A NSC-34 cell line-derived spheroid model: Potential and challenges for in vitro evaluation of neurodegeneration.

Three-dimensional (3D) spheroid models aim to bridge the gap between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models, created by self-clustering cells to mimic a 3D environment with surrounding extracellular framework, provide a valuable research tool. The NSC-34 cell line, generated by fusing mouse spinal cord motor neurons and neuroblastoma cells, is essential for studying neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), where abnormal protein accumulation, such as TAR-DNA-binding protein 43 (TDP-43), occurs in affected nerve cells. However, NSC-34 behavior in a 3D context remains underexplored, and this study represents the first attempt to create a 3D model to determine its suitability for studying pathology. We generated NSC-34 spheroids using a nonadhesive hydrogel-based template and characterized them for 6 days. Light microscopy revealed that NSC-34 cells in 3D maintained high viability, a distinct round shape, and forming stable membrane connections. Scanning electron microscopy identified multiple tunnel-like structures, while ultrastructural analysis highlighted nuclear bending and mitochondria alterations. Using inducible GFP-TDP-43-expressing NSC-34 spheroids, we explored whether 3D structure affected TDP-43 expression, localization, and aggregation. Spheroids displayed nuclear GFP-TDP-43 expression, albeit at a reduced level compared with 2D cultures and generated both TDP-35 fragments and TDP-43 aggregates. This study sheds light on the distinctive behavior of NSC-34 in 3D culture, suggesting caution in the use of the 3D model for ALS or TDP-43 pathologies. Yet, it underscores the spheroids' potential for investigating fundamental cellular mechanisms, cell adaptation in a 3D context, future bioreactor applications, and drug penetration studies. RESEARCH HIGHLIGHTS: 3D spheroid generation: NSC-34 spheroids, developed using a hydrogel-based template, showed high viability and distinct shapes for 6 days. Structural features: advanced microscopy identified tunnel-like structures and nuclear and mitochondrial changes in the spheroids. Protein dynamics: the study observed how 3D structures impact TDP-43 behavior, with altered expression but similar aggregation patterns to 2D cultures. Research implications: this study reveals the unique behavior of NSC-34 in 3D culture, suggests a careful approach to use this model for ALS or TDP-43 pathologies, and highlights its potential in cellular mechanism research and drug testing applications.

Electrophysiological and morphological modulation of neuronal-glial network by breast cancer and nontumorigenic mammary cell conditioned medium.

Breast cancer is a significant global health concern, with the overexpression of human epidermal growth factor receptor 2 (HER2/ERBB2) being a driver oncogene in 20%-30% of cases. Indeed, HER2/ERBB2 plays a crucial role in regulating cell growth, differentiation, and survival via a complex signaling network. Overexpression of HER2/ERBB2 is associated with more aggressive behavior and increased risk of brain metastases, which remains a significant clinical challenge for treatment. Recent research has highlighted the role of breast cancer secretomes in promoting tumor progression, including excessive proliferation, immune invasion, and resistance to anti-cancer therapy, and their potential as cancer biomarkers. In this study, we investigated the impact of ERBB2+ breast cancer SKBR-3 cell line compared with MCF10-A mammary non-tumorigenic cell conditioned medium on the electrophysiological activity and morphology of neural networks derived from neurons differentiated from human induced pluripotent stem cells. Our findings provide evidence of active modulation of neuronal-glial networks by SKBR-3 and MCF10-A conditioned medium. These results provide insights into the complex interactions between breast cancer cells and the surrounding microenvironment. Further research is necessary to identify the specific factors within breast cancer conditioned medium that mediate these effects and to develop targeted therapies that disrupt this interaction.

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells.

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

A biopolymeric mesh enriched with PLGA microparticles loaded with AT101 for localized glioblastoma treatment.

Current treatment strategies for glioblastoma (GBM) including surgical resection and adjuvant radio/chemotherapy result in a limited progression-free survival time of patients due to rapidly occurring tumor recurrences. The urgent need for more effective treatments has led to the development of different approaches for localized drug delivery systems (DDSs) offering the advantages of reduced systemic side effects. A promising candidate for the treatment of GBMs is AT101, the R-(-)-enantiomer of gossypol due to its ability to induce apoptosis or trigger autophagic cell death in tumor cells. Here, we present an alginate-based drug-releasing mesh ladened with AT101-loaded PLGA microspheres (AT101-GlioMesh). The AT101-loaded PLGA microspheres were fabricated using an oil-in-water emulsion solvent evaporation method obtaining a high encapsulation efficiency. The drug-loaded microspheres enabled the release of AT101 over several days at the tumor site. The cytotoxic effect of the AT101-loaded mesh was evaluated using two different GBM cell lines. Strikingly, encapsulation of AT101 in PLGA-microparticles and subsequent embedding in GlioMesh resulted in a sustained delivery and more efficient cytotoxic effect of AT101 on both GBM cell lines. Thus, such a DDS holds promise for GBM therapy likely by preventing the development of tumor recurrences.

A handheld bioprinter for multi-material printing of complex constructs.

In situbioprinting-the process of depositing bioinks at a defected area, has recently emerged as a versatile technology for tissue repair and restorationviasite-specific delivery of pro-healing constructs. The ability to print multiple materialsin situis an exciting approach that allows simultaneous or sequential dispensing of different materials and cells to achieve tissue biomimicry. Herein, we report a modular handheld bioprinter that deposits a variety of bioinksin situwith exquisite control over their physical and chemical properties. Combined stereolithography 3D printing and microfluidic technologies allowed us to develop a novel low-priced handheld bioprinter. The ergonomic design of the handheld bioprinter facilitate the shape-controlled biofabrication of multi-component fibers with different cross-sectional shapes and material compositions. Furthermore, the capabilities of the produced fibers in the local delivery of therapeutic agents was demonstrated by incorporating drug-loaded microcarriers, extending the application of the printed fibers to on-demand, temporal, and dosage-control drug delivery platforms. Also, the versatility of this platform to produce biosensors and wearable electronics was demonstrated via incorporating conductive materials and integrating pH-responsive dyes. The handheld printer's efficacy in generating cell-laden fibers with high cell viability for site-specific cell delivery was shown by producing single-component and multi-component cell-laden fibers. In particular, the multi-component fibers were able to model the invasion of cancer cells into the adjacent tissue.

A micropatterned thermoplasmonic substrate for neuromodulation of in vitro neuronal networks.

Understanding how the spatial organization of a neural network affects its activity represents a leading issue in neuroscience. Thanks to their accessibility and easy handling, in vitro studies remain an essential tool to investigate the relationship between the structure and function of a neuronal network. Among all the patterning techniques, ink-jet printing acquired great interest thanks to its direct-write approach, which allows the patterned substrate realization without mold, leading to a considerable saving of both cost and time. However, the inks commonly used give the possibility to control only the structure of a neuronal network, leaving aside the functional aspect. In this work, we synthesize a photosensitive ink combining the rheological and bioadhesive properties of chitosan with the plasmonic properties of gold nanorods, obtaining an ink able to control both the spatial organization of a two-dimensional neuronal network and its activity through photothermal effect. After the ink characterization, we demonstrate that it is possible to print, with high precision, different geometries on a microelectrode array. In this way, it is possible obtaining a patterned device to control the structure of a neuronal network, to record its activity and to modulate it via photothermal effect. Finally, to our knowledge, we report the first evidence of photothermal inhibition of human neurons activity. STATEMENT OF SIGNIFICANCE: Patterned cell cultures remain the most efficient and simple tool for linking structural and functional studies, especially in the neuronal field. Ink-jet printing is the technique with which it is possible to realize patterned structures in the fastest, simple, versatile and low-cost way. However, the inks currently used permit the control only of the neuronal network structure but do not allow the control-modulation of the network activity. In this study, we realize and characterize a photosensitive bioink with which it is possible to drive both the structure and the activity of a neuronal network. Moreover, we report the first evidence of activity inhibition by the photothermal effect on human neurons as far as we know.

Spheroid size influences cellular senescence and angiogenic potential of mesenchymal stromal cell-derived soluble factors and extracellular vesicles.

Introduction: The secretome of mesenchymal stromal cells (MSCs) serves as an innovative tool employed in the regenerative medicine approach. In this particular context, three-dimensional (3D) culture systems are widely utilized to better replicate in vivo conditions and facilitate prolonged cell maintenance during culture. The use of spheroids enables the preservation of the classical phenotypical characteristics of MSCs. However, the distinct microenvironment within the spheroid may impact the secretome, thereby enhancing the angiogenic properties of adult MSCs that typically possess a reduced angiogenic potential compared to MSCs derived from perinatal tissues due to the hypoxia created in the internal region of the spheroid. Methods: In this study, large spheroids (2,600 cells, ∼300 µm diameter) and small spheroids (1,000 cells, ∼200 µm diameter) were used to examine the role of spheroid diameter in the generation of nutrients and oxygen gradients, cellular senescence, and the angiogenic potential of secreted factors and extracellular vesicles (EVs). Results: In this study, we demonstrate that large spheroids showed increased senescence and a secretome enriched in pro-angiogenic factors, as well as pro-inflammatory and anti-angiogenic cytokines, while small spheroids exhibited decreased senescence and a secretome enriched in pro-angiogenic molecules. We also demonstrated that 3D culture led to a higher secretion of EVs with classical phenotypic characteristics. Soluble factors and EVs from small spheroids exhibited higher angiogenic potential in a human umbilical vein endothelial cell (HUVEC) angiogenic assay. Discussion: These findings highlighted the necessity of choosing the appropriate culture system for obtaining soluble factors and EVs for specific therapeutic applications.

A facile approach for the development of high mechanical strength 3D neuronal network scaffold based on chitosan and graphite nanoplatelets.

In this work, novel composite microparticles based on chitosan (CHI) and graphite nanoplatelets (GNP) were developed as 3D scaffolds for neuronal cells. The aim is to improve the scaffold strength while maintaining its ability to sustain cell adhesion and differentiation. An air-assisted jetting technique followed by physical crosslinking is employed to obtain CHI/GNP microparticles. Optical and Field Emission Scanning Electron Microscopy micrographs showed a uniform distribution of GNP within the CHI porous matrix. The presence of GNP turned out to improve the strength of the microparticles while conferring good electrical conductivity and ameliorating their stability in aqueous environment. The morphological and immunocytochemical characterization, combined with a preliminary electrophysiological analysis, evidenced the effectiveness of the developed composite microparticles as a scaffold for neuron growth. These scaffolds could be employed for the development of advanced 3D neuronal in vitro models for networks dynamics analysis and drug screening.

Assembly of chitosan-graphite oxide nanoplatelets core shell microparticles for advanced 3D scaffolds supporting neuronal networks growth.

This manuscript reports the development of functional 3D scaffolds based on chitosan (CHI) and graphite oxide nanoplatelets (GO) for neuronal network growth. To this aim, CHI microparticles, produced by alkaline gelation method, were coated with GO exploiting a simple template-assisted assembly based on the electrostatic attraction in an aqueous medium. The optimal deposition conditions were evaluated by optical microscopy and studied by quartz crystal microbalance. FE-SEM observations highlight the formation of a core-shell structure where the porous chitosan core is completely wrapped by a uniform GO layer. This outer shell protects the inner chitosan from enzymatic degradation thus potentially extending the scaffold viability for in vivo applications. The presence of hydrophilic oxygen-containing functionalities on the outermost layer of GO and its inner conductive graphitic core maintained the bioactivity of the scaffold and promoted neuronal cell adhesion and growth. The proposed approach to modify the surface of CHI microparticles makes it possible for the design of 3D scaffolds for advanced neuronal tissue engineering applications.

On an effective approach to improve the properties and the drug release of chitosan-based microparticles.

The work investigated the possibility to develop an easy scalable treatment capable of modifying only the surface of chitosan-based materials, limiting the degradation of the bulk and the burst release of a drug, without compromising the properties of the polymeric matrix. To this aim, microparticles of CHI were superficially coated with poly-(styrene-co-maleic anhydride) (PSMA), taking advantage of the potential reactivity of chitosan amino groups and maleic functionalities of PSMA. The specific reactions/interactions occurring between the two polymers were studied by IR measurements, while FE-SEM analysis evidenced the modification of the morphology of the particles contacted with PSMA. Contact angle measurements demonstrated the change of wettability in the modified systems and TGA analysis allowed to estimate the amount of the deposited PSMA. The above treatment turned out to improve the particle stability both in an acidic environment and in an enzymatic system. The release properties of the treated and of the untreated particles, over a period of 10 h, were tested using, as model drug, the protein Bovine Serum Albumin (BSA). Finally, the cytocompatibility of the developed composite microparticles was assessed on MCF-7 human breast cancer cells, which measurements demonstrated the non-toxicity of the treatment.