Initial insights into the interaction of antibodies radiolabeled with Lutetium-177 and Actinium-225 with tumor microenvironment in experimental human and canine osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is a prevalent primary bone cancer affecting both humans and canines. This study describes initial insights into the interaction of the human monoclonal antibody IF3 to an insulin-like growth factor 2 receptor (IGF2R) radiolabeled with either alpha-emitting Actinium-225 (225Ac) or beta-emitting Lutetium-177 (177Lu) radionuclides with the OS cells and tumor microenvironment (TME) in experimental human and canine OS. BASIC PROCEDURES: SCID mice bearing canine Gracie or human OS-33 OS tumors were treated with 177Lu- or 225Ac-labeled IF3 antibody, sacrificed at 24, 72 or 168 h post-treatment and their tumors were analyzed by immunohistochemistry (IHC) for the presence of OS cells, various elements of TME as well as for the double DNA strand breaks with γH2AX and caspase 3 assays. MAIN FINDINGS: IHC revealed a reduction in IGF2R-positive OS cells and OS stem cell populations post therapy with 225Ac- and 177Lu-labeled IF3 antibody. Notably, radiolabeled IF3 antibody effectively diminished pro-tumorigenic M2 macrophages, highlighting its therapeutic promise. The study also unveiled varied responses of natural killer (NK) cells and M1 macrophages, shedding light on the intricate TME interplay. Time-dependent increase in γ-H2AX staining in canine Gracie and human OS-33 tumors treated with [177Lu]Lu-IF3 and [225Ac]Ac-IF3 was observed at 24 and 72 h post-RIT. PRINCIPAL CONCLUSIONS: These findings suggest that radiolabeled antibodies offer a hopeful avenue for personalized OS treatment, emphasizing the importance of understanding their impact on the TME and potential synergies with immunotherapy.

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants. This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed. No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Measurement of gravitational and thermal effects in a liquid-actuated torsion pendulum.

We describe a proof-of-principle experiment aiming to investigate the inverse-square law of gravitation at the centimeter scale. The sensor is a two-stage torsion pendulum, while actuation is accomplished by a variable liquid mass. The time-varying gravitational force is related to the level of the circulating fluid in one or two containers at a short distance from the test mass, with all moving mechanical parts positioned at a large distance. We provide a description of the apparatus and present the first results. We identified a systematic effect of thermal origin, producing offsets of few fNm in torque and of about 10 pN in force. When this effect is neutralized, the measurements agree well with the predictions of simulations. We also discuss the upcoming instrument upgradations and the expected sensitivity improvement that will allow us to perform measurements with adequate accuracy to investigate the unexplored regions of the α-λ parameter space of a Yukawa-like deviation from the Newtonian potential.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis.

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Neurospheres obtained from the ciliary margin of the chicken eye possess positional values and retinal ganglion cells differentiated from them respond to EphA/ephrin-A system.

In the Central Nervous System (CNS) there are some niches of undifferentiated, neural progenitor/stem cells that produce active neurogenesis originating functionally integrated neurons. In the chicken eye, there is a neurogenic niche in the ciliary margin (CM) which has the ability to originate all the cell types of the neural retina. During retinal development, cells acquire positional values along the radial and tangential axes. These positional values are the necessary base for the formation of neural circuits. In this work, we have analyzed whether neural progenitor cells (NPCs) of CM have positional values regarding the radial and tangential axes, and if they have the potential to differentiate into retinal ganglion cells (RGCs) in vitro. Furthermore, we analyzed whether these RGCs preserve positional values along the tangential axis and respond to the Eph/ephrin axon guidance system. In order to answer these questions, we cultured NPCs obtained from the CM favoring the formation of neurospheres. Our results showed that the expanding neurospheres are polarized structures in which their cells have specific positional values along their radial axis, recapitulating the apical-basal polarity of the CM and the neuroepithelium. We also showed that NPCs obtained from CM possess positional values along the nasal-temporal retinal axis. When the neurospheres were submitted to differentiation conditions, we observed that NPCs can differentiate into RGCs. These RGCs present long axons that express different members of the Eph/ephrin system and they are competent to respond to this axon guidance cue system, recapitulating the axonal behavior during retinotectal neural map development. All these findings contribute to understand the cellular and molecular mechanisms involved in CNS development and regeneration.

Optic vesicle morphogenesis requires primary cilia.

Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b-/- eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.

Regulation of axonal EphA4 forward signaling is involved in the effect of EphA3 on chicken retinal ganglion cell axon growth during retinotectal mapping.

The Eph and ephrins are involved in the genesis of topographic ordered connections at the visual system. Previously we demonstrated that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) toward the caudal tectum preventing them from branching in the rostral tectum. Now we investigated whether tectal EphA3 plays this role by modulating the axonal EphA4 forward signaling or throughout axonal ephrin-As reverse signaling. For this purpose we used cultures of nasal retinal explants and dissociated retinal neurons from chicken embryos. We treated them with clustered EphA3-Fc, Fc (control), PI-PLC (sheds ephrin-As) or KYL (inhibits ephrin-As-mediated EphA4 activation). We achieved in vitro and in vivo electroporations of chicken embryo retinas with wild type EphA4, Ki-EphA4 (kinase inactive dominant negative EphA4) or EGFP in pMES expression vector. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrin-As system. Our results showed that: 1) shedding of ephrin-As and the inhibition of ephrin-A-mediated EphA4 activity increase axon length and decrease axonal interstitial filopodia density of nasal RGCs; and 2) a dominant negative form of EphA4 increases axon growth in vitro and induces nasal RGC axons to grow passing throughout their target area in the caudal tectum meanwhile overexpression of EphA4 produces the opposite effects. All together, these results demonstrate that ephrin-A-mediated EphA4 forward signaling decreases the level of axon growth and increases the density of axonal interstitial filopodia of nasal RGCs. Besides, our results showed that: 3) EphA3 ectodomain increases axon growth and decreases the density of axonal interstitial filopodia and branching in vitro and in vivo and 4) EphA3 ectodomain diminishes the ephrin-A2/EphA4 colocalization, and the EphA4 and ephexin1 phosphorylation. All together, these results show that the EphA3 ectodomain produces the opposite effects than the EphA4 forward signaling, by decreasing this signaling pathway throughout competing with EphA4 for ephrin-As binding. Furthermore, it is proposed that tectal EphA3 participates in the establishment of retinotectal mapping throughout this mechanism and that EphAs can regulate axon growth and branching by modulating other EphA receptors forward signaling.

Use of two gRNAs for CRISPR/Cas9 improves bi-allelic homologous recombination efficiency in mouse embryonic stem cells.

Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi-allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs. The bi-allelic recombination targeting efficiency is over 90% when using two gRNAs together with the inhibition of non-homologous end-joint repair. Moreover, this technique is compatible with the generation of knocked-in mice and the use of ESC-derived differentiation protocols, therefore facilitating and accelerating the gene targeting in mice and ESCs.

An Eye Organoid Approach Identifies Six3 Suppression of R-spondin 2 as a Critical Step in Mouse Neuroretina Differentiation.

Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.

Characterization of Tunneling Nanotubes in Wharton's jelly Mesenchymal Stem Cells. An Intercellular Exchange of Components between Neighboring Cells.

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.

[Using Twitter in oncology. Research, continuing education, and advocacy]. / L'uso di Twitter in oncologia. Ricerca, aggiornamento e advocacy.

Traditional mass media coverage has been enhanced by Twitter, an interactive, real-time media, useful in health care, and particularly in oncology. Social media such as Twitter are gaining increasing acceptance as tools for instantaneous scientific dialogue. Professional medical societies such as ASCO and ESMO are using microblogging to expand the reach of scientific communications at and around their scientific meetings. To widen the message and maximize the potential for word-of-mouth marketing using Twitter, organizations (such as AIOM, ASCO or ESMO) and industries need a strategic communications plan to ensure on-going social media conversations. Twitter is a very powerful tool indeed that amplifies the results of scientific meetings, and conference organisers should put in place strategies to capitalise on this. This review demonstrates that cancer patients also share information more and more via Twitter about their disease, including diagnosis, symptoms, and treatments. This information could prove useful to health care providers.

[Cancer on the big screen. How and when movies deal with oncological diseases]. / Il cancro sul grande schermo. Quanto, come e quando il cinema si occupa delle malattie oncologiche.

Films that feature characters with cancer have become a familiar sight for movie-goers. 148 movies treating tumors were selected, produced all over the world since the Thirties, in which cancer had "prompt", "relevant", or "plot" character. In order to clearly understand each film's peculiar message about cancer, we recollected data such as genealogy, year and country of production, main characters' age and gender, and kind of tumor. Movies deal with cancer through very relevant questions, as well as themes and contexts that have great influence on oncologist's mind and consciousness. Specially in recent years, films have tackled some of the most important issues around cancer, such as his epidemiology and environmental causes; the economic implications of therapies; the management of symptoms and side effects; the psychological dynamics; the care toward the ending of life. The most frequent treatment mentioned in the movies was chemotherapy followed by antalgic therapy. Very often the ill person on the screen doesn't get over the disease and his death is somehow useful to the plot's outcome. This pattern is so strongly standardized that it persists in spite of real progress of treatments. Movies use disease, and other tragedies, as a dramatic device, and since drama is what we expect of the medium, should we be concerned that there is a gap between fiction and reality? Movies represent an essential step of educational process, but their potential has been fully exploited only in recent times. By watching movies on cancer, oncologists could become more conscious of problems they are already facing in the therapeutic setting: cancer and sexuality, the relationship between the ill person and the medical staff, side effects of therapies. Some films simply make us reflect upon the meaning of life and death. This is useful for the sharing of cancer care, from personal or familiar problems to issues of collective relevance.

Anaglyph of retinal stem cells and developing axons: selective volume enhancement in microscopy images.

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells.

uPA-uPAR molecular complex is involved in cell signaling during neuronal migration and neuritogenesis.

BACKGROUND: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase-type plasminogen activator (uPA)-in association with its receptor-triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA-uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. RESULTS: In response to uPA, neuronal migration and neuritogenesis are promoted in a dose-dependent manner. After stimulation, uPAR interacts with α5- and ß1-integrin subunits, which may constitute an αß-heterodimer that acts as a uPA-uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA-promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. CONCLUSIONS: uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks.

EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Optic tectum morphogenesis: a step-by-step model based on the temporal-spatial organization of the cell proliferation. Significance of deterministic and stochastic components subsumed in the spatial organization.

BACKGROUND: Cell proliferation plays an important morphogenetic role. This work analyzes the temporal-spatial organization of cell proliferation as an attempt to understand its contribution to the chick optic tectum (OT) morphogenesis. RESULTS: A morphogenetic model based on space-dependent differences in cell proliferation is presented. Step1: a medial zone of high mitotic density (mZHMD) appears at the caudal zone. Step2: the mZHMD expands cephalically forming the dorsal curvature and then duplicates into two bilateral ZHMDs (bZHMD). Step3: the bZHMDs move toward the central region of each hemitectum. Step4: the planar expansion of both bZHMD and a relative decrement in the dorsal midline growth produces a dorsal medial groove separating the tectal hemispheres. Step5: a relative caudal displacement of the bZHMDs produces the OT caudal curvature. Numerical sequences derived from records of mitotic cells spatial coordinates, analyzed as stochastic point processes, show that they correspond to 1/f((ß)) processes. The spatial organization subsumes deterministic and stochastic components. CONCLUSIONS: The deterministic component describes the presence of a long-range influence that installs an asymmetric distribution of cell proliferation, i.e., an asymmetrically located ZHMD that print space-dependent differences onto the tectal corticogenesis. The stochastic component reveals short-range anti-correlations reflecting spatial clusterization and synchronization between neighboring cells.

Measurement of the optical parameters of the Virgo interferometer.

The Virgo interferometer, aimed at detecting gravitational waves, is now in a commissioning phase. Measurements of its optical properties are needed for the understanding of the instrument. We present the techniques developed for the measurement of the optical parameters of Virgo. These parameters are compared with the Virgo specifications.