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Automated Scoring to Assess RAD51-Mediated Homologous Recombination in Ovarian Patient-Derived Tumor Organoids.
Poly(ADP-ribose) polymerase inhibitors (PARPi) have been shown to improve progression-free survival, particularly in homologous recombination-deficient ovarian cancers. Identifying patients eligible for PARPi is currently based on next-generation sequencing, but the persistence of genomic scars in tumors after restoration of homologous recombination (HR) or epigenetic changes can be a limitation. Functional assays could thus be used to improve this profiling and faithfully identify homologous recombination-deficient tumors. The repair capacity (RECAP) test assesses the formation of RAD51 foci in proliferating cells after irradiation and can be used on tumors as well as on patient-derived tumor organoids (PDTO). However, RAD51 foci scoring is often performed manually without standardization. The purpose of this translational study was to develop an automated tool for scoring RAD51-mediated HR based on whole slide imaging of ovarian PDTO. To that end, we quantified Cyclin A2 and RAD51 immunofluorescence on 9 PDTO models derived from 8 ovarian cancer patients, and next, we compared the RECAP test results to genome instability score and to the patient clinical response. We therefore developed a standardized and automatized quantitative histoimaging tool allowing a comparative RAD51 foci evaluation and thus to define the HR status in PDTO. Our RECAP-based classification was correlated to the genome instability score, offering a new opportunity for standardization of HR assessment in PDTO. This new automated tool to score HR status, which remains to be validated on a large cohort of patients, may thus be used as a complement to next-generation sequencing-based tests in order to improve the identification of the number of patients eligible for PARPi.
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Batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells.
Batrachotoxin has been reported to inhibit fast axonal transport. We have examined the effect of batrachotoxin on saltatory organelle movements in N115 neuroblastoma cells (a model of fast axonal transport) using time-lapse video intensification microscopy. Batrachotoxin (0.1-1.0 microM) inhibits saltatory organelle movement. Contrary to previously published hypotheses, this inhibition of intra-axonal movement depends upon the action of batrachotoxin on action potential Na+ channels. Evidence for this conclusion is: (1) the range of concentrations of batrachotoxin which inhibit saltatory organelle movement is consistent with the dose-response curve for the activation of action potential Na+ channels by batrachotoxin in N18 neuroblastoma cells; (2) tetrodotoxin, which blocks action potential Na+ channels, prevents the inhibition of organelle movements by batrachotoxin; (3) batrachotoxin has no effect on saltatory movement in cells, including some neuroblastoma cell lines, which lack action potential Na+ channels; and (4) in Na+-free or low Na+ media, batrachotoxin does not block organelle movement. We suggest that changes in internal ion concentrations which follow the influx of Na+ are responsible for the inhibition of fast axonal transport by batrachotoxin.
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Response to Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment".
Maricic et al. performed an undisclosed in silico–only whole-exome sequencing analysis of our data and found genomic alterations previously undetected in some clones. Some of the predicted alterations, if true, could change the original genotype of the clones. We failed to experimentally validate all but one of these genomic alterations, which did not affect our previous results or data interpretation.
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Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle.
The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.
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Self-renewal and solid tumor stem cells.
Solid tumors arise in organs that contain stem cell populations. The tumors in these tissues consist of heterogeneous populations of cancer cells that differ markedly in their ability to proliferate and form new tumors. In both breast cancers and central nervous system tumors, cancer cells differ in their ability to form tumors. While the majority of the cancer cells have a limited ability to divide, a population of cancer stem cells that has the exclusive ability to extensively proliferate and form new tumors can be identified based on marker expression. Growing evidence suggests that pathways that regulate the self-renewal of normal stem cells are deregulated in cancer stem cells resulting in the continuous expansion of self-renewing cancer cells and tumor formation. This suggests that agents that target the defective self-renewal pathways in cancer cells might lead to improved outcomes in the treatment of these diseases.
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Development of Non-Invasive miRNA Markers for Assessing the Quality of Human Induced Pluripotent Stem Cell-Derived Retinal Organoids.
Human retinal organoids (ROs) have emerged as valuable tools for studying retinal development, modeling human retinal diseases, and screening drugs. However, their application is limited primarily due to time-intensive generation, high costs, and low reproducibility. Quality assessment of RO differentiation is crucial for their application in research. However, traditional methods such as morphological evaluation and immunohistochemical analysis have limitations due to their lack of precision and invasiveness, respectively. This study aims to identify non-invasive biomarkers for RO differentiation quality using exosomal microRNAs (miRNAs), which are known to reflect cell-specific functions and development in the retina. We differentiated ROs from human induced pluripotent stem cells (hiPSCs) and classified them into 'superior' and 'inferior' groups based on morphological and immunohistochemical criteria. Exosomes from the conditioned media were isolated and analyzed for miRNA content. Our findings revealed distinct miRNA profiles between superior and inferior ROs, with superior ROs exhibiting higher miRNA diversity and specifically up- or down-regulated miRNAs. Gene ontology and pathway enrichment analyses indicated that the target genes of these miRNAs are involved in neuron proliferation and differentiation. The study suggests the potential of exosomal hsa-miR-654-3p and hsa-miR-451a as non-invasive biomarkers for real-time monitoring of RO quality, facilitating the development of standardized, efficient, and cost-effective culture methods.
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Short term effects of gamma radiation on endothelial barrier function: uncoupling of PECAM-1.
A limiting factor in the treatment of cancer with radiotherapy is the damage to surrounding normal tissue, particularly the vasculature. Vessel pathologies are a major feature of the side effects of radiotherapy and little is known about early events that could initiate subsequent diseases. We tested the hypothesis that gamma radiation has early damaging effects on the human endothelial barrier. Two models were used; Human Brain Microcapillary Endothelial Cells (HBMEC), and Human Umbilical Vein Endothelial Cells (HUVEC). Endpoints included Trans-Endothelial Electrical Resistance (TEER), barrier permeability to 10 kDa and 70 kDa tracer molecules, and the localization of F-actin, and junction proteins and the Platelet Endothelial Cell Adhesion Molecule (PECAM-1). Radiation induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the radiotherapy doses. This dip in resistance correlated to the transient loss of PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Redistribution of PECAM-1 was also seen in 3-D human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as evidenced by an increased permeability over 24 h. This is the first demonstration of a transient and rapid effect of gamma radiation on human endothelial barriers that involves cell detachment and the loss of PECAM-1. Considering the association of cell adhesion molecules with vasculopathies, such an effect has the potential to be clinically relevant to the longer-term effects of radiotherapy.
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Organelle dynamics in lobster axons: anterograde and retrograde particulate organelles.
Particulate organelles in isolated axons from the walking legs of the lobster were detected with differential interference contrast optics and video microscopic techniques. The motion of the organelles was studied in normal axons, in axons whose surface membrane was rendered permeable with saponin, and in axoplasm extruded from the axons. In normal axons at 20-22 degrees C, organelles moved more rapidly in the anterograde direction than in the retrograde direction (respective mean velocities 1.73 micron/s and 0.63 micron/s). The instantaneous velocities of both sets of organelles were variable: those of the anterograde organelles varied less than those of retrograde organelles. The variation in instantaneous velocity was patterned; all organelles studied had velocities that fluctuated slowly with a major frequency at about 0.1 Hz. Some organelles oscillated about a fixed position at a similar major frequency. In axons with a permeabilized surface membrane there was no organelle motion unless adenosine 5'-triphosphate (ATP) was present in the bathing medium. Organelle motion reactivated with ATP was patterned in a way similar to that in normal intact axons. In extruded axoplasm in the presence of ATP, organelles moved along transport filaments that were assumed to be microtubules. Movement of organelles from one transport filament to another was not accompanied by changes in motion that could explain the normal fluctuation in velocity. The evidence indicates that the variable, or oscillatory, character of organelle motion in lobster axons is caused by an active component of the mechanisms of axonal transport.
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Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.
Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.
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Harnessing three-dimensional (3D) cell culture models for pulmonary infections: State of the art and future directions.
Pulmonary infections have been a leading etiology of morbidity and mortality worldwide. Upper and lower respiratory tract infections have multifactorial causes, which include bacterial, viral, and rarely, fungal infections. Moreover, the recent emergence of SARS-CoV-2 has created havoc and imposes a huge healthcare burden. Drug and vaccine development against these pulmonary pathogens like respiratory syncytial virus, SARS-CoV-2, Mycobacteria, etc., requires a systematic set of tools for research and investigation. Currently, in vitro 2D cell culture models are widely used to emulate the in vivo physiologic environment. Although this approach holds a reasonable promise over pre-clinical animal models, it lacks the much-needed correlation to the in vivo tissue architecture, cellular organization, cell-to-cell interactions, downstream processes, and the biomechanical milieu. In view of these inadequacies, 3D cell culture models have recently acquired interest. Mammalian embryonic and induced pluripotent stem cells may display their remarkable self-organizing abilities in 3D culture, and the resulting organoids replicate important structural and functional characteristics of organs such the kidney, lung, gut, brain, and retina. 3D models range from scaffold-free systems to scaffold-based and hybrid models as well. Upsurge in organs-on-chip models for pulmonary conditions has anticipated encouraging results. Complexity and dexterity of developing 3D culture models and the lack of standardized working procedures are a few of the setbacks, which are expected to be overcome in the coming times. Herein, we have elaborated the significance and types of 3D cell culture models for scrutinizing pulmonary infections, along with the in vitro techniques, their applications, and additional systems under investigation.