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CONTEXT: Endocrine therapy combined with CDK4/6 inhibitor is the standard frontline treatment for the vast majority of HR+/HER2- metastatic breast cancer (MBC) patients. Despite an overall survival benefit, patients eventually progress and mechanisms of resistance to this combination are not well identified. Non-invasive monitoring of the efficacy of treatment could help into tailoring therapeutic regimen. Extracellular vesicles (EVs) are a group of heterogeneous nanosized bioparticles (30-1000nm), released by almost all cell types - including tumor, platelets, and immune cells – carrying informative biological material emanating from the mother cell and circulating in the blood stream. In this project, we ambition to assess whether the vesiclemia, i.e. plasmatic extracellular vesicle concentration can be used as a clinic-biological parameter to assist in the monitoring of patients. METHODS: EPICURE is an ongoing pilot prospective cohort study of heterogeneous and massive data integration, i.e. multi-omics approaches in MBC patients. The present study focused on patients with HR+/HER2- MBC receiving frontline endocrine therapy+iCDK4/6. Plasma samples were drawn every 3 months during the treatment until progression. The workflow for the enrichment of circulating EVs was developed from frozen plasma. A semi-automatized isolation procedure using size exclusion chromatography combined with the newly developed interference light microscopy apparatus Videodrop allowed to routinely separate plasmatic EVs and quantify vesiclemia in a fast and reliable manner for a large number of samples (longitudinal follow-up of MBC patients). RESULTS: 26 patients were included and monitored for vesiclemia. Median age was 58.5y (±13.7). Metastatic disease sites were distributed as follows: bone metastases (21; 84%), liver (21; 84%), thoracic (10; 40%), node (2; 8%), brain (1; 4%), others (2; 8%). Endocrine therapy included aromatase inhibitors (20; 77%), fulvestrant (6; 29%), GnRH agonists (5; 19%) and CDK4/6 inhibitors palbocilib (15; 57%), abemaciclib (8; 31%) and ribociclib (3; 11%). With a median follow-up time of 22.1 months (95%CI 21.2 - not reached), median progression free survival was 21.8 months (95%CI 18.1 - not reached). Median time on treatment was 19.4 months (IQR 11.6 - 22.1). Objective response rate was confirmed in 12 patients (6 with complete response). Out of the 26 patients, 12 stopped the frontline treatment due to disease progression. 135 longitudinal follow-up samples were analyzed for EVs. All patients who progressed rapidly after treatment initiation (i.e median PFS< 6 months) had an increased vesiclemia at the months 2 and 6 after inclusion. Conversely, patients who had a median PFS >18m had a stable vesiclemia during this period. CONCLUSION This study aims to use circulating EVs as a therapeutic companion to anticipate treatment resistance and tumor progression in MBC patients. Our preliminary data suggest that the vesiclemia could be used as a predictive tool to anticipate treatment failure, and thus might be used to tailor treatments in time. Citation Format: Mathilde Richard, Jean Sebastien FRENEL, Laurent Mathiot, Mario Campone, Mathilde Colombie, Marie Robert, Anne Patsouris, Frederic Bigot, Julie Gavard, Gwennan André-Grégoire, Laetitia Guevel. Extracellular vesicle-based biomarker assay for the monitoring of the efficacy of frontline endocrine therapy + CDK4-6 inhibitors in metastatic breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-09.

Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness and resistance, as they carry and disseminate oncogenic material in the local tumor microenvironment and at distance. However, whether their quality and quantity reflect individual health status and changes in homeostasis is still not fully elucidated. Here, we separated EVs from plasma collected at different time points alongside with the clinical management of GBM patients. Our findings confirm that plasmatic EVs could be separated and characterized with standardized protocols, thereby ensuring the reliability of measuring vesiclemia, i.e. extracellular vesicle concentration in plasma. This unveils that vesiclemia is a dynamic parameter, which could be reflecting tumor burden and/or response to treatments. Further label-free liquid chromatography tandem mass spectrometry unmasks the von Willebrand Factor (VWF) as a selective protein hallmark for GBM-patient EVs. Our data thus support the notion that EVs from GBM patients showed differential protein cargos that can be further surveyed in circulating EVs, together with vesiclemia.

Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness and resistance, as they carry and disseminate oncogenic material in the local tumor microenvironment and at distance. However, whether their quality and quantity reflect individual health status and changes in homeostasis is still not fully elucidated. Here, we separated EVs from plasma collected at different time points alongside with the clinical management of GBM patients. Our findings confirm that plasmatic EVs could be separated and characterized with standardized protocols, thereby ensuring the reliability of measuring vesiclemia, i.e. extracellular vesicle concentration in plasma. This unveils that vesiclemia is a dynamic parameter, which could be reflecting tumor burden and/or response to treatments. Further label-free liquid chromatography tandem mass spectrometry unmasks the von Willebrand Factor (VWF) as a selective protein hallmark for GBM-patient EVs. Our data thus support the notion that EVs from GBM patients showed differential protein cargos that can be further surveyed in circulating EVs, together with vesiclemia.

International audience; Although rare, glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment, and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness, and resistance, as they carry oncogenic material. Moreover, EVs have been shown to communicate locally in a paracrine way but also at remote throughout the organism. Indeed, recent reports demonstrated the presence of brain tumor-derived EVs into body fluids such as plasma and cerebrospinal fluid. Fluid-associated EVs have indeed been suspected to reflect quantitative and qualitative information about the status and fate of the tumor and can potentially act as a resource for noninvasive biomarkers that might assist in diagnosis, treatment, and follow-up of glioblastoma patients. Here, we coined the name vesiclemia to define the concentration of plasmatic EVs, an intuitive term to be directly transposed in the clinical jargon.

International audience; Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis ofDMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1R). Subsequently, we were able to showed that PGC1-R expression is dramatically reduced inGRMDcompared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

The alpha subunit (alphaTm) of Thermotoga maritima RNA polymerase has been characterized to investigate its role in transcriptional regulation in one of the few known anaerobic hyperthermophilic bacteria. The highly thermostable alphaTm shares 54% similarity with its Escherichia coli analogue (alphaEc). The T. maritima rpoA gene coding the alpha subunit does not complement the thermosensitive rpoA112 mutation of E. coli. However, alphaTm and alphaEc show similar folding patterns as determined by circular dichroism. Purified alphaTm binds to the T. maritima PargGo promoter region (probably to a UP-element) and Arg282 appears to be crucial for DNA binding. The thermostable protein is also able to interact with transcription regulatory proteins, like ArgR from T. neapolitana or CRP from E. coli. These data indicate that the RNA polymerase alpha subunit might play a crucial role in the modulation of gene expression in hyperthermophiles.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

During recent years, considerable effort has been made to develop proteomics technologies, with the aim of providing a complementary approach to the genomics tools already used in biomedical settings. This development has been extremely fast, and a number of emerging methodological proteomics tools now allow scientists to study the variable aspects of proteins in particular cell types, tissues or disease states. These tools include antibody arrays, two-dimensional-gel electrophoresis (2D-GE) and mass spectrometry (MS), the latter knowing an increasing use. In particular, candidate or non-candidate-based analyses of cell signalling represent powerful approaches for the investigation of the answers developed by cells in response to genetic modifications. Signalling molecules are key players in the regulation of the numerous and various biological processes occurring in a cell, and the alteration of signalling pathways has been associated with multiple diseases. Alterations in individual signalling pathways have been described in neuromuscular disorders, however, little information is available regarding their putative implication in Duchenne Muscular Dystrophy (DMD).

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

International audience; dult skeletal muscle possesses remarkable regenerative capacity that has conventionally been attributed to the satellite cells. These precursor cells were thought to contain distinct populations with varying myogenic potential. Recently, the identification of multipotent stem cells capable of new myofiber formation has expanded the general view on the muscle regenerative process. Here we examined the characteristics of turkey skeletal muscle-derived cell (MDC) populations that were separated according to their adhesion abilities. We sought to determine whether these abilities could be a potential tool for separating cells with different myogenic commitment. Using the preplate technique, we showed that MDCs display a wide range of adhesion ability, allowing us to isolate a marginal fraction with initial adhesion defect. Methodological investigations revealed that this defect represents an intrinsic and well-established biological feature for these cells. In vitro behavioral and morphological analyses showed that late adherent cells (LACs) share several primitive cell characteristics. Phenotypic assessment indicated that LACs contain early stage myogenic cells and immature progenitors of satellite cells, whereas early adherent cells consist mainly of fully committed precursors. Overall, our findings demonstrate for the first time in an avian model that differential MDC adhesion properties could be used to efficiently purify cells with varying myogenic commitment, including immature progenitor cells. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus . In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus P argCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the α4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the α4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.