarray should prompt consideration of testing for a KMT2A fusion by another method to avoid misclassification.

Temporal changes in childhood cancer incidence and survival by stage at diagnosis in Australia, 2000-2017.

BACKGROUND: The Toronto Paediatric Cancer Stage Guidelines are a compendium of staging systems developed to facilitate collection of consistent and comparable data on stage at diagnosis for childhood cancers by cancer registries. MATERIAL AND METHODS: This retrospective, observational cohort study investigated changes in stage-specific incidence and survival for children diagnosed between 2000-2008 compared to 2009-2017 using the population-based Australian Childhood Cancer Registry. Information on mortality for each patient was available to 31st December 2020. Shifts in incidence by stage were evaluated using chi-square tests, and differences in stage-specific five-year observed survival for all causes of death over time were assessed using flexible parametric models. RESULTS: Stage was assigned according to the Toronto Guidelines for 96% (n = 7944) of the total study cohort (n = 8292). Changes in the distribution of incidence by stage between the two diagnosis periods were observed for retinoblastoma, with stage 0 increasing from 26% to 37% of cases (p = 0.02), and hepatoblastoma, with metastatic disease increasing from 22% to 39% of cases (p = 0.04). There were large gains in stage-specific survival over time for stage IV rhabdomyosarcoma (five-year adjusted mortality hazard ratio for 2009-2017 compared to 2000-2008 of 0.38, 95% CI 0.19-0.77; p = 0.01), stage M3 for medulloblastoma (HR = 0.41, 95% CI 0.21-0.79; p = 0.01) and metastatic neuroblastoma excluding stage MS (HR = 0.61, 95% CI 0.44-0.84; p < 0.01). CONCLUSION: These results indicate that improvements in childhood cancer survival in Australia are most likely due to refined management rather than changes in stage at diagnosis, particularly for metastatic solid tumours. Wide international uptake of the Toronto Guidelines will allow comprehensive evaluation of differences in survival between countries.

Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Childhood cancer survival and avoided deaths in Australia, 1983-2016.

BACKGROUND: Large improvements in childhood cancer survival have been reported over recent decades. Data from cancer registries have the advantage of providing a 'whole of population' approach to gauge the success of cancer control efforts. OBJECTIVES: The aim of this study was to investigate recent survival estimates for children diagnosed with cancer Australia and to examine the extent of changes in survival over the last 35 years. For the first time, we also estimated the number of deaths among Australian children that were potentially avoided due to improvements in survival. METHODS: A retrospective, population-based cohort study design was used. Case information was extracted from the Australian Childhood Cancer Registry for 1983-2016, with follow-up to 31 December 2017. Eligible children were aged 0-14 with a basis of diagnosis other than autopsy or death certificate only. Five-year relative survival was calculated using the semi-complete cohort method for three diagnosis periods (1983-1994, 1995-2006 and 2007-2016), and changes in survival over time were assessed via flexible parametric models. Avoided deaths within 5 years for those diagnosed between 1995 and 2016 were estimated under the assumption that survival rates remained the same as for 1983-1994. RESULTS: Overall 5-year survival within the study cohort (n = 20,871) increased from 72.8% between 1983 and1994 to 86.1% between 2007 and 2016, equating to an adjusted excess mortality hazard ratio of 1.82 (95% confidence interval 1.67, 1.97). Most cancers showed improvements in survival; other gliomas, hepatoblastoma and osteosarcoma were exceptions. Among children diagnosed between 1995 and 2016, 38.7% of expected deaths within 5 years of diagnosis (n = 1537 of 3970) were avoided due to temporal improvements in survival. CONCLUSIONS: Survival for childhood cancer has continued to improve over recent years, thanks mainly to ongoing progress in treatment development combined with improved supportive care. Providing innovative measures of survival, such as avoided deaths, may assist with understanding outcome data produced by cancer registries.

Structural Diversity within the Endoplasmic Reticulum-From the Microscale to the Nanoscale.

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions.

BACKGROUND: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. METHODS: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. RESULTS: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001). CONCLUSIONS: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Changes in cancer incidence and survival among Aboriginal and Torres Strait Islander children in Australia, 1997-2016.

BACKGROUND: This study reports cancer incidence and survival among Aboriginal and Torres Strait Islander children and other Australian children, and assesses changes over time. PROCEDURE: Data were from the population-based Australian Childhood Cancer Registry. The study comprised children aged under 15 diagnosed between 1997 and 2016 and with mortality follow-up until 31 December 2017. Incidence trends were analysed using JoinPoint regression. Five-year cancer-specific survival was calculated using the semi-complete approach with survival comparisons made using multivariable flexible parametric models. RESULTS: Aboriginal and Torres Strait Islander children accounted for 506 of 13,299 eligible cases (3.8%). Incidence rates for Aboriginal and Torres Strait Islander children across the study period increased by 2.3% annually (95% confidence interval [CI]: +0.6% to +4.0%) and for other Australian children increased by 0.6% annually (95% CI: +0.3% to +0.9%; p = .05). Nonetheless, cancer incidence was consistently lower for Aboriginal and Torres Strait Islander children, with an incidence rate ratio of 0.73 (95% CI: 0.62-0.85; p < .01) between 2012 and 2016. Survival for Aboriginal and Torres Strait Islander children with solid tumours was 70.6% (95% CI: 62.5%-77.3%) and for other Australian children was 83.5% (95% CI: 82.3%-84.7%; p < .01), with indications of this difference diminishing in recent years. CONCLUSIONS: Improvements in identification, particularly in urban areas, most likely accounts for the greater increase in cancer incidence rates among Aboriginal and Torres Strait Islander children. Examination of data on stage at diagnosis and treatment may provide important insights into survival for children with solid tumours.

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.