RESUMO
Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling.
Assuntos
Sobrevivência Celular , Fator de Transcrição STAT3 , Esferoides Celulares , Neoplasias da Bexiga Urinária , Fator de Transcrição STAT3/metabolismo , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Humanos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologiaRESUMO
BACKGROUND: Osteosarcoma (OS) survival rates and outcome have not improved in 50 years since the advent of modern chemotherapeutics. Thus, there is a critical need for an improved understanding of the tumor microenvironment to identify better therapies. Extracellular matrix (ECM) deposition and hypoxia are known to abrogate the efficacy of various chemical and cell-based therapeutics. Here, we aim to mechanistically investigate the combinatorial effects of hypoxia and matrix deposition with the use of OS spheroids. METHODS: We use two murine OS cell lines with differential metastatic potential to form spheroids. We form spheroids of two sizes, use ascorbate-2-phosphate supplementation to enhance ECM deposition, and study cell response under standard (21% O2) and physiologic (5% O2) oxygen tensions. Finally, we examine chemotherapeutic responses to doxorubicin treatment. RESULTS: ECM production and oxygen tension are key determinants of spheroid size through cell organization based on nutrient and oxygen distribution. Interestingly, highly metastatic OS is more susceptible to chemotherapeutics compared to less metastatic OS when matrix production increases. Together, these data suggest that dynamic interactions between ECM production and oxygen diffusion may result in distinct chemotherapeutic responses despite inherent tumor aggressiveness. CONCLUSION: This work establishes OS spheroids as a valuable tool for early OS tumor formation investigation and holds potential for novel therapeutic target and prognostic indicator discovery.
Assuntos
Matriz Extracelular , Osteossarcoma , Oxigênio , Esferoides Celulares , Microambiente Tumoral , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Esferoides Celulares/efeitos dos fármacos , Matriz Extracelular/metabolismo , Animais , Camundongos , Oxigênio/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
To accurately study human organ function and disease 'in the dish', it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue.
Assuntos
Sistema Enzimático do Citocromo P-450 , Fígado , Humanos , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Engenharia Tecidual/métodos , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacosRESUMO
In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies.
Assuntos
Diferenciação Celular , Deleção Cromossômica , Cromossomos Humanos Par 9 , Células-Tronco Pluripotentes Induzidas , Esferoides Celulares , Humanos , Cromossomos Humanos Par 9/genética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/efeitos dos fármacos , Anormalidades Craniofaciais/patologia , Anormalidades Craniofaciais/metabolismo , Deficiência Intelectual/metabolismo , Proliferação de Células/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Receptores de N-Metil-D-Aspartato/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/efeitos dos fármacos , Células Cultivadas , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Rotenona/toxicidade , Cardiopatias Congênitas , Proteínas do Tecido NervosoRESUMO
The 3D multicellular tumor spheroid (MTS) model exhibits enhanced fidelity in replicating the tumor microenvironment and demonstrates exceptional resistance to clinical drugs compared to the 2D monolayer model. In this study, we used multiomics (transcriptome, proteomics, and metabolomics) tools to explore the molecular mechanisms and metabolic differences of the two culture models. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways revealed that the differentially expressed genes between the two culture models were mainly enriched in cellular components and biological processes associated with extracellular matrix, extracellular structural organization, and mitochondrial function. An integrated analysis of three omics data revealed 11 possible drug resistance targets. Among these targets, seven genes, AKR1B1, ALDOC, GFPT2, GYS1, LAMB2, PFKFB4, and SLC2A1, exhibited significant upregulation. Conversely, four genes, COA7, DLD, IFNGR1, and QRSL1, were significantly downregulated. Clinical prognostic analysis using the TCGA survival database indicated that high-expression groups of SLC2A1, ALDOC, and PFKFB4 exhibited a significant negative correlation with patient survival. We further validated their involvement in chemotherapy drug resistance, indicating their potential significance in improving prognosis and chemotherapy outcomes. These results provide valuable insights into potential therapeutic targets that can potentially enhance treatment efficacy and patient outcomes.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Transportador de Glucose Tipo 1 , Glicólise , Fosfofrutoquinase-2 , Esferoides Celulares , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Esferoides Celulares/efeitos dos fármacos , Glicólise/genética , Glicólise/efeitos dos fármacos , Células HeLa , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Antineoplásicos/farmacologiaRESUMO
Maintaining the differentiated phenotype and function of primary hepatocytes in vitro and in vivo represents a distinct challenge. Our paper describes microcapsules comprised of a bioactive polymer and overcoated with an ultrathin film as a means of maintaining the function of entrapped hepatocytes for at least two weeks. We previously demonstrated that heparin (Hep)-based microcapsules improved the function of entrapped primary hepatocytes by capturing and releasing cell-secreted inductive signals, including hepatocyte growth factor (HGF). Further enhancement of hepatic function could be gained by loading exogenous HGF into microcapsules. In this study, we demonstrate that an ultrathin coating of tannic acid (TA) further enhances endogenous HGF signaling for entrapped hepatocytes and increases by 2-fold the rate of uptake of exogenous HGF by Hep microcapsules. Hepatocytes in overcoated microcapsules exhibited better function and hepatic gene expression than in capsules without a TA coating. Our study showcases the potential application of ultrathin coatings to modulate the bioactivity of microcapsules and may enable the use of encapsulated hepatocytes for modeling drug toxicity or treating liver diseases.
Assuntos
Cápsulas , Heparina , Hepatócitos , Hepatócitos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Cápsulas/química , Animais , Heparina/química , Heparina/farmacologia , Taninos/química , Taninos/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Humanos , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , CamundongosRESUMO
Ex vivo assessment of drug response with conventional cell viability assays remains the standard practice for guiding initial therapeutic choices. However, such ensemble approaches fail to capture heterogeneities in treatment response and cannot identify early markers of response. Here, we leverage Raman spectroscopy (RS) as an accurate, low-cost, extraction-free, and label-free approach to track metabolic changes in cancer cells, spheroids, and organoids in response to cisplatin treatment. We identified 12 statistically significant metabolites in cells and 19 metabolites in spheroids and organoids as a function of depth. We show that the cisplatin treatment of 4T1 cells and spheroids results in a shift in metabolite levels; metabolites including nucleic acids such as DNA, 783 cm-1 with p = 0.00021 for cells; p = 0.02173 for spheroids, major amino acids such as threonine, 1338 cm-1 with p = 0.00045 for cells; p = 0.01022 for spheroids, proteins such as amide III, 1248 cm-1 with p = 0.00606 for cells; p = 0.00511 for spheroids serve as early predictors of response. Our RS findings were also applicable to canine-derived organoids, showing spatial variations in metabolic changes as a function of organoid depth in response to cisplatin. Further, the metabolic pathways such as tricarboxylic acid (TCA)/citric acid cycle and glyoxylate and dicarboxylate metabolism that drive drug response showed significant differences based on organoid depth, replicating the heterogeneous treatment response seen in solid tumors where there is a difference from the periphery to the tumor core. Our study showcases the versatility of RS as a predictive tool for treatment response applicable from cells to organotypic cultures, that has the potential to decrease animal burden and readout time for preclinical drug efficacy.
Assuntos
Antineoplásicos , Cisplatino , Organoides , Análise Espectral Raman , Esferoides Celulares , Cisplatino/farmacologia , Organoides/metabolismo , Organoides/efeitos dos fármacos , Organoides/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Animais , Cães , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Camundongos , HumanosRESUMO
Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.
Assuntos
RNA Interferente Pequeno , Esferoides Celulares , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Humanos , RNA Interferente Pequeno/genética , Técnicas de Cultura de Células em Três Dimensões/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Interferência de RNARESUMO
Metastasis contributes to the increased mortality rate of cancer, but the intricate mechanisms remain unclear. Cancer cells from a primary tumor invade nearby tissues and access the lymphatic or circulatory system. If these cells manage to survive and extravasate from the vasculature into distant tissues and ultimately adapt to survive, they will proliferate and facilitate malignant tumor formation. Traditional two-dimensional (2D) cell cultures offer a rapid and convenient method for validating the efficacy of anticancer drugs within a reasonable cost range, but their utility is limited because of tumors' high heterogeneity in vivo and spatial complexities. Three-dimensional (3D) cell cultures that mimic the physiological conditions of cancer cells in vivo have gained considerable interest. In these cultures, cells assemble into spheroids through gravity, magnetic forces, or their low-adhesion to the plates. Although these approaches address some of the limitations of 2D cultures, they often require a considerable amount of time and cost. Therefore, this study aims to enhance the effectiveness of 3D culture techniques by using microfluidic systems to provide a high-throughput and sensitive pipeline for drug screening. Using these systems, we studied the effects of lanthanide elements, which have garnered interest in cancer treatment, on spheroid formation and cell spreading. Our findings suggest that these elements alter the compactness of cell spheroids and decrease cell mobility.
Assuntos
Elementos da Série dos Lantanídeos , Esferoides Celulares , Esferoides Celulares/efeitos dos fármacos , Humanos , Elementos da Série dos Lantanídeos/toxicidade , Elementos da Série dos Lantanídeos/farmacologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
Increasing evidence is implicating roles for platelets in the development and progression of ovarian cancer, a highly lethal disease that can arise from the fallopian tubes, and has no current method of early detection or prevention. Thrombosis is a major cause of mortality of ovarian cancer patients suggesting that the cancer alters platelet behavior. The objective of this study was to develop a cell culture model of the pathological interactions of human platelets and ovarian cancer cells, using normal FT epithelial cells as a healthy control, and to test effects of the anti-platelet dihomo-gamma-linolenic acid (DGLA) in the model. Both healthy and cancer cells caused platelet aggregation, however platelets only affected spheroid formation by cancer cells and had no effect on healthy cell spheroid formation. When naturally-formed spheroids of epithelial cells were exposed to platelets in transwell inserts that did not allow direct interactions of the two cell types, platelets caused increased size of the spheroids formed by cancer cells, but not healthy cells. When cancer cell spheroids formed using magnetic nanoshuttle technology were put in direct physical contact with platelets, the platelets caused spheroid condensation. In ovarian cancer cells, DGLA promoted epithelial-to-mesenchymal (EMT) transition at doses as low as 100 µM, and inhibited metabolic viability and induced apoptosis at doses ≥150 µM. DGLA doses ≤150 µM used to avoid direct DGLA effects on cancer cells, had no effect on the pathological interactions of platelets and ovarian cancer cells in our models. These results demonstrate that the pathological interactions of platelets with ovarian cancer cells can be modeled in cell culture, and that DGLA has no effect on these interactions, suggesting that targeting platelets is a rational approach for reducing cancer aggressiveness and thrombosis risk in ovarian cancer patients, however DGLA is not an appropriate candidate for this strategy.
Assuntos
Ácido 8,11,14-Eicosatrienoico , Plaquetas , Células Epiteliais , Neoplasias Ovarianas , Esferoides Celulares , Humanos , Plaquetas/efeitos dos fármacos , Feminino , Células Epiteliais/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Ácido 8,11,14-Eicosatrienoico/farmacologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Esferoides Celulares/efeitos dos fármacos , Linhagem Celular Tumoral , Agregação Plaquetária/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Comunicação Celular/efeitos dos fármacosRESUMO
The precision of previous cancer research based on tumor spheroids, especially the microgel-encapsulating tumor spheroids, was limited by the high heterogeneity in the tumor spheroid size and shape. Here, we reported a user-friendly solenoid valve-based sorter to reduce this heterogeneity. The artificial intelligence algorithm was employed to detect and segmentate the tumor spheroids in real-time for the size and shape calculation. A simple off-chip solenoid valve-based sorting actuation module was proposed to sort out target tumor spheroids with the desired size and shape. Utilizing the developed sorter, we successfully uncovered the drug response variations on cisplatin of lung tumor spheroids in the same population but with different sizes and shapes. Moreover, with this sorter, the precision of drug testing on the spheroid population level was improved to a level comparable to the precise but complex single spheroid analysis. The developed sorter also exhibits significant potential for organoid morphology and sorting for precision medicine research.
Assuntos
Técnicas Biossensoriais , Microgéis , Esferoides Celulares , Humanos , Esferoides Celulares/patologia , Esferoides Celulares/efeitos dos fármacos , Microgéis/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Desenho de Equipamento , Linhagem Celular Tumoral , Inteligência ArtificialRESUMO
Drug resistance is one of the biggest challenges in the fight against cancer. In particular, in the case of glioblastoma, the most lethal brain tumour, resistance to temozolomide (the standard of care drug for chemotherapy in this tumour) is one of the main reasons behind treatment failure and hence responsible for the poor prognosis of patients diagnosed with this disease. In this work, we combine the power of three-dimensional in vitro experiments of treated glioblastoma spheroids with mathematical models of tumour evolution and adaptation. We use a novel approach based on internal variables for modelling the acquisition of resistance to temozolomide that was observed in experiments for a group of treated spheroids. These internal variables describe the cell's phenotypic state, which depends on the history of drug exposure and affects cell behaviour. We use model selection to determine the most parsimonious model and calibrate it to reproduce the experimental data, obtaining a high level of agreement between the in vitro and in silico outcomes. A sensitivity analysis is carried out to investigate the impact of each model parameter in the predictions. More importantly, we show how the model is useful for answering biological questions, such as what is the intrinsic adaptation mechanism, or for separating the sensitive and resistant populations. We conclude that the proposed in silico framework, in combination with experiments, can be useful to improve our understanding of the mechanisms behind drug resistance in glioblastoma and to eventually set some guidelines for the design of new treatment schemes.
Assuntos
Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Modelos Biológicos , Temozolomida , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Antineoplásicos Alquilantes/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Esferoides Celulares/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Dacarbazina/farmacologia , Simulação por Computador , Adaptação FisiológicaRESUMO
Plasmonic photothermal therapy (PPTT) employing plasmonic gold nanorods (GNRs) presents a potent strategy for eradication of tumors including aggressive brain gliomas. Despite its promise, there is a pressing need for a more comprehensive evaluation of PPTT using sophisticated in vitro models that closely resemble tumor tissues, thereby facilitating the elucidation of therapeutic mechanisms. In this study, we exposed 3D glioma spheroids (tumoroids) to (16-mercaptohexadecyl)trimethylammonium bromide-functionalized gold nanorods (MTAB-GNRs) and a near-infrared (NIR) laser. We demonstrate that the photothermal effect can be fine-tuned by adjusting the nanoparticle concentration and laser power. Depending on the selected parameters, the laser can trigger either regulated or non-regulated cell death (necrosis) in both mouse GL261 and human U-87 MG glioma cell lines, accompanied by translocation of phosphatidylserine in the membrane. Our investigation into the mechanism of regulated cell death induced by PPTT revealed an absence of markers associated with classical apoptosis pathways, such as cleaved caspase 3. Instead, we observed the presence of cleaved caspase 1, gasdermin D, and elevated levels of NLRP3 in NIR-irradiated tumoroids, indicating the activation of pyroptosis. This finding correlates with previous observations of lysosomal accumulation of MTAB-GNRs and the known lysosomal pathway of pyroptosis activation. We further confirmed the absence of toxic breakdown products of GNRs using electron microscopy, which showed no melting or fragmentation of gold nanoparticles under the conditions causing regulated cell death. In conclusion, PPTT using coated gold nanorods offers significant potential for glioma cell elimination occurring through the activation of pyroptosis rather than classical apoptosis pathways.
Assuntos
Glioma , Ouro , Nanotubos , Piroptose , Ouro/química , Ouro/farmacologia , Nanotubos/química , Glioma/patologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Camundongos , Animais , Piroptose/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Terapia Fototérmica , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Cátions/química , Cátions/farmacologia , Células Tumorais Cultivadas , Sobrevivência Celular/efeitos dos fármacos , Nanopartículas Metálicas/químicaRESUMO
The distinct chemical structure of thiourea derivatives provides them with an advantage in selectively targeting cancer cells. In our previous study, we selected the most potent compounds, 2 and 8, with 3,4-dichloro- and 3-trifluoromethylphenyl substituents, respectively, across colorectal (SW480 and SW620), prostate (PC3), and leukemia (K-562) cancer cell lines, as well as non-tumor HaCaT cells. Our research has demonstrated their anticancer potential by targeting key molecular pathways involved in cancer progression, including caspase 3/7 activation, NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells) activation decrease, VEGF (Vascular Endothelial Growth Factor) secretion, ROS (Reactive Oxygen Species) production, and metabolite profile alterations. Notably, these processes exhibited no significant alterations in HaCaT cells. The effectiveness of the studied compounds was also tested on spheroids (3D culture). Both derivatives 2 and 8 increased caspase activity, decreased ROS production and NF-κB activation, and suppressed the release of VEGF in cancer cells. Metabolomic analysis revealed intriguing shifts in cancer cell metabolic profiles, particularly in lipids and pyrimidines metabolism. Assessment of cell viability in 3D spheroids showed that SW620 cells exhibited better sensitivity to compound 2 than 8. In summary, structural modifications of the thiourea terminal components, particularly dihalogenophenyl derivative 2 and para-substituted analog 8, demonstrate their potential as anticancer agents while preserving safety for normal cells.
Assuntos
Antineoplásicos , NF-kappa B , Espécies Reativas de Oxigênio , Tioureia , Fator A de Crescimento do Endotélio Vascular , Humanos , Tioureia/farmacologia , Tioureia/análogos & derivados , Antineoplásicos/farmacologia , Antineoplásicos/química , Espécies Reativas de Oxigênio/metabolismo , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 3/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Relação Estrutura-AtividadeRESUMO
Multilayer conformal coatings have been shown to provide a nanoscale barrier between cells and their environment with adequate stability, while regulating the diffusion of nutrition and waste across the cell membrane. The coating method aims to minimize capsule thickness and implant volume while reducing the need for immunosuppressive drugs, making it a promising approach for islet cell encapsulation in clinical islet transplantation for the treatment of Type 1 diabetes. This study introduces an immunoprotective nanocoating obtained through electrostatic interaction between quaternized phosphocholine-chitosan (PC-QCH) and tetrahydropyran triazole phenyl-alginate (TZ-AL) onto mouse ß-cell spheroids. First, successful synthesis of the proposed polyelectrolytes was confirmed with physico-chemical characterization. A coating with an average thickness of 540 nm was obtained with self-assembly of 4-bilayers of PC-QCH/TZ-AL onto MIN6 ß-cell spheroids. Surface coating of spheroids did not affect cell viability, metabolic activity, or insulin secretion, when compared to non-coated spheroids. The exposure of the polyelectrolytes to THP-1 monocyte-derived macrophages lead to a reduced level of TNF-α secretion and exposure of coated spheroids to RAW264.7 macrophages showed a decreasing trend in the secretion of TNF-α and IL-6. In addition, coated spheroids were able to establish normoglycemia when implanted into diabetic NOD-SCID mice, demonstrating in vivo biocompatibility and cellular function. These results demonstrate the ability of the PC-QCH/TZ-AL conformal coating to mitigate pro-inflammatory responses from macrophages, and thus can be a promising candidate towards nanoencapsulation for cell-based therapy, particularly in type 1 diabetes, where the insulin secreting ß-cells are subjected to inflammation and immune cell attack.
Assuntos
Alginatos , Quitosana , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Esferoides Celulares , Animais , Camundongos , Alginatos/química , Alginatos/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Quitosana/química , Quitosana/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/citologia , Células RAW 264.7 , Humanos , Sobrevivência Celular/efeitos dos fármacos , Insulina/metabolismo , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologiaRESUMO
Three-dimensional (3D) bioprinting culture models capable of reproducing the pathological architecture of diseases are increasingly advancing. In this study, 3D scaffolds were created using extrusion-based bioprinting method with alginate, gelatin, and hyaluronic acid to investigate the effects of hyaluronic acid on the physical properties of the bioscaffold as well as on the formation of liver cancer spheroids. Conformational analysis, rheological characterization, and swelling-degradation tests were performed to characterize the scaffolds. After generating spheroids from hepatocellular carcinoma cells on the 3D scaffolds, cell viability and proliferation assays were performed. Flow cytometry and immunofluorescence microscopy were used into examine the expression of albumin, CD44, and E-cadherin to demonstrate functional capability and maturation levels of the spheroid-forming cells. The results show that hyaluronic acid in the scaffolds correlates with spheroid formation and provides high survival rates. It is also associated with an increase in CD44 expression and a decrease in E-cadherin, while there is no significant change in the albumin expression in the cells. Overall, the findings demonstrate that hyaluronic acid in a 3D hydrogel scaffold supports spheroid formation and may induce stemness. We present a promising 3D scaffold model for enhancing liver cancer spheroid formation and mimicking solid tumors. This model also has the potential for further studies to examine stem cell properties in 3D models.
Assuntos
Receptores de Hialuronatos , Ácido Hialurônico , Células-Tronco Neoplásicas , Esferoides Celulares , Alicerces Teciduais , Ácido Hialurônico/farmacologia , Ácido Hialurônico/química , Humanos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Alicerces Teciduais/química , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Bioimpressão/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Gelatina/química , Hidrogéis/química , Hidrogéis/farmacologiaRESUMO
Osteosarcoma is the most common primary bone malignancy with a challenging prognosis marked by a high rate of metastasis. The limited success of current treatments may be partially attributed to an incomplete understanding of osteosarcoma pathophysiology and to the absence of reliable in vitro models to select the best molecules for in vivo studies. Among the natural compounds relevant for osteosarcoma treatment, Licochalcone A (Lic-A) and chalcone derivatives are particularly interesting. Here, Lic-A and selected derivatives have been evaluated for their anticancer effect on multicellular tumor spheroids from MG63 and 143B osteosarcoma cell lines. A metabolic activity assay revealed Lic-A, 1i, and 1k derivatives as the most promising candidates. To delve into their mechanism of action, caspase activity assay was conducted in 2D and 3D in vitro models. Notably, apoptosis and autophagic induction was generally observed for Lic-A and 1k. The invasion assay demonstrated that Lic-A and 1k possess the ability to mitigate the spread of osteosarcoma cells within a matrix. The effectiveness of chalcone as a natural scaffold for generating potential antiproliferative agents against osteosarcoma has been demonstrated. In particular, chalcones exert their antiproliferative activity by inducing apoptosis and autophagy, and in addition they are capable of reducing cell invasion. These findings suggest Lic-A and 1k as promising antitumor agents against osteosarcoma cells.
Assuntos
Apoptose , Autofagia , Neoplasias Ósseas , Chalconas , Osteossarcoma , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Humanos , Chalconas/farmacologia , Autofagia/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Proliferação de Células/efeitos dos fármacos , Invasividade Neoplásica , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Antineoplásicos/farmacologiaRESUMO
Emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) pose potential health risks to humans through dietary exposure. However, research into their mechanisms of toxicity is limited, with a lack of comprehensive toxicological data. This study investigates from a hepatic lipid metabolism perspective, establishing a more precise and reliable 3D HepaRG hepatocyte spheroid model as an alternative for toxicity assessment. Utilizing physiological indices, histopathological analyses, lipidomics, and molecular docking techniques, it comprehensively elucidates the effects of ENNs and BEA on hepatic lipid homeostasis and their molecular toxicological mechanisms. Our findings indicate that ENNs and BEA impact cellular viability and biochemical functions, significantly altering lipid metabolism pathways, particularly those involving glycerophospholipids and sphingolipids. Molecular docking has demonstrated strong binding affinity of ENNs and BEA with key enzymes in lipid metabolism such as Peroxisome Proliferator-Activated Receptor α (PPARα) and Cytosolic Phospholipase A2 (cPLA2), revealing the mechanistic basis for their hepatotoxic effects and potential to impair liver function and human health. These insights enhance our understanding of the potential hepatotoxicity of such fungal toxins and lay a foundation for the assessment of their health risks.
Assuntos
Depsipeptídeos , Hepatócitos , Metabolismo dos Lipídeos , Depsipeptídeos/toxicidade , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Simulação de Acoplamento Molecular , Micotoxinas/toxicidade , Micotoxinas/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Up to 80 % of chemotherapeutic drugs induce myelosuppression in patients. Chemotherapy not only impairs of hematopoietic stem cells (HSCs) but also damages bone marrow niches (vascular and endosteal). Current treatments for myelosuppression overlook these chemotherapy-induced damages to bone marrow niches and the critical role of niche restoration on hematopoietic regeneration. Ginsenoside protopanaxatriol (PPT) protects vascular endothelium from injury, while icariin (ICA) promotes osteogenic differentiation. The combination of PPT and ICA aims to restore damaged vascular and endosteal niches, thus rejuvenating HSCs for treating myelosuppression. PURPOSE: This study aims to develop effective, bone marrow niche-directed PPT/ICA therapies for treating chemotherapy-induced myelosuppression. METHODS: 3D cell spheroids were used to investigate the effects of PPT/ICA on cell-cell interactions in vascular niches, osteogenesis, and extracellular matrix (ECM) secretion in endosteal niches. In vitro mimic niche models were designed to access the drug combination's efficacy in rejuvenating and mobilizing in HSCs within bone marrow niches. The delivery capability of PPT/ICA to key niche cell types (mesenchymal stromal cells (MSCs), endothelial cells (ECs), and osteoblasts (OBs)) via nanocarriers has been determined. DSS6 peptide-modified nanoparticles (DSS6-NPs) were prepared for specific co-delivery of PPT/ICA into key niche cell populations in vivo. RESULTS: PPT can prevent vascular niche injury by restoring vascular EC cell-cell adhesion and the intercellular interactions between ECs and MSCs in 5-fluorouracil (5-FU)-damaged cell spheroids. ICA repaired 5-FU-damaged endosteal niches by promoting osteogenesis and ECM secretion. The combination of PPT and ICA restores key HSC niche factor gene expressions, normalizing HSC differentiation and mobilization. The in vitro cellular uptake efficiency of nanocarriers in a mimic niche is positively correlated with their in vivo delivery into bone marrow niche cells. DSS6-NPs greatly enhance the delivery of PPT/ICA into MSCs and OBs within bone marrow niches. Co-loading of PPT/ICA into DSS6-NPs effectively repairs damaged bone marrow niches and promotes HSC rejuvenation in vivo. CONCLUSION: The combination of PPT and ICA effectively prevents injury to the vascular and endosteal niches, thereby promoting hematopoietic regeneration in the bone marrow. This study provides novel niche-directed PPT/ICA therapies for managing chemotherapy-induced myelosuppression.
Assuntos
Células-Tronco Hematopoéticas , Sapogeninas , Nicho de Células-Tronco , Células-Tronco Hematopoéticas/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Sapogeninas/farmacologia , Osteogênese/efeitos dos fármacos , Humanos , Animais , Esferoides Celulares/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Camundongos , Antineoplásicos/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fluoruracila/farmacologiaRESUMO
Regulating the complex microenvironment after tooth extraction to promote alveolar bone regeneration is a pressing challenge for restorative dentistry. In this study, through modulating the mechanical properties of the cellular matrix, we guided various types of cells by self-organizing to form multicellular spheroids (MCSs) and hybridized MCSs with Prussian Blue nanoparticles (PBNPs) in the process. The constructed Prussian Blue nanohybridized multicellular spheroids (PBNPs@MCSs) with empowered antioxidant functions effectively reduced cell apoptosis under peroxidative conditions and exhibited enhanced ability to regulate the microenvironment and promote bone repair both in vitro and in vivo. In addition, the PBNPs@MCSs exhibited enhanced photoacoustic imaging ability to trace low doses of PBNPs. Therefore, the constructed PBNPs@MCSs based on the biomimetic hydrogel can be used as a form of an engraftment building block, with a greater potential for pro-bone repair application in the complex microenvironment of the oral cavity.