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Absolute specificity for retrograde fast axonal transport displayed by lipid droplets originating in the axon of an identified Aplysia neuron in vitro.
Lipid droplets were found to form all along the axon of the giant cerebral neuron (GCN) of the sea hare Aplysia californica when the cell was placed in culture. The emission of yellow fluorescence by the droplets after exposure of the neuron to Nile red and their uniformly dark appearance in electron micrographs of axons fixed with glutaraldehyde and osmium tetroxide identified them as lipid droplets. In contrast to lipid droplets in fat cells and certain other cell types, these droplets were bounded by a membrane, indicating that the lipid droplet is a type of organelle that is membranated in some situations but not others. As observed by video-enhanced contrast-differential interference contrast microscopy, the droplets grew manyfold in place in the axon to diameters of 1-3 micron within 2-3 days. Often they formed coherent tandem arrays of 3-15 droplets. Droplets were usually essentially stationary but occasionally moved tens of microns by fast axonal transport, the largest spherical organelles to have been observed to undergo transport. They usually moved as singlets, sometimes as tandem arrays. The direction of transport was always retrograde (towards the cell body). Thus, an organelle need neither originate nor be modified in the axon terminal to be specified for retrograde transport. Whether or not an organelle is formed in the cell body might determine directionality. Alternatively, size might be a determining factor, with large organelles specified for retrograde transport.
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Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.
Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.
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The OVAREX study: Establishment of ex vivo ovarian cancer models to validate innovative therapies and to identify predictive biomarkers.
BACKGROUND: Ovarian cancer is the first cause of death from gynecological malignancies mainly due to development of chemoresistance. Despite the emergence of PARP inhibitors, which have revolutionized the therapeutic management of some of these ovarian cancers, the 5-year overall survival rate remains around 45%. Therefore, it is crucial to develop new therapeutic strategies, to identify predictive biomarkers and to predict the response to treatments. In this context, functional assays based on patient-derived tumor models could constitute helpful and relevant tools for identifying efficient therapies or to guide clinical decision making. METHOD: The OVAREX study is a single-center non-interventional study which aims at investigating the feasibility of establishing in vivo and ex vivo models and testing ex vivo models to predict clinical response of ovarian cancer patients. Patient-Derived Xenografts (PDX) will be established from tumor fragments engrafted subcutaneously into immunocompromised mice. Explants will be generated by slicing tumor tissues and Ascites-Derived Spheroids (ADS) will be isolated following filtration of ascites. Patient-derived tumor organoids (PDTO) will be established after dissociation of tumor tissues or ADS, cell embedding into extracellular matrix and culture in specific medium. Molecular and histological characterizations will be performed to compare tumor of origin and paired models. Response of ex vivo tumor-derived models to conventional chemotherapy and PARP inhibitors will be assessed and compared to results of companion diagnostic test and/or to the patient's response to evaluate their predictive value. DISCUSSION: This clinical study aims at generating PDX and ex vivo models (PDTO, ADS, and explants) from tumors or ascites of ovarian cancer patients who will undergo surgical procedure or paracentesis. We aim at demonstrating the predictive value of ex vivo models for their potential use in routine clinical practice as part of precision medicine, as well as establishing a collection of relevant ovarian cancer models that will be useful for the evaluation of future innovative therapies. TRIAL REGISTRATION: The clinical trial has been validated by local research ethic committee on January 25th 2019 and registered at ClinicalTrials.gov with the identifier NCT03831230 on January 28th 2019, last amendment v4 accepted on July 18, 2023.
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Endocrine cells do not exist in rat Brunner's glands. An electron microscopic study.
It was revealed by light microscopic experiments that in Brunner's glands and the epithelium of the human and mammalian duodenum, cells were present, with basally lying granulations. These granules could be identified at the ultrastructural level as accumulations of pleomorphe electron-dense granules ('argentaffin', 'chromaffin' and 'osmiophilic' granules). Depending on the light and electron microscopic staining techniques used, these cells were classified as 'mucous', 'serous', or 'endocrine' ('enteroendocrine', 'paracrine' and 'enterochromaffin') cells. In the present study, the following ultrastructural morphological criteria of Brunner's gland cells were correlated: size, shape and electron density of their nuclei and nucleoli with morphological changes of their cytoplasmic and intranuclear organelles, which serve the synthesis of mucopeptides. This proved that the synthesis of mucopeptides occurred in 3 phases. The first phase served the intranuclear synthesis of organelles, which latterly were used for the cytoplasmic mucopeptide synthesis. This phase was characterized by the appearance of an electron-lucent and euchromatic nucleus, which also contained an euchromatic and electron-lucent nucleolus, and a thin electron-dense nucleus membrane (euchromatic phase). Only a few freely lying ribosomes, membrane-bound ribosomes (rough endoplasmic reticulum, RER) and mitochondria were dispersed throughout the cytoplasm. Therefore, the cytoplasm appeared electron-lucent and nearly empty. The second phase was characterized by a round but heterochromatical nucleus containing also a heterochromatic nucleolus, and the appearance of receptosome- (endosome, endocytotic vesicle) and lysosome-like structures near to the cytoplasmic membrane of the basal cell portion, as well as in the basal and lateral perinuclear region (early heterochromatic phase). The receptosome-like structures were round-oval and contained a single electron-lucent inclusion vesicle, which had an empty space or an electron-opaque granule. Both inclusion structures were located eccentrically on the inner surface of the membrane of the receptosome-like structure. Here and there, it was possible to visualize great receptosome-like structures (multivesicular bodies) containing numerous membranous electron-lucent and electron-opaque inclusion vesicles. Electron-dense pleomorphe lysosome-like structures, bearing 1-4 electron-lucent inclusion vesicles with faint grains in their spaces, were also recognized in the basal cell portion, as well as in the basal and lateral perinuclear region.(ABSTRACT TRUNCATED AT 400 WORDS)
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Organelle dynamics in lobster axons: anterograde, retrograde and stationary mitochondria.
Mitochondria in isolated motor axons from the walking legs of lobster were observed with differential interference contrast optics and video microscopic techniques. Movements of the mitochondria were analyzed in time-lapse videotape records. The mean velocity of transport in the retrograde direction (1.33 +/- 0.64 micron/s) was greater than the mean velocity of transport in the anterograde direction (0.72 +/- 0.26 micron/s). The mean lengths of the mitochondria moving in the retrograde and anterograde directions were only slightly different (6.9 microns and 5.5 microns, respectively). No correlation was found between mitochondrial length and average velocity or reciprocal velocity. The instantaneous velocities of mitochondria were distributed over a range of approximately 3 micron/s; both the anterograde and retrograde distributions contained a small proportion of values whose sign was opposite to the modal value. The variation in instantaneous velocity took place at frequencies close to 0.1 Hz. Some mitochondria displayed longitudinally oriented oscillatory movements of a similar low frequency. While the movement of most mitochondria was parallel to the axis of the axon, transverse deviations and complex circular paths were sometimes observed. Some mitochondria reversed their orientation and continued in the same direction, so that the end which had been the leading end became the trailing end. Many mitochondria immediately beneath the plasma membrane were stationary and adhered strongly to the plasma membrane when the axoplasmic structure was disrupted. In electron micrographs, fine strands connected peripheral mitochondria and the plasma membrane. These strands may anchor the stationary mitochondria to the plasma membrane.
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Harnessing Next-Generation 3D Cancer Models to Elucidate Tumor-Microbiome Crosstalk.
The tumor microenvironment (TME) is a complex and dynamic ecosystem increasingly recognized for its interplay with the microbiome. In colorectal, breast, lung, liver, and brain cancers, bacterial communities and their metabolites are shown to influence tumor progression, immune responses, and therapeutic outcomes. To study these interactions in physiologically relevant contexts, advanced 3D in vitro models have emerged, including spheroids, organoids, microfluidic organ-on-a-chip platforms, and 3D-bioprinted constructs. These systems provide spatial organization, mechanical cues, and co-culture capabilities that facilitate investigation of host-microbiome-tumor cross-talk. Incorporation of live bacteria, their metabolites, and immune components into these platforms has yielded new insights into how the microbiome shapes cancer behavior, inflammation, and drug resistance. This review outlines recent advances in 3D model development for studying tumor-microbiome interactions, highlighting organ-specific applications, extracellular matrix-mimicking hydrogels, and biofabrication strategies. It also addresses key challenges, including maintaining microbiome viability, modeling temporal dynamics, and integrating immune complexity. Overcoming these limitations requires interdisciplinary approaches that merge bioengineering, microbiology, and oncology. Evolving 3D platforms offer powerful tools for microbiome-informed cancer modeling and hold significant promise for advancing therapeutic screening and precision oncology.
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Adenovirus-mediated delivery of antisense gene to urokinase-type plasminogen activator receptor suppresses glioma invasion and tumor growth.
The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.
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Organs-on-chips technologies - A guide from disease models to opportunities for drug development.
Current in-vitro 2D cultures and animal models present severe limitations in recapitulating human physiopathology with striking discrepancies in estimating drug efficacy and side effects when compared to human trials. For these reasons, microphysiological systems, organ-on-chip and multiorgans microdevices attracted considerable attention as novel tools for high-throughput and high-content research to achieve an improved understanding of diseases and to accelerate the drug development process towards more precise and eventually personalized standards. This review takes the form of a guide on this fast-growing field, providing useful introduction to major themes and indications for further readings. We start analyzing Organs-on-chips (OOC) technologies for testing the major drug administration routes: (1) oral/rectal route by intestine-on-a-chip, (2) inhalation by lung-on-a-chip, (3) transdermal by skin-on-a-chip and (4) intravenous through vascularization models, considering how drugs penetrate in the bloodstream and are conveyed to their targets. Then, we focus on OOC models for (other) specific organs and diseases: (1) neurodegenerative diseases with brain models and blood brain barriers, (2) tumor models including their vascularization, organoids/spheroids, engineering and screening of antitumor drugs, (3) liver/kidney on chips and multiorgan models for gastrointestinal diseases and metabolic assessment of drugs and (4) biomechanical systems recapitulating heart, muscles and bones structures and related diseases. Successively, we discuss technologies and materials for organ on chips, analyzing (1) microfluidic tools for organs-on-chips, (2) sensor integration for real-time monitoring, (3) materials and (4) cell lines for organs on chips. (Nano)delivery approaches for therapeutics and their on chip assessment are also described. Finally, we conclude with a critical discussion on current significance/relevance, trends, limitations, challenges and future prospects in terms of revolutionary impact on biomedical research, preclinical models and drug development.
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A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell.
Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF alpha-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).
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Translational Models for Glioblastoma: Revolutionizing Drug Development and Personalized Medicine through Clinical Insights.
Glioblastoma (GBM) remains one of the most aggressive and treatment resistant brain tumors and continues to present major challenges for effective therapeutic development. The failure of numerous late-stage clinical trials highlights the limited predictive value of conventional preclinical models. Although established cell lines, two-dimensional cultures, and animal models have been extensively employed, existing platforms fail to adequately recapitulate the complex tumor microenvironment, blood brain barrier function, and interpatient heterogeneity that drive therapeutic resistance in GBM. To address this translational limitation, advanced experimental systems have been developed to more accurately reproduce key features of the human GBM microenvironment through the integration of microengineering approaches, biomaterials, and patient derived cells. This review focuses on recent advances in microfluidic GBM chip models and three-dimensional bioprinted GBM platforms, while also summarizing a broad range of in vitro and in vivo model systems extending from conventional 2D cultures and organoids to animal-based platforms. Microfluidic and 3D bioprinting technologies enable controlled reconstruction of critical biophysical and biochemical features of the GBM microenvironment. These systems allow regulation of oxygen availability, drug exposure, and cellular interactions within engineered tumor constructs. As a result, patient specific GBM models with heterogeneous architectures can be generated with improved biological relevance and closer resemblance to in vivo tumor behavior. In parallel, in vivo systems remain indispensable for capturing systemic pharmacokinetics, host immune responses, and organ level toxicity. Among these, mouse models encompassing syngeneic, xenograft, and genetically engineered mouse models (GEMMs) continue to serve as the foundation of preclinical GBM research, while larger animal models have been explored to better approximate the physiological and immunological features of human disease. Overall, this review discusses recent advances in GBM model development across microengineered in vitro platforms and in vivo animal systems and examines their potential to enhance translational accuracy, accelerate drug discovery, and support personalized therapeutic strategies. By emphasizing the technological scope and clinical relevance of these platforms, this work aims to provide practical guidance for the rational selection and integration of GBM model systems in the discovery and optimization of next generation therapeutics.