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Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.
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Técnicas de Cultivo de Célula/métodos , Hematopoyesis , Macrófagos/fisiología , Neuronas/fisiología , Células Madre Pluripotentes/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NeurogénesisRESUMEN
Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.
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Neuronas , Células de Schwann , Animales , Ratones , Ratas , Técnicas de Cocultivo , Axones , Animales Modificados GenéticamenteRESUMEN
At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.
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Ascitis , Fibrina , Fibrinógeno , Integrina alfa5beta1 , Neoplasias Ováricas , Esferoides Celulares , Microambiente Tumoral , Humanos , Femenino , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Ascitis/patología , Ascitis/metabolismo , Integrina alfa5beta1/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Fibrinógeno/metabolismo , Fibrina/metabolismo , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/patología , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Línea Celular Tumoral , Receptores de Vitronectina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adhesión Celular , Peritoneo/patología , Peritoneo/metabolismo , Epitelio/metabolismo , Epitelio/patología , Cadherinas/metabolismo , Células Tumorales CultivadasRESUMEN
Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.
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Lesión Pulmonar Aguda , Células Madre Mesenquimatosas , Neumonía , Ratones , Animales , Macrófagos Alveolares/metabolismo , Lipopolisacáridos/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/terapia , Pulmón/metabolismo , Macrófagos/metabolismo , Modelos Animales de Enfermedad , Inflamación/terapia , Inflamación/metabolismo , Organoides/metabolismoRESUMEN
Solar-driven bioelectrosynthesis represents a promising approach for converting abundant resources into value-added chemicals with renewable energy. Microorganisms powered by electrochemical reducing equivalents assimilate CO2, H2O, and N2 building blocks. However, products from autotrophic whole-cell biocatalysts are limited. Furthermore, biocatalysts tasked with N2 reduction are constrained by simultaneous energy-intensive autotrophy. To overcome these challenges, we designed a biohybrid coculture for tandem and tunable CO2 and N2 fixation to value-added products, allowing the different species to distribute bioconversion steps and reduce the individual metabolic burden. This consortium involves acetogen Sporomusa ovata, which reduces CO2 to acetate, and diazotrophic Rhodopseudomonas palustris, which uses the acetate both to fuel N2 fixation and for the generation of a biopolyester. We demonstrate that the coculture platform provides a robust ecosystem for continuous CO2 and N2 fixation, and its outputs are directed by substrate gas composition. Moreover, we show the ability to support the coculture on a high-surface area silicon nanowire cathodic platform. The biohybrid coculture achieved peak faradaic efficiencies of 100, 19.1, and 6.3% for acetate, nitrogen in biomass, and ammonia, respectively, while maintaining product tunability. Finally, we established full solar to chemical conversion driven by a photovoltaic device, resulting in solar to chemical efficiencies of 1.78, 0.51, and 0.08% for acetate, nitrogenous biomass, and ammonia, correspondingly. Ultimately, our work demonstrates the ability to employ and electrochemically manipulate bacterial communities on demand to expand the suite of CO2 and N2 bioelectrosynthesis products.
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Dióxido de Carbono , Firmicutes , Fijación del Nitrógeno , Fotosíntesis , Rhodopseudomonas , Acetatos/metabolismo , Amoníaco , Dióxido de Carbono/metabolismo , Técnicas de Cocultivo , Ecosistema , Firmicutes/crecimiento & desarrollo , Firmicutes/metabolismo , Nitrógeno/metabolismo , Rhodopseudomonas/crecimiento & desarrollo , Rhodopseudomonas/metabolismoRESUMEN
Tumor mass comprises not only cancer cells but also heterogeneous populations of immune and stromal cells, along with the components of the extracellular matrix, collectively called the tumor microenvironment (TME). This diverse population of cells can communicate with each other, which can positively or negatively affect tumor growth and progression to malignancy. The most common type of immune cells in the TME are macrophages. Macrophages continuously differentiate into a broad landscape of tumor-associated macrophages (TAMs) in response to numerous signals from the TME, which makes studies on TAMs quite challenging. Therefore, implementing reliable protocols is a milestone for drawing consistent conclusions about the interactions between cancer cells and TAMs. Here, we provide the details for the polarization of a human leukemia monocytic cell line, THP-1, into M0, M1 and M2 macrophages. We also present a step-by-step protocol for a transwell co-culture using a human breast cancer cell line, HCC1806, and THP-1-derived macrophages. Finally, we describe the colony formation and migration assays performed on the breast cancer cells after the co-culture with macrophages to measure the influence of macrophages on the oncogenic features of cancer cells. In summary, our co-culture-based protocols can be a valuable resource for investigating the interactions between macrophages and cancer cells.
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Neoplasias de la Mama , Humanos , Femenino , Técnicas de Cocultivo , Neoplasias de la Mama/patología , Macrófagos/metabolismo , Línea Celular , Microambiente Tumoral , Línea Celular TumoralRESUMEN
Mechanical impact-induced primary injury after traumatic brain injury (TBI) leads to acute microglial pro-inflammatory activation and consequently mediates neurodegeneration, which is a major secondary brain injury mechanism. However, the detailed pathologic cascades have not been fully elucidated, partially because of the pathologic complexity in animal TBI models. Although there are several in vitro TBI models, none of them closely mimic post-TBI microglial activation. In the present study, we aimed to establish an in vitro TBI model, specifically reconstituting the pro-inflammatory activation and associated neurodegeneration following TBI. We proposed three sets of experiments. First, we established a needle scratch injured neuron-induced microglial activation and neurodegeneration in vitro model of TBI. Second, we compared microglial pro-inflammatory cytokines profiles between the in vitro TBI model and TBI in male mice. Additionally, we validated the role of injured neurons-derived damage-associated molecular patterns in amplifying microglial pro-inflammatory pathways using the in vitro TBI model. Third, we applied the in vitro model for the first time to characterize the cellular metabolic profile of needle scratch injured-neuron-activated microglia, and define the role of metabolic reprogramming in mediating pro-inflammatory microglial activation and mediated neurodegeneration. Our results showed that we successfully established a novel in vitro TBI model, which closely mimics primary neuronal injury-triggered microglial pro-inflammatory activation and mediated neurodegeneration after TBI. This in vitro model provides an advanced and highly translational platform for dissecting interactions in the pathologic processes of neuronal injury-microglial activation-neuronal degeneration cascade, and elucidating the detailed underlying cellular and molecular insights after TBI.SIGNIFICANCE STATEMENT Microglial activation is a key component of acute neuroinflammation that leads to neurodegeneration and long-term neurologic outcome deficits after TBI. However, it is not feasible to truly dissect primary neuronal injury-induced microglia activation, and consequently mediated neurodegeneration in vivo Furthermore, there is currently lacking of in vitro TBI models closely mimicking the TBI primary injury-mediated microglial activation. In this study, we successfully established and validated a novel in vitro TBI model of microglial activation, and for the first time, characterized the cellular metabolic profile of microglia in this model. This novel microglial activation in vitro TBI model will help in elucidating microglial inflammatory activation and consequently associated neurodegeneration after TBI.
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Lesiones Traumáticas del Encéfalo , Microglía , Ratones , Masculino , Animales , Microglía/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Macrófagos/metabolismo , Neuronas/metabolismo , Ratones Endogámicos C57BLRESUMEN
Cell-cell interactions, which allow cells to communicate with each other through molecules in their microenvironment, are critical for the growth, health, and functions of cells. Previous studies show that drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level, which is partially responsible for cancer recurrence. Studying protein targets and pathways involved in cell-cell communication provides essential information for fundamental cell biology studies and therapeutics of human diseases. In the current studies, we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication. Comparative studies were carried out using monoculture cells. Shotgun bottom-up proteomics results indicate that the P53 signaling pathway has a strong association with drug resistance mechanisms, and multiple TP53-related proteins were upregulated in both direct and indirect coculture systems. In addition, cell-cell communication pathways, including the phagosome and the HIF-signaling pathway, contribute to both direct and indirect coculture systems. Consequently, AK3 and H3-3A proteins were identified as potential targets for cell-cell interactions that are relevant to drug resistance mechanisms. We propose that the P53 signaling pathway, in which mitochondrial proteins play an important role, is responsible for inducing drug resistance through communication between drug-resistant and drug-sensitive cancer cells.
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Comunicación Celular , Técnicas de Cocultivo , Resistencia a Antineoplásicos , Proteómica , Transducción de Señal , Proteína p53 Supresora de Tumor , Humanos , Proteómica/métodos , Comunicación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológicoRESUMEN
The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.
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Microbiota , Proteoma , Técnicas de Cocultivo , Peso Molecular , ProteómicaRESUMEN
Colorectal cancer (CRC) contains considerable heterogeneity; therefore, models of the disease must also reflect the multifarious components. Compared to traditional 2D models, 3D cellular models, such as tumor spheroids, have the utility to determine the drug efficacy of potential therapeutics. Monoculture spheroids are well-known to recapitulate gene expression, cell signaling, and pathophysiological gradients of avascularized tumors. However, they fail to mimic the stromal cell influence present in CRC, which is known to perturb drug efficacy and is associated with metastatic, late-stage colorectal cancer. This study seeks to develop a cocultured spheroid model using carcinoma and noncancerous fibroblast cells. We characterized the proteomic profile of cocultured spheroids in comparison to monocultured spheroids using data-independent acquisition with gas-phase fractionation. Specifically, we determined that proteomic differences related to translation and mTOR signaling are significantly increased in cocultured spheroids compared to monocultured spheroids. Proteins related to fibroblast function, such as exocytosis of coated vesicles and secretion of growth factors, were significantly differentially expressed in the cocultured spheroids. Finally, we compared the proteomic profiles of both the monocultured and cocultured spheroids against a publicly available data set derived from solid CRC tumors. We found that the proteome of the cocultured spheroids more closely resembles that of the patient samples, indicating their potential as tumor mimics.
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Técnicas de Cocultivo , Proteómica , Transducción de Señal , Esferoides Celulares , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteómica/métodos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Línea Celular Tumoral , Biosíntesis de Proteínas , Fibroblastos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteoma/análisis , Proteoma/metabolismoRESUMEN
As an advance laboratory model, three-dimensional (3D) organoid culture has recently been recruited to study development, physiology and abnormality of kidney tissue. Micro-tissues derived from primary renal cells are composed of 3D epithelial structures representing the main characteristics of original tissue. In this research, we presented a simple method to isolate mouse renal clonogenic mesenchymal (MLCs) and epithelial-like cells (ELCs). Then we have done a full characterization of MLCs using flow cytometry for surface markers which showed that more than 93% of cells expressed these markers (Cd44, Cd73 and Cd105). Epithelial and stem/progenitor cell markers characterization also performed for ELC cells and upregulating of these markers observed while mesenchymal markers expression levels were not significantly increased in ELCs. Each of these cells were cultured either alone (ME) or in combination with human umbilical vein endothelial cells (HUVECs) (MEH; with an approximate ratio of 10:5:2) to generate more mature kidney structures. Analysis of 3D MEH renal micro-tissues (MEHRMs) indicated a significant increase in renal-specific gene expression including Aqp1 (proximal tubule), Cdh1 (distal tubule), Umod (loop of Henle), Wt1, Podxl and Nphs1 (podocyte markers), compared to those groups without endothelial cells, suggesting greater maturity of the former tissue. Furthermore, ex ovo transplantation showed greater maturation in the constructed 3D kidney.
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Células Endoteliales de la Vena Umbilical Humana , Riñón , Animales , Riñón/metabolismo , Riñón/citología , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Organoides/metabolismo , Organoides/citología , Células Epiteliales/metabolismo , Células Epiteliales/citología , Diferenciación Celular , Biomarcadores/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Técnicas de Cultivo Tridimensional de Células/métodosRESUMEN
Recurrent hormone receptor-positive (HR+) breast cancer kills more than 600,000 women annually. Although HR+ breast cancers typically respond well to therapies, approximately 30% of patients relapse. At this stage, the tumors are usually metastatic and incurable. Resistance to therapy, particularly endocrine therapy is typically thought to be tumor intrinsic (e.g., estrogen receptor mutations). However, tumor-extrinsic factors also contribute to resistance. For example, stromal cells, such as cancer-associated fibroblasts (CAFs), residing in the tumor microenvironment, are known to stimulate resistance and disease recurrence. Recurrence in HR+ disease has been difficult to study due to the prolonged clinical course, complex nature of resistance, and lack of appropriate model systems. Existing HR+ models are limited to HR+ cell lines, a few HR+ organoid models, and xenograft models that all lack components of the human stroma. Therefore, there is an urgent need for more clinically relevant models to study the complex nature of recurrent HR+ breast cancer, and the factors contributing to treatment relapse. Here, we present an optimized protocol that allows a high take-rate, and simultaneous propagation of patient-derived organoids (PDOs) and matching CAFs, from primary and metastatic HR+ breast cancers. Our protocol allows for long-term culturing of HR+ PDOs that retain estrogen receptor expression and show responsiveness to hormone therapy. We further show the functional utility of this system by identifying CAF-secreted cytokines, such as growth-regulated oncogene α , as stroma-derived resistance drivers to endocrine therapy in HR+ PDOs.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Recurrencia Local de Neoplasia/patología , Fibroblastos/metabolismo , Organoides/metabolismo , Microambiente TumoralRESUMEN
Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A1R, A2AR, A2BR, and A3R). We previously demonstrated that the Gs-coupled A2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (IK) as well as transient (IA) outward K+ currents. To deepen the unclear role of this receptor subtype in neuron-OL interplay and in myelination process, we tested the effects of different A2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A2BR agonist, BAY60-6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A2BR antagonists, PSB-603 and MRS1706. To clarify this unexpected data, we wondered whether A2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A2BRs. Their selective activation by BAY60-6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB-603. Throughout this A2BR-dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A2BR in DRGNs alone prevents the increased myelination induced by BAY60-6583 in cocultures. In conclusion, our data suggest a different role of A2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.
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Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain that binds to GABA receptors and hyperpolarizes the postsynaptic neuron. Gabazine acts as a competitive antagonist to type A GABA receptors (GABAAR), thereby causing diminished neuronal hyperpolarization and GABAAR-mediated inhibition. However, the biochemical effects and the potential regulatory role of astrocytes in this process remain poorly understood. To address this, we investigated the neuronal responses of gabazine in rat cortical cultures containing varying ratios of neurons and astrocytes. Electrophysiological characterization was performed utilizing microelectrode arrays (MEAs) with topologically controlled microcircuit cultures that enabled control of neuronal network growth. Biochemical analysis of the cultures was performed using traditional dissociated cultures on coverslips. Our study indicates that, upon gabazine stimulation, astrocyte-rich neuronal cultures exhibit elevated electrophysiological activity and tyrosine phosphorylation of tropomyosin receptor kinase B (TrkB; receptor for brain-derived neurotrophic factor), along with distinct cytokine secretion profiles. Notably, neurons lacking proper astrocytic support were found to experience synapse loss and decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, astrocytes contributed to neuronal viability, morphology, vascular endothelial growth factor (VEGF) secretion, and overall neuronal network functionality, highlighting the multifunctional role of astrocytes.
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The tumor microenvironment (TME) includes immune and stromal cells and noncellular extracellular matrix (ECM) components. Tumor-associated macrophages (TAMs) are the most important immune cells in TME and are crucial for carcinomas' progression. The purpose was to analyze direct and indirect interactions in co-culture of tumor cells with monocytes/macrophages and, additionally, to indicate which interactions are more important for cancer development. Cytokines, reactive oxygen species, nitric oxide level, tumor cell cycle and changes in tumor cell morphology after human tumor cells (Hep-2 and RK33 cell lines) with human monocyte/macrophage (THP-1 cell line) interactions were tested. Morphology and cytoskeleton organization of tumor cells did not change after co-culture with macrophages. In co-culture of tumor cells with human monocyte, changes in the percentage of tumor cells in cell cycle phases was observed. No significant changes in reactive oxygen species (ROS) were found in the co-culture as compared to the tumor cell mono-culture. Monocytes produced about three times higher ROS than tumor cells. In co-cultures, a lower nitric oxide (NOx) level was found as compared to the sum of the production by both mono-cultures. Co-culture conditions limited the production of cytokines (IL-4, IL-10 and IL-13) as compared to the sum of their level in mono-cultures. In conclusion, macrophages influence tumor cell growth and functions. Mutual (direct and paracrine) interactions between tumor cells and macrophages changed cytokine production and tumor cell cycle profile. The data obtained may allow us to initially indicate which kind of interactions may have a greater impact on cancer development processes.
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Colorectal cancer, the third most commonly occurring tumor worldwide, poses challenges owing to its high mortality rate and persistent drug resistance in metastatic cases. We investigated the tumor microenvironment, emphasizing the role of cancer-associated fibroblasts in the progression and chemoresistance of colorectal cancer. We used an indirect co-culture system comprising colorectal cancer organoids and cancer-associated fibroblasts to simulate the tumor microenvironment. Immunofluorescence staining validated the characteristics of both organoids and fibroblasts, showing high expression of epithelial cell markers (EPCAM), colon cancer markers (CK20), proliferation markers (KI67), and fibroblast markers (VIM, SMA). Transcriptome profiling was conducted after treatment with anticancer drugs, such as 5-fluorouracil and oxaliplatin, to identify chemoresistance-related genes. Changes in gene expression in the co-cultured colorectal cancer organoids following anticancer drug treatment, compared to monocultured organoids, particularly in pathways related to interferon-alpha/beta signaling and major histocompatibility complex class II protein complex assembly, were identified. These two gene groups potentially mediate drug resistance associated with JAK/STAT signaling. The interaction between colorectal cancer organoids and fibroblasts crucially modulates the expression of genes related to drug resistance. These findings suggest that the interaction between colorectal cancer organoids and fibroblasts significantly influences gene expression related to drug resistance, highlighting potential biomarkers and therapeutic targets for overcoming chemoresistance. Enhanced understanding of the interactions between cancer cells and their microenvironment can lead to advancements in personalized medical research..
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Despite significant progress in treating chronic lymphocytic leukaemia (CLL), resistance to therapy remains challenging. NOTCH1 activation, common in CLL, confers adverse prognosis. This study explores the impact of NOTCH1 signalling on venetoclax sensitivity in vitro. Although NOTCH1 activation minimally impaired the susceptibility of CLL cells to venetoclax, ex vivo cell competition studies reveal that cells with constitutive NOTCH1 activation outgrew their wild-type counterparts in the presence of ongoing venetoclax exposure. Our findings suggest that while NOTCH1 activation is insufficient to confer venetoclax refractoriness, there is enhanced potential for cells with NOTCH1 activation to escape and thus become fully resistant to venetoclax.
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Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2-4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.
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Carcinoma de Pulmón de Células no Pequeñas , Técnicas de Cocultivo , Inmunoconjugados , Neoplasias Pulmonares , Receptor ErbB-2 , Trastuzumab , Humanos , Trastuzumab/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Inmunoconjugados/farmacología , Receptor ErbB-2/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Éteres Corona/farmacología , Antineoplásicos Inmunológicos/farmacología , Camptotecina/análogos & derivadosRESUMEN
Astrocytes transfer extracellular functional mitochondria into neurons to rescue injured neurons after a stroke. However, there are no reports on drugs that interfere with intercellular mitochondrial transfer. Chrysophanol (CHR) was an effective drug for the treatment of cerebral ischemia-reperfusion injury (CIRI) and was selected as the test drug. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model and the middle cerebral artery occlusion animal model were established to investigate the effect of CHR on CIRI. The result showed that astrocytes could act as mitochondrial donors to ameliorate neuronal injury. Additionally, the neuroprotective effect of astrocytes was enhanced by CHR, the CHR improved the neuronal mitochondrial function, decreased the neurological deficit score and infarction volume, recovered cell morphology in ischemic penumbra. The mitochondrial fluorescence probe labeling technique has shown that the protective effect of CHR is associated with accelerated astrocytic mitochondrial transfer to neurons. The intercellular mitochondrial transfer may be an important way to ameliorate ischemic brain injury and be used as a key target for drug treatment.
Asunto(s)
Antraquinonas , Isquemia Encefálica , Daño por Reperfusión , Ratas , Animales , Isquemia Encefálica/metabolismo , Astrocitos/metabolismo , Daño por Reperfusión/metabolismo , Neuronas/metabolismo , MitocondriasRESUMEN
The growing spread of infectious diseases has become a potential global health threat to human beings. According to WHO reports, in this study, we investigated the impact of co-cultivating the isolated endophytic fungus Aspergillus sp. CO2 and Bacillus sp. COBZ21 as a method to stimulate the production of natural bioactive substances. (GC/MS)-based metabolomics profiling of two sponge-associated microbes, Aspergillus sp. CO2 and Bacillus sp. COBZ21, revealed that the co-culture of these two isolates induced the accumulation of metabolites that were not traced in their axenic cultures. By detection of different activities of extracts of Bacillus sp. COBZ21 and Aspergillus sp. CO2 and coculture between Bacillus sp. COBZ21 and Aspergillus sp. CO2. It was noted that the coculture strategy was the reason for a notable increase in some different activities, such as the antimicrobial activity, which showed potent activity against Escherichia coli ATCC 25,922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC 10,231. The antibiofilm activity showed significant biofilm inhibitory activity toward Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10,145, and Staph aureus NRRLB-767, with activity up to 53.66, 71.17, and 47.89%, while it showed low activity against E. coli ATCC 25,922, while the antioxidant activity based on the DPPH assay showed maximum activity (75.25%). GC-MS investigations revealed the presence of variable chemical constituents belonging to different chemical categories, which reflected their chemical diversity. The main components are (+-) cis-Deethylburnamine (2.66%), Bis(3,6,9,12-tetraoxapentaethylene) crowno-N,N,N',N'-tetra methylpphanediamine (2.48%), and 11-phenyl-2,4,6,8-tetra(2-thienyl)-11-aza-5,13-dithiaeteracyclo[7.3.0.1(2,8)0.0(3,7)] trideca-3,6-diene-10,12,13-trione (3.13%), respectively, for Bacillus sp. axenic culture, Aspergillus sp. CO2, Aspergillus sp. CO2, and Bacillus sp. COBZ21 coculture. By studying the ADME-related physicochemical properties of coculture extract, the compound showed log Po/w values above 5 (8.82). The solubility of the substance was moderate. In order to provide a comprehensive definition of medicinal chemistry and leadlikness, it is important to note that the latter did not meet the criteria outlined in the rule of three (RO3). The toxicity prediction of the coculture extract was performed using the ProTox II web server, which showed that the selected compound has no pronounced toxicity.