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The PENDOR study: establishment of a panel of patient-derived tumor organoids from endometrial cancer to assess efficacy of PARP inhibitors.
BACKGROUND: Combination of chemotherapy and immunotherapy is the current standard of care for advanced endometrial cancer. However, survival outcome remains poor, highlighting the urgent need for new treatments and reliable tools to identify patients who will benefit from them. Patient-Derived Tumor Organoids (PDTO) are three-dimensional structures established from patient tumors, and are closely mimicking the features of the tumor of origin. Moreover, more and more evidences show that PTDOs hold promises as predictive tools for the response to treatment of patients. METHOD: The PENDOR study is a monocentric observational study designed to assess the feasibility of generating and testing PDTOs derived from endometrial cancer for evaluating treatment sensitivity. PDTOS will be established from surgical specimens not required for anatomopathological diagnosis. Tumor cells will be dissociated, embedded in extracellular matrix, and cultured in a medium supplemented with growth factors and signaling pathways inhibitors. Molecular and histological analyses will be conducted to validate the resemblance of PDTO to the original tumor. Response of PDTO to conventional chemotherapy and PARP inhibitors will be evaluated and compared to clinical response and to the results of an academic HRD test Genomic Instability Scar (GIScar), respectively, to assess their predictive value. DISCUSSION: This pilot study aims to validate the feasibility to develop PDTOs from endometrial cancer from patients who will undergo surgical resection. We aim to provide a proof of concept regarding the predictive value of these models for their potential application into routine clinical practice as part of precision medicine. This approach could therefore facilitate the identification of patients who could benefit from PARP inhibitors. TRIAL REGISTRATION: This clinical trial (N°ID-RCB: 2024-A01206-41) has been validated by local research ethic committee on July 16th 2024 and registered at ClinicalTrials.gov with the identifier NCT06603506 on September 6th 2024, version 1.
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Human Organoids and Organs-on-Chips for Addressing COVID-19 Challenges.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an imminent threat to our lives. Although animal models and monolayer cell cultures are utilized for pathogenesis studies and the development of COVID-19 therapeutics, models that can more accurately reflect human-relevant responses to this novel virus are still lacking. Stem cell organoids and bioengineered organs-on-chips have emerged as two cutting-edge technologies used to construct biomimetic in vitro three-dimensional (3D) tissue or organ models. In this review, the key features of these two model systems that allow them to recapitulate organ physiology and function are introduced. The recent progress of these technologies for virology research is summarized and their utility in meeting the COVID-19 pandemic is highlighted. Future opportunities and challenges in the development of advanced human organ models and their potential to accelerate translational applications to provide vaccines and therapies for COVID-19 and other emerging epidemics are also discussed.
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Recent advances in 3D models of the nervous system for neural regeneration research and drug development.
The development of drugs for nervous diseases poses distinctive difficulties owing to the incomplete understanding of the physiology and complex pathogenesis of the multifaceted central (CNS) and peripheral (PNS) nervous systems. Conventional animal tests and in vitro two-dimensional (2D) cell cultures fail to reproduce the sophisticated structure of natural human tissues, hindering the new drug discovery process. The emerging three-dimensional (3D) neural tissue models, including organoids, organ-on-chip and 3D-printed neural scaffolds, can provide an improved reproduction of the critical features, structural complexity, biological functions, dynamic circulation micro-environment and cell-matrix/cell interactions of the nervous systems. This review examines state-of-the-art 3D models for neural physiology/pathology, emphasizing their drug development applications. Fundamental advantages of various in vitro 3D neural models for investigating the mechanisms of nerve regeneration and disorders in both the CNS and PNS are compared in terms of the different modeling techniques. In addition, the applications of 3D neural models in drug development are summarized covering a range of areas such as disease modeling for basic research, pharmacokinetic and pharmacodynamic testing for drug screening and drug safety evaluation. Furthermore, current challenges and future outlook of biomimetic models and the existing bottlenecks hindering their successful translation into clinical use are discussed. STATEMENT OF SIGNIFICANCE: This review highlights the groundbreaking potential of 3D neural models-organoids, organ-on-chip, and 3D-printed scaffolds-to revolutionize neurological research and drug development. Unlike conventional methods, these models replicate the intricate structure and function of the human nervous system, enabling precise study of diseases like Alzheimer's, spinal injuries, and brain tumors. By summarizing and discussing recent advancements, the review compares techniques, their applications in drug screening and personalized medicine, and addresses challenges in model accuracy and scalability. Bridging neuroscience, engineering, and pharmacology, this work provides a roadmap for researchers to innovate therapies. The insights presented are critical for accelerating drug discovery and improving treatment outcomes, making 3D neural models essential for scientists and clinicians tackling neurological disorders.
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The Future of Protozoan Infection Research: 3D Cell Culture and beyond.
Protozoan infections remain a major challenge to human health, owing to the substantial impact these parasites exert on their hosts. In vitro culture systems are fundamental for elucidating the host-parasite interactions underlying these infections; however, traditional two-dimensional models are often limited and fail to recapitulate key aspects of human tissue architecture and function. In this context, advanced in vitro cell culture models have emerged as powerful tools, offering increased morphofunctional resemblance to human tissues. This review critically examines recent studies employing advanced platforms, including Transwell systems, spheroids, organoids, scaffolds and biomaterials, microfluidic devices, and organ-on-chip technologies. Collectively, these approaches enable the modeling of diverse human tissues, such as the intestine, brain, heart, and vascular compartments, and support the investigation of multiple pathogenic protozoa, including Trypanosoma cruzi, Plasmodium spp., and Giardia duodenalis. By integrating advanced in vitro models into protozoan infection research, it is possible to achieve a more faithful representation of the human host environment, simulating infection processes and to foster the development of more effective and targeted pharmacological interventions.
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Three-dimensional visualization of coated vesicle formation in fibroblasts.
Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep-etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.
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Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
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Aberrant neurodevelopment in human iPS cell-derived models of Alexander disease.
Alexander disease (AxD) is a rare and severe neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP). While the exact disease mechanism remains unknown, previous studies suggest that mutant GFAP influences many cellular processes, including cytoskeleton stability, mechanosensing, metabolism, and proteasome function. While most studies have primarily focused on GFAP-expressing astrocytes, GFAP is also expressed by radial glia and neural progenitor cells, prompting questions about the impact of GFAP mutations on central nervous system (CNS) development. In this study, we observed impaired differentiation of astrocytes and neurons in co-cultures of astrocytes and neurons, as well as in neural organoids, both generated from AxD patient-derived induced pluripotent stem (iPS) cells with a GFAPR239C mutation. Leveraging single-cell RNA sequencing (scRNA-seq), we identified distinct cell populations and transcriptomic differences between the mutant GFAP cultures and a corrected isogenic control. These findings were supported by results obtained with immunocytochemistry and proteomics. In co-cultures, the GFAPR239C mutation resulted in an increased abundance of immature cells, while in unguided neural organoids and cortical organoids, we observed altered lineage commitment and reduced abundance of astrocytes. Gene expression analysis revealed increased stress susceptibility, cytoskeletal abnormalities, and altered extracellular matrix and cell-cell communication patterns in the AxD cultures, which also exhibited higher cell death after stress. Overall, our results point to altered cell differentiation in AxD patient-derived iPS-cell models, opening new avenues for AxD research.
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Sensory supply of the anterior uvea: a light and electron microscope study.
We have studied the ultrastructural features of sensory nerve fibers in the ciliary body and the iris and their parent trigeminal ganglion cells, using intra-axonally transported horseradish peroxidase as a tracer. Unmyelinated nerve fibers of ipsilateral trigeminal origin were found in stroma of both the ciliary body and the iris. Most appeared in bundles of nerve fibers; but some were found singly. Two distinct types of varicosities were found, one containing both a few irregularly shaped vesicles and mitochondria, the other containing mitochondria only. The thin profiles of the axons between these varicosities or endings contained neurotubules and filaments. The trigeminal ganglion cells supplying the anterior eye were of a relatively small size (15-50 micron in diameter) and confined to the anteromedial part of the ipsilateral ganglion. These cells could not be classified according to the arrangement of their subcellular organelles. The morphological characteristics of the sensory nerve fibers and ganglion cells supplying the anterior uvea are consistent with the view that pain may be the predominant sensation mediated by these neurons.
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Suppression of ferroptosis by vitamin A or radical-trapping antioxidants is essential for neuronal development.
The development of functional neurons is a complex orchestration of multiple signaling pathways controlling cell proliferation and differentiation. Because the balance of antioxidants is important for neuronal survival and development, we hypothesized that ferroptosis must be suppressed to gain neurons. We find that removal of antioxidants diminishes neuronal development and laminar organization of cortical organoids, which is fully restored when ferroptosis is inhibited by ferrostatin-1 or when neuronal differentiation occurs in the presence of vitamin A. Furthermore, iron-overload-induced developmental growth defects in C. elegans are ameliorated by vitamin E and A. We determine that all-trans retinoic acid activates the Retinoic Acid Receptor, which orchestrates the expression of anti-ferroptotic genes. In contrast, retinal and retinol show radical-trapping antioxidant activity. Together, our study reveals an unexpected function of vitamin A in coordinating the expression of essential cellular gatekeepers of ferroptosis, and demonstrates that suppression of ferroptosis by radical-trapping antioxidants or by vitamin A is required to obtain mature neurons and proper laminar organization in cortical organoids.
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β-Arrestin 2 and Epac2 Cooperatively Mediate DRD1-Stimulated Proliferation of Human Neural Stem Cells and Growth of Human Cerebral Organoids.
G-protein-coupled receptors (GPCRs) reportedly relay specific signals, such as dopamine and serotonin, to regulate neurogenic processes although the underlying signaling pathways are not fully elucidated. Based on our previous work, which demonstrated dopamine receptor D1 (DRD1) effectively induces the proliferation of human neural stem cells, here we continued to show the knockout of β-arrestin 2 by CRISPR/Cas9 technology significantly weakened the DRD1-induced proliferation and neurosphere growth. Furthermore, inhibition of the downstream p38 MAPK by its specific inhibitors or small hairpin RNA mimicked the weakening effect of β-arrestin 2 knockout. In addition, blocking of Epac2, a PKA independent signal pathway, by its specific inhibitors or small hairpin RNA also significantly reduced DRD1-induced effects. Simultaneous inhibition of β-arrestin 2/p38 MAPK and Epac2 pathways nearly abolished the DRD1-stimulated neurogenesis, indicating the cooperative contribution of both pathways. Consistently, the expansion and folding of human cerebral organoids as stimulated by DRD1 were also mediated cooperatively by both β-arrestin 2/p38 MAPK and Epac2 pathways. Taken together, our results reveal that GPCRs apply at least 2 different signal pathways to regulate neurogenic processes in a delicate and balanced manners.