Culture Model Research Articles

Proteomic analysis of murine norovirus-infected Raw 264.7 cells reveals the induction of Ras GTPases

The human norovirus (abbreviated as HuNV) is the most common agent responsible for acute viral gastroenteritis. Despite being recognized as a water-borne pathogenic virus for a long time, the cellular tropism of norovirus has not yet been clearly explained. The main reason is the lack of appropriate cell culture and animal model systems for HuNV infection. Murine norovirus (abbreviated as MNV) is often used as a proxy for human norovirus when trying to understand the expression profiles left behind by norovirus infection in a host. In the current study, the host response to MNV was examined using the macrophage Raw 264.7 in terms of the altered host proteomes. After MNV infection, host Raw 264.7 cell lysates were collected for proteome profiling at the time points of 0.5 hpi (early phase, control), 16 hpi (mid-phase), and 24 hpi (late phase). LC–MS analysis was employed for label-free shotgun proteomics on the host cell proteomes. The progression of MNV infection status was monitored using an immunofluorescence-conjugated noroviral capsid protein VP1 and a confocal microscope. The up-regulation of Ras GTPases such Rab5A and Rab6A was found to be implicated in norovirus gastroenteritis, as revealed by proteomic profiling. Consequently, the recognition of Ras-related proteins can lead to a better understanding of how noroviral infection affects the immune system of the host cell.

Cultural Sensitivity in AI Language Learning: Using NLP to Enhance Language Understanding Across Cultural Contexts

The aim of this paper is to demonstrate the integration of cultural awareness into AI language learning systems using Natural Language Processing (NLP) models. Recognising that the traditional methods of language learning tend not to capture cultural variations, the studies focus on how culturally relevant NLP models (GPT-3, BERT, RNNs) promote language acquisition and cross-cultural integration. Data preprocessing, cultural data augmentation and model training was used to encode cultural variables such as greetings, politeness, and contextually relevant expressions into training datasets. Experimental results suggest that culturally sensitive models outperform the generic model on tasks that involve cultural awareness, such as politeness detection, cultural phrase recognition, and tone sensitivity. GPT-3 for example improved the accuracy of politeness detection tasks from 72.4% to 85.7%. These results highlight the need to bring cultural awareness into AI to develop engaging, context-sensitive language learning. It concludes that culturally based NLP models enhance learner capacity for real-world communication and foster global cultural awareness. Future work should focus on expanding culturally diverse datasets and refining AI models to address subtle cultural complexities

Phytomedicine Potential of Oroxylum indicum Root and Its Constituents: Targeting Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative condition characterized by a gradual decline in cognitive function, for which few effective treatments exist. This study investigated the neuroprotective potential of Oroxylum indicum root extract and its key constituents (baicalein, chrysin, oroxylin A) against AD hallmarks. The extract and its constituents exhibited antioxidant activity in the DPPH assay. They inhibited β-amyloid aggregation as measured by the thioflavin T assay and acetylcholinesterase activity using the Ellman method. In cell culture models, O. indicum extract showed an ability to protect neurons from the toxic effects of H2O2. Western blot analysis revealed the extract and its major active component, baicalein, downregulated pro-apoptotic markers (cleaved caspase-3, and BAX) upon H2O2 exposure. Furthermore, they reduced the expression of amyloidogenic proteins (BACE1) and phosphorylated tau. These findings suggest that O. indicum root extract, particularly baicalein, possesses multifaceted neuroprotective properties, targeting various aspects of AD pathogenesis, including oxidative stress, cholinergic dysfunction, β-amyloid formation, aggregation, and apoptosis. O. indicum root thus warrants further investigation as a promising source of therapeutic agents for AD.

Therapeutic Potential of Targeting Ferroptosis in Periprosthetic Osteolysis Induced by Ultra-High-Molecular-Weight Polyethylene Wear Debris

Background/Objectives: Periprosthetic osteolysis is the primary cause of arthroplasty failure in the majority of patients. Mechanistically, wear debris released from the articulating surfaces of a prosthesis initiates local inflammation and several modes of regulated cell death programs, such as ferroptosis, which represents a promising therapeutic target in various chronic inflammatory diseases. Thus, the current study aimed at exploring the therapeutic potential of targeting ferroptosis in a polyethylene-wear-debris-induced osteolysis model. Methods: Inverted cell culture model was used for stimulating the cells with wear debris in vitro, and calvarial osteolysis model was used for evaluating the therapeutic effects of inhibitors in vivo. Results: The immunostaining of periprosthetic bone tissues demonstrated a number of osteocytes expressing ferroptosis markers. Likewise, the expressions of ferroptosis markers were confirmed in polyethylene-wear-debris-stimulated osteocyte-like cells and primary osteoblasts in a direct stimulation model but not in an indirect stimulation model. Furthermore, polyethylene wear debris was implanted onto calvarial bone and mice were treated with the ferroptosis inhibitors DFO and Fer-1. These treatments alleviated the inflammatory and pathological bone resorption induced by the wear debris implantation. Conclusions: Our data broaden the knowledge of the pathogenesis of periprosthetic osteolysis and highlight ferroptosis as a promising therapeutic target.

Visible and invisible cultural patterns influencing women’s use of maternal health services among Igala women in Nigeria: a focused ethnographic study

BackgroundExplicit and implicit cultural patterns are critical cultural norms, beliefs, and practices that determine women’s health-seeking behaviour. These cultural patterns could limit women’s use of maternal health services, resulting in maternal health complications. The study aims to provide an in-depth understanding of explicit and implicit cultural patterns, their meanings and how they influence women’s use of maternal health services among Igala women in Nigeria.MethodsRoper and Shapira’s (2000) focused ethnography was employed with 43 women aged 18–43 years recruited using the purposive and snowballing technique. The study was conducted with 21 women for one-on-one interviews and two focus group discussions with six women in the rural area and seven women in the urban area. In addition, participant observation of nine women from the third trimester to birth was conducted, yielding 189 h of observation in two primary healthcare facilities in rural and urban areas. Data analysis was conducted using Roper and Shapira’s (2000) method.ResultThree themes were generated using the PEN3 cultural model: perceptions, enabler, and nurturers. Subthemes generated under the theme of perception were, Belief in witchcraft, Pregnancy announces itself, I cannot tell people I am in labour, and Unspoken acquiescence to the culture. Under the theme of nurturer, Home birth was found to limit access to maternal health services. The theme of enabler yielded subthemes such as You are not woman enough; I want my placenta and Rising matriarchs.ConclusionImplicit and explicit cultural patterns significantly influence women’s use of maternal health services. Given the complexity of culture and its influence on women’s use of maternal health services, multifaceted strategies tailored to the cultural needs of communities are needed to enhance the realization of Sustainable Development Goal #3:1 in Nigeria.

Improving 3D deep learning segmentation with biophysically motivated cell synthesis

Biomedical research increasingly relies on three-dimensional (3D) cell culture models and artificial-intelligence-based analysis can potentially facilitate a detailed and accurate feature extraction on a single-cell level. However, this requires for a precise segmentation of 3D cell datasets, which in turn demands high-quality ground truth for training. Manual annotation, the gold standard for ground truth data, is too time-consuming and thus not feasible for the generation of large 3D training datasets. To address this, we present a framework for generating 3D training data, which integrates biophysical modeling for realistic cell shape and alignment. Our approach allows the in silico generation of coherent membrane and nuclei signals, that enable the training of segmentation models utilizing both channels for improved performance. Furthermore, we present a generative adversarial network (GAN) training scheme that generates not only image data but also matching labels. Quantitative evaluation shows superior performance of biophysical motivated synthetic training data, even outperforming manual annotation and pretrained models. This underscores the potential of incorporating biophysical modeling for enhancing synthetic training data quality.

Poly(curcumin-co-poly(ethylene glycol)) films provide neuroprotection following reactive oxygen species insult in vitro.

Curcumin is an antioxidant and anti-inflammatory molecule that may provide neuroprotection following central nervous system (CNS) injury. However, curcumin is hydrophobic, limiting its ability to be loaded and then released from biomaterials for neural applications. We previously developed polymers containing curcumin, and these polymers may be applied to neuronal devices or to neural injury to promote neuroprotection. Thus, our objective was to evaluate two curcumin polymers as potential neuroprotective materials for neural applications. 
Approach: For each curcumin polymer, we created three polymer solutions by varying the weight percentage of curcumin polymer in solvent. These solutions were subsequently coated onto glass coverslips, and the thickness of the polymer was assessed using profilometry. Polymer degradation and dissolution was assessed using brightfield microscopy, scanning electron microscopy, and gel permeation chromatography. The ability of the polymers to protect cortical neurons from free radical insult was assessed using an in vitro cortical culture model.
Main Results: The P50 curcumin polymer (containing greater poly(ethylene glycol) content than the P75 polymer), eroded readily in solution, with erosion dependent on the weight percentage of polymer in solvent. Unlike the P50 polymer, the P75 polymer did not undergo erosion. Since the P50 polymer underwent erosion, we expected that the P50 polymer would more readily protect cortical neurons from free radical insult. Unexpectedly, even though P75 films did not erode, P75 polymers protected neurons from free radical insult, suggesting that erosion is not necessary for these polymers to enable neuroprotection.
Significance: This study is significant as it provides a framework to evaluate polymers for future neural applications. Additionally, we observed that some curcumin polymers do not require dissolution to enable neuroprotection. Future work will assess the ability of these materials to enable neuroprotection within in vivo models of neural injury.&#xD.

Phenotypic analysis of complex bioengineered 3D models.

With advances in underlying technologies such as complex multicellular systems, synthetic materials, and bioengineering techniques, we can now generate in vitro miniaturized human tissues that recapitulate the organotypic features of normal or diseased tissues. Importantly, these 3D culture models have increasingly provided experimental access to diverse and complex tissues architectures and their morphogenic assembly in vitro. This review presents an analytical toolbox for biological researchers using 3D modeling technologies through which they can find a collation of currently available methods to phenotypically assess their 3D models in their normal state as well as their response to therapeutic or pathological agents.

3D Cell Culture Models as a Platform for Studying Tumor Progression, Testing Treatment Responses, and Discovering Biomarkers.

In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.

A Comparative Study of Teacher Training Systems in China and Nigeria

This paper explores the historical background and cultural influences of the teacher training systems in China and Nigeria and the characteristics of their training systems. First, it analyses the traditional culture and modern management model of teacher training in China, emphasising the combination of professional ethics and professional competence. Subsequently, the impact of Nigeria's multicultural background on its teacher training is explored, pointing out its complex training system in the context of geographical and cultural differences. Finally, by comparing the differences between the two countries in terms of objectives, methods and contents of teacher training, corresponding recommendations are made, such as the suggestion that Chinese teacher training can add more excellent traditional culture and encourage teachers to incorporate it into their daily lessons. Teacher training in Nigeria should be more targeted, taking into account the specific local cultural background, infrastructure, etc., to improve the professionalism and quality of teachers and provide a reference for improving the professional level of teachers and the quality of education.

Doxorubicin and topotecan resistance in ovarian cancer: Gene expression and microenvironment analysis in 2D and 3D models.

This study explores the mechanisms underlying chemotherapy resistance in ovarian cancer (OC) using doxorubicin (DOX) and topotecan (TOP)-resistant cell lines derived from the drug-sensitive A2780 ovarian cancer cell line. Both two-dimensional (2D) monolayer cell cultures and three-dimensional (3D) spheroid models were employed to examine the differential drug responses in these environments. The results revealed that 3D spheroids demonstrated significantly higher resistance to DOX and TOP than 2D cultures, suggesting a closer mimicry of in vivo tumour conditions. Molecular analyses identified overexpression of essential drug resistance-related genes, including MDR1 and BCRP, and extracellular matrix (ECM) components, such as MYOT and SPP1, which were more pronounced in resistant cell lines. MDR1 and BCRP overexpression contribute to chemotherapy resistance in OC by expelling drugs like DOX and TOP. Targeting these transporters with inhibitors or gene silencing could improve drug efficacy, making them key therapeutic targets to enhance treatment outcomes for drug-resistant OC. The study further showed that EMT-associated markers, including VIM, SNAIL1, and SNAIL2, were upregulated in the 3D spheroids, reflecting a more mesenchymal phenotype. These findings suggest that factors beyond gene expression, such as spheroid architecture, cell-cell interactions, and drug penetration, contribute to the enhanced resistance observed in 3D cultures. These results highlight the importance of 3D cell culture models for a more accurate representation of tumour drug resistance mechanisms in ovarian cancer, providing valuable insights for therapeutic development.

Synergistic Effects of Mistletoe Lectin and Cisplatin on Triple-Negative Breast Cancer Cells: Insights from 2D and 3D In Vitro Models.

Triple-negative breast cancer (TNBC) remains a challenging subtype due to its aggressive nature and limited treatment options. This study investigated the potential synergistic effects of Korean mistletoe lectin (Viscum album L. var. coloratum agglutinin, VCA) and cisplatin on MDA-MB-231 TNBC cells using both 2D and 3D culture models. In 2D cultures, the combination of VCA and cisplatin synergistically inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase. Also, the combination treatment significantly reduced cell migration and invasion. Gene expression analysis showed significant changes in specific genes related to apoptosis (Bax, Bcl-2), metastasis (MMP-2, MMP-9), and EMT (E-cadherin, N-cadherin). Three-dimensional spheroid models corroborated these findings, demonstrating enhanced cytotoxicity and reduced invasion with the combination treatment. Significantly, the 3D models exhibited differential drug sensitivity compared to 2D cultures, emphasizing the importance of utilizing physiologically relevant models in preclinical studies. The combination treatment also reduced the expression of angiogenesis-related factors VEGF-A and HIF-1α. This comprehensive study provides substantial evidence for the potential of VCA and cisplatin combination therapy in TNBC treatment and underscores the significance of integrating 2D and 3D models in preclinical cancer research.

Using the Conserved Hexapeptide in HOX Proteins as an Antagonist of HOX/PBX Interactions.

The HOX and PBX genes encode transcription factors that have key roles in development and cancer, both independently and as a heterodimer within a complex of proteins that recognizes specific sequences in DNA and can both activate and repress transcription of target genes. Due to functional redundancy amongst HOX proteins, knock down or knock out studies of individual genes often do not result in an altered phenotype. An alternative approach is to target the interaction between HOX and PBX proteins, which is dependent on a conserved hexapeptide region within HOX. To this end, several peptides have been developed based on the hexapeptide sequence which act as competitive antagonists of HOX/PBX binding, including HXR9 and HTL001. Here, we review the methodology that has been used in these studies, including peptide syntheses, cell culture, assays, and mouse models.

S6K1 is a Targetable Vulnerability in Tumors Exhibiting Plasticity and Therapy Resistance.

Background: Most tumors initially respond to treatment, yet refractory clones subsequently develop owing to resistance mechanisms associated with cancer cell plasticity and heterogeneity. Methods: We used a chemical biology approach to identify protein targets in cancer cells exhibiting diverse driver mutations and representing models of tumor lineage plasticity and therapy resistance. An unbiased screen of a drug library was performed against cancer cells followed by synthesis of chemical analogs of the most effective drug. The cancer subtype target range of the leading drug was determined by PRISM analysis of over 900 cancer cell lines at the Broad Institute, MA. RNA-sequencing and enrichment analysis of differentially expressed genes, as well as computational molecular modeling and pull-down with biotinylated small molecules were used to identify and validate RPS6KB1 (p70S6K or S6K1) as an essential target. Genetic restoration was used to test the functional role of S6K1 in cell culture and xenograft models. Results: We identified a novel derivative of the antihistamine drug ebastine, designated Super-ebastine (Super-EBS), that inhibited the viability of cancer cells representing diverse KRAS and EGFR driver mutations and models of plasticity and treatment resistance. Interestingly, PRISM analysis indicated that over 95% of the diverse cancer cell lines tested were sensitive to Super-EBS and the predicted target was the serine/threonine kinase S6K1. S6K1 is upregulated in various cancers relative to counterpart normal/benign tissues and phosphorylated-S6K1 predicts poor prognosis for cancer patients. We noted that inhibition of S6K1 phosphorylation was necessary for tumor cell growth inhibition, and restoration of phospho-S6K1 rendered tumor cells resistant to Super-EBS. Inhibition of S6K1 phosphorylation by Super-EBS induced caspase-2 dependent apoptosis via inhibition of the Cdc42/Rac-1/p-PAK1 pathway that led to actin depolymerization and caspase-2 activation. The essential role of S6K1 in the action of Super-EBS was recapitulated in xenografts, and knockout of S6K1 abrogated tumor growth in mice. Conclusion: S6K1 is a therapeutic vulnerability in tumors exhibiting intrinsic and/or acquired resistance to treatment.

Exploring bovine three-dimensional chondrocyte culture models in osteoarthritis research: A systematic review

Background: The use of different animal species for chondrocyte culture has been employed to investigate the diseases that affect cartilage, including osteoarthritis. Bovine cartilage and chondrocytes can be used to establish three-dimensional cell cultures, which offer a more dependable in vitro model when compared to conventional monolayer cultures. However, bovine chondrocytes in three-dimensional cultures have not been widely implemented, losing a potential source of mammal tissue that could prove valuable for preclinical studies on osteoarthritis. Objective: The objective of this study was to conduct a comprehensive review of the existing scientific literature that employs three-dimensional cultures of bovine cartilage to investigate osteoarthritis. Methods: A systematic search was performed using the electronic databases PubMed and Scopus, to identify clinical studies using 3D cell culture for osteoarthritis. Search terms included: ´3D culture’, ‘3D cell culture’, ‘bovine cartilage’ and ‘chondrocyte’. A total of 59 articles were gathered, and after screening, 12 articles were included in the final analysis. Risk of bias assessment was conducted categorizing each of the studies as having a 'low,' 'medium,' or 'high' risk of bias. Results: Analysis of the articles included in this review highlighted the increased variability in harvesting sites involving carpal, metacarpal, and knee joints, as well as variation in culture methods utilizing cell passages ranging from passage zero to passage nine. Moreover, medium, and high risk of bias were detected in all the articles probably due to challenges in randomization and blinding of the studies. In summary, this review critically examines three-dimensional cell culture for the investigation of cartilage disorders, with a particular emphasis on bovine cartilage. Conclusions: Future studies should include consistent methods across the in vitro phase of the study, such as uniform harvest sites, as well as using early chondrocyte passages to preserve cellular phenotype. Furthermore, comparison of relevant translational models should include age-matched conditions to avoid further confounding factors.

Gelatin nanofibers coated with hyaluronic acid as a mesenchymal stromal cell scaffold for corneal regeneration

Electrospun gelatin nanofibers coated with hyaluronic acid (GelNF-HA) were synthesized as a scaffold for delivering human corneal mesenchymal stromal cells (C-MSCs) directly to deep corneal injuries. Aligned GelNFs were produced by electrospinning, crosslinked using vapor of glutaraldehyde, coated with HA, and crosslinked with EDC/NHS. The GelNF-HA was characterized by SEM, mechanical, and optical properties. It was then investigated as a substrate for C-MSC proliferation and migration in vitro and in a rabbit cornea culture model. The expression of α-smooth muscle actin (α-SMA) was determined in the ex vivo model. SEM showed that the GelNF-HA scaffold was composed of aligned GelNFs with 75 % of the fibers oriented against the same angle. It exhibited a Young’s modulus of 1.66 ± 0.59 MPa and approximately 93 % transmittance of visible light. The GelNF-HA membranes supported C-MSC proliferation in vitro. In a scratch migration assay, it facilitated complete wound closure after 48 h in culture. C-MSC-laden GelNF-HA scaffolds supported corneal wound healing in an ex vivo model as well, expressing a lower percentage of stromal α-SMA compared to both the no-treatment keratectomy-only and C-MSC groups (p < 0.05). The C-MSC-supportive GelNF-HA scaffolds hold therapeutic potential for stromal regeneration in the treatment of deep corneal defects.

The Role of H3K27 acetylation in oxygen-glucose deprivation-induced spinal cord injury and potential for neuroprotective therapies

ObjectiveSpinal cord injury (SCI) is a debilitating condition that often results in paralysis and lifelong medical challenges. Research has shown that epigenetic modifications, particularly histone acetylation, play a role in neuroprotection following hypoxic-ischemic events in SCI. The objective of this study was to explore the effects of histone H3K27 acetylation, along with its underlying mechanisms, on the tolerance to hypoxia and ischemia in SCI. MethodsThis study employed an organotypic spinal cord slice culture model subjected to oxygen-glucose deprivation (OGD). We assessed cell apoptosis and changes in cellular type patterns under these conditions. Following hypoxia and ischemia, we analyzed the expression and distribution of H3K27ac across various nerve cell types. To identify key downstream genes, we integrated ChIP-seq and RNA-seq analyses, investigating molecular mechanisms driving the response to OGD in this model. ResultsOGD stimulation increased cell apoptosis and induced time-dependent changes in the expression patterns of neurons, astrocytes, microglia, and oligodendrocytes in organotypic spinal cord slices, accompanied by a significant reduction in H3K27ac levels. Integrated ChIP-seq and RNA-seq analyses revealed that H3K27ac downregulation under hypoxic and ischemic conditions contributes to spinal cord damage by promoting neuroinflammation and disrupting gene regulation. Furthermore, we identified key downstream targets, including Apoc1, Spp1, Aff1, Brd4, KCNN3, and Rgma, which may represent promising therapeutic targets for SCI. ConclusionOur data underscore the pivotal role of H3K27ac in the organotypic spinal cord slice culture model following OGD exposure, offering promising avenues for neuroprotective therapies via epigenetic-immune regulation.

CEA-Induced PI3K/AKT Pathway Activation through the Binding of CEA to KRT1 Contributes to Oxaliplatin Resistance in Gastric Cancer

BackgroundThe serum level of carcinoembryonic antigen (CEA) has prognostic value in patients with gastric cancer (GC) receiving oxaliplatin-based chemotherapy. As the molecular functions of CEA are increasingly uncovered, its role in regulating oxaliplatin resistance in GC attracts attention. MethodsThe survival analysis adopted the KaplanMeier method. Effects of CEA on proliferative capacity were investigated using CCK8, colony formation, and xenograft assays. Oxaliplatin sensitivity was identified through IC50 detection, apoptosis analysis, comet assay, organoid culture model, and xenograft assay. Multi-omics approaches were utilized to explore CEA’s downstream effects. The binding of CEA to KRT1 was confirmed through proteomic analysis and Co-IP, GST pull-down, and immunofluorescence colocalization assays. Furthermore, small molecule inhibitors were identified using virtual screening and surface plasmon resonance. ResultsStarting from clinical data, we confirmed that CEA demonstrated superior ability to predict the prognosis of patients with GC who received oxaliplatin-based chemotherapy, particularly in predicting recurrence-free survival based on serum CEA level. In vitro and in vivo experiments revealed CEAhigh GC cells presented increased proliferative capacity and decreased oxaliplatin sensitivity. The resistance phenotype was transmitted through secreted CEA. Multi-omics analysis revealed that CEA activated the PI3K/AKT pathway by binding to KRT1, leading to oxaliplatin resistance. Finally, the small molecule inhibitor evacetrapib, which competitively inhibits the CEA-KRT1 interaction, was identified and validated in vitro. ConclusionsIn summary, the CEA-KRT1-PI3K/AKT axis regulates oxaliplatin sensitivity in GC cells. Treatment with small molecule inhibitors such as evacetrapib to inhibit this interaction constitutes a novel therapeutic strategy.

Nanoparticle shape is the game-changer for blood-brain barrier crossing and delivery through tunneling nanotubes among glioblastoma cells.

Tunneling nanotubes (TNTs) are thin, dynamic, long membrane protrusions that allow intercellular exchanges of signaling clues, molecules and organelles. The presence of TNTs and their involvement as drug delivery channels have been observed in several types of cancer, including glioblastoma. Recently, increased attention has been directed toward nanoparticles (NPs) that can be transported in TNTs. However, few data are available on the role of physical parameters of nanoparticles, such as size, shape, charge and flexibility, in determining their transfer efficiency between cells by TNTs. Here, we focused our attention on NP shape, manufacturing spherical, discoidal and deformable negatively charged lipid-based NPs with sizes <120 nm and similar stiffness. The TNT-mediated transfer of NPs was investigated in 2D and 3D culture models of human glioblastoma cells. The permeability and biocompatibility of the blood-brain barrier (BBB) were also assessed. Results showed that discoidal NPs displayed the highest TNT-mediated transfer efficiency between cancer cells, with a maximum velocity of 69 nm s-1, and a higher endothelial permeability (1.29 × 10-5 cm min-1) across the BBB in an in vitro model. This depends on the NP shape because discoidal NPs have a larger surface area exposed to the flow along the TNT channel. Overall, the results suggest that the shape of NPs is the game-changer for more efficient TNT-mediated transfer between cancer cells, thus introducing a sustainable solution to improve the diffusion rate at which the NPs spread in the tumour microenvironment, opening the possibility of ameliorating drug distribution to difficult-to-reach cancer cell populations.

MAPK14/p38α shapes the molecular landscape of endometrial cancer and promotes tumorigenic characteristics.

The molecular underpinnings of high-grade endometrial carcinoma (HGEC) metastatic growth and survival are poorly understood. Here, we show that ascites-derived and primary tumor HGEC cell lines in 3D spheroid culture faithfully recapitulate key features of malignant peritoneal effusion and exhibit fundamentally distinct transcriptomic, proteomic, and metabolomic landscapes compared with conventional 2D monolayers. Using a genetic screening platform, we identify MAPK14 (which encodes the protein kinase p38α) as a specific requirement for HGEC in spheroid culture. MAPK14/p38α has broad roles in programming the phosphoproteome, transcriptome, and metabolome of HGEC spheroids, yet has negligible impact on monolayer cultures. MAPK14 promotes tumorigenicity invivo and is specifically required to sustain a sub-population of spheroid cells that is enriched in cancer stemness markers. Therefore, spheroid growth of HGEC activates unique biological programs, including p38α signaling, that cannot be captured using 2D culture models and are highly relevant to malignant disease pathology.