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Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks-vasculogenic mimicry-that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Proteínas Inhibidoras de la Diferenciación , Melanoma , Melanoma/genética , Melanoma/patología , Melanoma/metabolismo , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteína 1 Inhibidora de la Diferenciación/genética , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Línea Celular Tumoral , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Fenotipo , Diferenciación Celular/genética , Epigénesis Genética , Combinación de Medicamentos , Colágeno , Proteoglicanos , Laminina , Proteínas de Neoplasias

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Intravenously administered perfluorocarbon nanoemulsion (PFC) are taken up by phagocytic immune cells which enables the non-invasive visualization of inflammatory hot spots by combined 1H/19F magnetic resonance imaging (MRI). However, little is known about the influence of inflammatory stimuli on cellular uptake and biodistribution of PFCs. Here, we systematically investigated the impact of inflammation induced by subcutaneous implantation of Matrigel/lipopolysaccharide (Matrigel/LPS) or myocardial infarction (MI; 50 minutes ischemia reperfusion) on PFC-uptake and biodistribution in C57BL/6J mice. We detected strong 19F signals in Matrigel/LPS plugs and infarcted hearts, which were completely absent in controls. Cellular uptake of PFCs was increased in neutrophils isolated from the blood and Matrigel/LPS plugs, whereas uptake by monocytes was only slightly elevated. In contrast, MI caused only a moderate early increase of PFC-uptake in monocytes and neutrophils. Interestingly, the inflammatory model did also affect the biodistribution of the PFCs. The blood half-life of PFCs was slightly increased after Matrigel/LPS implantation, whereas it was reduced after MI. Compared to controls, the 19F signal of the liver was significantly stronger in Matrigel/LPS, but not in MI animals. Interestingly, stimulation of primary immune cells and RAW264.7 macrophages with LPS had no effect on PFC-uptake, whereas CRP-incubation elevated internalization of PFCs at least in RAW264.7 cells. In conclusion, we show that the cellular PFC-uptake can differ between individual inflammatory conditions. This is an important aspect that has to be considered for the proper interpretation of 1H/19F MRI data obtained from inflammatory hot spots.

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Introduction: Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. Methods: We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. Results: We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1ß treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Discussion: Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

Asunto(s)

Matriz Extracelular , Homeostasis , Laminina , Neuroglía , Animales , Matriz Extracelular/metabolismo , Neuroglía/metabolismo , Neuroglía/inmunología , Ratones , Laminina/metabolismo , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/inmunología , Células Cultivadas , Combinación de Medicamentos , Colágeno/metabolismo , Ratones Endogámicos C57BL , Proteoglicanos/metabolismo

RESUMEN

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

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The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

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The absence of effective extracellular matrix to mimic the natural tumor microenvironment remains a significant obstacle in cancer research. Matrigel, abundant in various biological matrix components, is limited in its application due to its high cost. This has prompted researchers to explore alternative matrix substitutes. Here, we have investigated the effects of the extracellular matrix derived from pig small intestinal submucosa (ECM-SIS) in xenograft tumor modeling. Our results showed that the pig-derived ECM-SIS effectively promotes the establishment of xenograft tumor models, with a tumor formation rate comparable to that of Matrigel. Furthermore, we showed that the pig-derived ECM-SIS exhibited lower immune rejection and fewer infiltrating macrophages than Matrigel. Gene sequencing analysis demonstrated only a 0.5% difference in genes between pig-derived ECM-SIS and Matrigel during the process of tumor tissue formation. These differentially expressed genes primarily participate in cellular processes, biological regulation, and metabolic processes. These findings emphasize the potential of pig-derived ECM-SIS as a cost-effective option for tumor modeling in cancer research.

Asunto(s)

Matriz Extracelular , Laminina , Animales , Matriz Extracelular/metabolismo , Porcinos , Ratones , Humanos , Proteoglicanos , Colágeno/química , Microambiente Tumoral , Mucosa Intestinal/metabolismo , Combinación de Medicamentos , Línea Celular Tumoral , Intestino Delgado , Geles , Neoplasias

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Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.

RESUMEN

Due to their ability to replicate the in vivo microenvironment through cell interaction and induce cells to stimulate cell function, three-dimensional cell culture models can overcome the limitations of two-dimensional models. Organoids are 3D models that demonstrate the ability to replicate the natural structure of an organ. In most organoid tissue cultures, matrigel made of a mouse tumor extracellular matrix protein mixture is an essential ingredient. However, its tumor-derived origin, batch-to-batch variation, high cost, and safety concerns have limited the usefulness of organoid drug development and regenerative medicine. Its clinical application has also been hindered by the fact that organoid generation is dependent on the use of poorly defined matrices. Therefore, matrix optimization is a crucial step in developing organoid culture that introduces alternatives as different materials. Recently, a variety of substitute materials has reportedly replaced matrigel. The purpose of this study is to review the significance of the latest advances in materials for cell culture applications and how they enhance build network systems by generating proper cell behavior. Excellence in cell behavior is evaluated from their cell characteristics, cell proliferation, cell differentiation, and even gene expression. As a result, graphene oxide as a matrix optimization demonstrated high potency in developing organoid models. Graphene oxide can promote good cell behavior and is well known for having good biocompatibility. Hence, advances in matrix optimization of graphene oxide provide opportunities for the future development of advanced organoid models.

Asunto(s)

Grafito , Organoides , Organoides/efectos de los fármacos , Organoides/citología , Animales , Grafito/química , Grafito/farmacología , Humanos , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Combinación de Medicamentos , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo Tridimensional de Células/métodos , Ratones , Laminina/química , Laminina/farmacología , Colágeno , Proteoglicanos

RESUMEN

Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.

Asunto(s)

Diferenciación Celular , Proliferación Celular , Colágeno , Combinación de Medicamentos , Laminina , Células-Madre Neurales , Polilisina , Proteoglicanos , Polilisina/química , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/efectos de los fármacos , Laminina/química , Laminina/farmacología , Colágeno/química , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteoglicanos/química , Proteoglicanos/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ratones

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Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

RESUMEN

BACKGROUND: Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS: Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS: UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS: Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

RESUMEN

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

Asunto(s)

Colágeno , Combinación de Medicamentos , Laminina , Células Madre Mesenquimatosas , Proteoglicanos , Ratas Sprague-Dawley , Andamios del Tejido , Cicatrización de Heridas , Animales , Laminina/química , Proteoglicanos/química , Colágeno/química , Humanos , Ratas , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Diferenciación Celular , Proliferación Celular , Encía/citología , Técnicas de Cultivo Tridimensional de Células/métodos , Células Cultivadas , Ingeniería de Tejidos/métodos , Masculino , Mucosa Bucal/citología

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The recent FDA decision to eliminate animal testing requirements emphasises the role of cell models, such as spheroids, as regulatory test alternatives for investigations of cellular behaviour, drug responses, and disease modelling. The influence of environment on spheroid formation are incompletely understood, leading to uncertainty in matrix selection for scaffold-based 3D culture. This study uses atomic force microscopy-based techniques to quantify cell adhesion to Matrigel and cellulose nanofibrils (CNF), and cell-cell adhesion forces, and their role in spheroid formation of hepatocellular carcinoma (HepG2) and induced pluripotent stem cells (iPS(IMR90)-4). Results showed different cell behaviour in CNF and Matrigel cultures. Both cell lines formed compact spheroids in CNF but loose cell aggregates in Matrigel. Interestingly, the type of cell adhesion protein, and not the bond strength, appeared to be a key factor in the formation of compact spheroids. The gene expression of E- and N-cadherins, proteins on cell membrane responsible for cell-cell interactions, was increased in CNF culture, leading to formation of compact spheroids while Matrigel culture induced integrin-laminin binding and downregulated E-cadherin expression, resulting in looser cell aggregates. These findings enhance our understanding of cell-biomaterial interactions in 3D cultures and offer insights for improved 3D cell models, culture biomaterials, and applications in drug research.

RESUMEN

Background: Primary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing ß-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.

RESUMEN

Endothelial wound-healing processes are fundamental for the maintenance and restoration of the circulatory system and are greatly affected by the factors present in the blood. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) induces the proinflammatory activation of endothelial cells and is able to cooperate with other proinflammatory activators. Our aim was to investigate the combined effect of mechanical wounding and MASP-1 on endothelial cells. Transcriptomic analysis showed that MASP-1 alters the expression of wound-healing-related and angiogenesis-related genes. Both wounding and MASP-1 induced Ca2+ mobilization when applied individually. However, MASP-1-induced Ca2+ mobilization was inhibited when the treatment was preceded by wounding. Mechanical wounding promoted CREB phosphorylation, and the presence of MASP-1 enhanced this effect. Wounding induced ICAM-1 and VCAM-1 expression on endothelial cells, and MASP-1 pretreatment further increased VCAM-1 levels. MASP-1 played a role in the subsequent stages of angiogenesis, facilitating the breakdown of the endothelial capillary network on Matrigel®. Our findings extend our general understanding of endothelial wound healing and highlight the importance of complement MASP-1 activation in wound-healing processes.

Asunto(s)

Células Endoteliales , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Molécula 1 de Adhesión Celular Vascular , Cicatrización de Heridas , Proteínas del Sistema Complemento

RESUMEN

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.

Asunto(s)

Diferenciación Celular , Matriz Extracelular Descelularizada , Hidrogeles , Células Madre Pluripotentes Inducidas , Organoides , Placenta , Médula Espinal , Humanos , Organoides/citología , Organoides/metabolismo , Organoides/efectos de los fármacos , Femenino , Placenta/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Embarazo , Hidrogeles/química , Hidrogeles/farmacología , Médula Espinal/citología , Médula Espinal/metabolismo , Diferenciación Celular/efectos de los fármacos , Matriz Extracelular Descelularizada/farmacología , Matriz Extracelular Descelularizada/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Laminina/farmacología , Laminina/química

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Prostate cancer (PCa) displays diverse intra-tumoral traits, impacting its progression and treatment outcomes. This study aimed to refine PCa cell culture conditions for dynamic monitoring of androgen receptor (AR) activity at the single-cell level. We introduced an extracellular matrix-Matrigel (ECM-M) culture model, enhancing cellular tracking during bioluminescence single-cell imaging while improving cell viability. ECM-M notably tripled the traceability of poorly adherent PCa cells, facilitating robust single-cell tracking, without impeding substrate permeability or AR response. This model effectively monitored AR modulation by antiandrogens across various PCa cell lines. Single-cell imaging unveiled heterogeneous antiandrogen responses within populations, correlating non-responsive cell proportions with drug IC50 values. Integrating ECM-M culture with the PSEBC-TSTA biosensor enabled precise characterization of ARi responsiveness within diverse cell populations. Our ECM-M model stands as a promising tool to assess heterogeneous single-cell treatment responses in cancer, offering insights to link drug responses to intracellular signaling dynamics. This approach enhances our comprehension of the nuanced and dynamic nature of PCa treatment responses.

Asunto(s)

Matriz Extracelular , Neoplasias de la Próstata , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Matriz Extracelular/metabolismo , Masculino , Línea Celular Tumoral , Antagonistas de Andrógenos/farmacología , Receptores Androgénicos/metabolismo , Análisis de la Célula Individual , Microscopía , Técnicas Biosensibles , Mediciones Luminiscentes

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Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Asunto(s)

Técnicas de Cultivo Tridimensional de Células , Colágeno , Combinación de Medicamentos , Células Epiteliales , Hidrogeles , Laminina , Péptidos , Proteoglicanos , Laminina/farmacología , Laminina/química , Hidrogeles/química , Hidrogeles/farmacología , Proteoglicanos/farmacología , Proteoglicanos/química , Colágeno/química , Colágeno/farmacología , Péptidos/farmacología , Péptidos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/citología , Humanos , Femenino , Técnicas de Cultivo Tridimensional de Células/métodos , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Glándulas Mamarias Humanas/citología , Organoides/efectos de los fármacos , Organoides/citología , Técnicas de Cultivo de Célula/métodos

RESUMEN

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

Asunto(s)

Detección Precoz del Cáncer , Neoplasias de la Próstata , Masculino , Humanos , Evaluación Preclínica de Medicamentos/métodos , Neoplasias de la Próstata/patología , Organoides

RESUMEN

Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules-both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody-marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients' colons, monitoring the biomarkers, and customizing their treatments. Key features • Analysis of molecular markers of cell behavior. • Protocol to visualize proteins in 3D cell culture. • This protocol requires colonoids isolated from mice colon grown on matrigel support. • Protocol requires at least eight days to complete.