RESUMO
BACKGROUND: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.
Assuntos
COVID-19 , Cálcio , Corantes Fluorescentes , SARS-CoV-2 , Chlorocebus aethiops , Células Vero , Cálcio/metabolismo , Cálcio/análise , Animais , COVID-19/virologia , COVID-19/metabolismo , Corantes Fluorescentes/química , Humanos , FótonsRESUMO
Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.
Assuntos
Técnicas de Cocultura , Células-Tronco Mesenquimais , Esferoides Celulares , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Humanos , Esferoides Celulares/efeitos dos fármacos , Linhagem Celular Tumoral , Microambiente Tumoral , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Tolerância a Medicamentos , Citocinas/metabolismoRESUMO
The resistance of tumors against anticancer drugs is a major impediment for chemotherapy. Tumors often develop multidrug resistance as a result of the cellular efflux of chemotherapeutic agents by ABC transporters such as P-glycoprotein (ABCB1/P-gp), Multidrug Resistance Protein 1 (ABCC1/MRP1), or Breast Cancer Resistance Protein (ABCG2/BCRP). By screening a chemolibrary comprising 140 compounds, we identified a set of naturally occurring aurones inducing higher cytotoxicity against P-gp-overexpressing multidrug-resistant (MDR) cells versus sensitive (parental, non-P-gp-overexpressing) cells. Follow-up studies conducted with the P-gp inhibitor tariquidar indicated that the MDR-selective toxicity of azaaurones is not mediated by P-gp. Azaaurone analogs possessing pronounced effects were then designed and synthesized. The knowledge gained from structure-activity relationships will pave the way for the design of a new class of anticancer drugs selectively targeting multidrug-resistant cancer cells.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Benzofuranos/química , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Madin Darby de Rim Canino , Espectroscopia de Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
Membrane transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The cellular accumulation of many drugs is the result of the net function of efflux and influx transporters. Efflux transporters such as P-glycoprotein/ABCB1 have been shown to confer multidrug resistance in cancer. Although expression of uptake transporters has been confirmed in cancer cells, their role in chemotherapy response has not been systematically investigated. In the present study we have adapted a fluorescence-based cytotoxic assay to characterize the influence of key drug-transporters on the toxicity of approved anticancer drugs. Co-cultures of fluorescently labeled parental and transporter-expressing cells (expressing ABCB1, ABCG2 or OATP2B1) were screened against 101 FDA-approved anticancer drugs, using a novel, automated, triple fluorescence-based cytotoxicity assay. By measuring the survival of parental and transporter-expressing cells in co-cultures, we identify those FDA-approved anticancer drugs, whose toxicity is influenced by ABCB1, ABCG2 or OATP2B1. In addition to confirming known substrates of ABCB1 and ABCG2, the fluorescence-based cytotoxicity assays identified anticancer agents whose toxicity was increased in OATP2B1 expressing cells. Interaction of these compounds with OATP2B1 was verified in dedicated transport assays using cell-impermeant fluorescent substrates. Understanding drug-transporter interactions is needed to increase the efficacy of chemotherapeutic agents. Our results highlight the potential of the fluorescence-based HT screening system for identifying transporter substrates, opening the way for the design of therapeutic approaches based on the inhibition or even the exploitation of transporters in cancer cells.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Luminescentes/genética , Transportadores de Ânions Orgânicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Simulação por Computador , Citometria de Fluxo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Humanos , Transportadores de Ânions Orgânicos/genética , Especificidade por Substrato , Transdução Genética , Proteína Vermelha FluorescenteRESUMO
ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Análise de Célula Única/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/isolamento & purificação , Adenosina Trifosfatases/genética , Benzimidazóis/química , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Corantes Fluorescentes/química , Regulação Neoplásica da Expressão Gênica , Humanos , Especificidade por SubstratoRESUMO
Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Ser(i)-DL-Ala(m))] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss(®) bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.
Assuntos
Tecido Adiposo/citologia , Substitutos Ósseos/metabolismo , Transplante Ósseo , Células-Tronco Mesenquimais/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Tecido Adiposo/efeitos dos fármacos , Adulto , Animais , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Transplante Ósseo/métodos , Bovinos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Modelos Biológicos , Osseointegração/efeitos dos fármacos , Osseointegração/fisiologia , Polímeros/farmacologia , Propriedades de Superfície/efeitos dos fármacosRESUMO
Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC(hTERT), ASC(Bmi-1), ASC(Bmi-1+hTERT) and ASC(SV40T+hTERT) were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC(Bmi-1) had limited replicative potential, while the rapidly proliferating ASC(SV40T+hTERT) acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC(hTERT) and ASC(hTERT+Bmi-1), on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC(hTERT) also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC(hTERT) are prone to transformation during extensive subculturing. The combination of Bmi-1 and hTERT successfully immortalized human ASCs without significantly perturbing their phenotype or biological behavior.
Assuntos
Tecido Adiposo/fisiologia , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Senescência Celular/fisiologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Telomerase/genética , Tecido Adiposo/citologia , Tecido Adiposo/patologia , Animais , Proliferação de Células , Senescência Celular/genética , Técnicas de Transferência de Genes , Humanos , Cariótipo , Lentivirus , Camundongos , Complexo Repressor Polycomb 1 , Células Estromais/citologia , Células Estromais/patologia , Células Estromais/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.
Assuntos
Antineoplásicos , Células-Tronco Mesenquimais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Carcinogênese/patologia , DNA/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Microambiente TumoralRESUMO
Neuronal plasticity has been shown to be causally linked to coincidence detection through dendritic spikes (dSpikes). We demonstrate the existence of SPW-R-associated, branch-specific, local dSpikes and their computational role in basal dendrites of hippocampal PV+ interneurons in awake animals. To measure the entire dendritic arbor of long thin dendrites during SPW-Rs, we used fast 3D acousto-optical imaging through an eccentric deep-brain adapter and ipsilateral local field potential recording. The regenerative calcium spike started at variable, NMDA-AMPA-dependent, hot spots and propagated in both direction with a high amplitude beyond a critical distance threshold (~150 µm) involving voltage-gated calcium channels. A supralinear dendritic summation emerged during SPW-R doublets when two successive SPW-R events coincide within a short temporal window (~150 ms), e.g., during more complex association tasks, and generated large dSpikes with an about 2.5-3-fold amplitude increase which propagated down to the soma. Our results suggest that these doublet-associated dSpikes can work as a dendritic-level temporal and spatial coincidence detector during SPW-R-related network computation in awake mice.
Assuntos
Interneurônios , Parvalbuminas , Camundongos , Animais , Potenciais de Ação/fisiologia , Interneurônios/fisiologia , Dendritos/fisiologia , Neurônios/fisiologia , Hipocampo/fisiologia , Células Piramidais/fisiologiaRESUMO
Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.
Assuntos
Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Animais , Transporte Biológico , Corantes/metabolismo , Cães , Interações Medicamentosas , Células HEK293 , Humanos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Células Madin Darby de Rim Canino , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Células Sf9 , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/antagonistas & inibidoresRESUMO
The role of extracellular vesicles (EVs) in mediating the immunosuppressory properties of mesenchymal stem cells (MSCs) has recently attracted remarkable scientific interest. The aim of this work was to analyze the transport mechanisms of membrane and cytoplasmic components between T lymphocytes and adipose tissue-derived MSCs (AD-MSCs), by focusing on the role of distinct populations of EVs, direct cell-cell contacts, and the soluble mediators per se in modulating T lymphocyte function. We found that neither murine thymocytes and human primary T cells nor Jurkat lymphoblastoid cells incorporated appreciable amounts of MSC-derived microvesicles (MVs) or exosomes (EXOs). Moreover, these particles had no effect on the proliferation and IFN-γ production of in vitro-stimulated primary T cells. In contrast, AD-MSCs incorporated large amounts of membrane components from T cells as an intensive uptake of EXOs and MVs could be observed. Interestingly, we found a bidirectional exchange of cytoplasmic components between human AD-MSCs and primary T lymphocytes, mediated by tunneling nanotubes (TNTs) derived exclusively from the T cells. In contrast, TNTs couldn't be observed between AD-MSCs and the Jurkat cells. Our results reveal a novel and efficient way of intercellular communication between MSCs and T cells, and may help a better understanding of the immunomodulatory function of MSCs.
Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/citologia , Nanotubos/química , Linfócitos T/citologia , Tecido Adiposo/citologia , Adulto , Animais , Membrana Celular/metabolismo , Pré-Escolar , Técnicas de Cocultura , Citoplasma/metabolismo , Exossomos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunomodulação , Células Jurkat , Ativação Linfocitária/imunologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal , Linfócitos T/metabolismoRESUMO
Bone tissue regeneration is a major, worldwide medical need, and several strategies have been developed to support the regeneration of extensive bone defects, including stem cell based bone grafts. In addition to the application of stem cells with high osteogenic potential, it is important to maintain proper blood flow in a bone graft to avoid inner graft necrosis. Mesenchymal stem cells (MSCs) may form both osteocytes and endothelial cells; therefore we examined the combined in vitro osteogenic and endothelial differentiation capacities of MSCs derived from adipose tissue, Wharton's jelly, and periodontal ligament. Based on a detailed characterization presented here, MSCs isolated from adipose tissue and periodontal ligament may be most appropriate for generating vascularized bone grafts.
RESUMO
Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth.