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1.
Mol Psychiatry ; 27(9): 3739-3748, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35501409

RESUMO

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.


Assuntos
Proteínas 14-3-3 , Encéfalo , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Proteínas 14-3-3/genética , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
2.
BMC Bioinformatics ; 21(1): 529, 2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203360

RESUMO

BACKGROUND: Antibodies revolutionized cancer treatment over the past decades. Despite their successfully application, there are still challenges to overcome to improve efficacy, such as the heterogeneous distribution of antibodies within tumors. Tumor microenvironment features, such as the distribution of tumor and other cell types and the composition of the extracellular matrix may work together to hinder antibodies from reaching the target tumor cells. To understand these interactions, we propose a framework combining in vitro and in silico models. We took advantage of in vitro cancer models previously developed by our group, consisting of tumor cells and fibroblasts co-cultured in 3D within alginate capsules, for reconstruction of tumor microenvironment features. RESULTS: In this work, an experimental-computational framework of antibody transport within alginate capsules was established, assuming a purely diffusive transport, combined with an exponential saturation effect that mimics the saturation of binding sites on the cell surface. Our tumor microenvironment in vitro models were challenged with a fluorescent antibody and its transport recorded using light sheet fluorescence microscopy. Diffusion and saturation parameters of the computational model were adjusted to reproduce the experimental antibody distribution, with root mean square error under 5%. This computational framework is flexible and can simulate different random distributions of tumor microenvironment elements (fibroblasts, cancer cells and collagen fibers) within the capsule. The random distribution algorithm can be tuned to follow the general patterns observed in the experimental models. CONCLUSIONS: We present a computational and microscopy framework to track and simulate antibody transport within the tumor microenvironment that complements the previously established in vitro models platform. This framework paves the way to the development of a valuable tool to study the influence of different components of the tumor microenvironment on antibody transport.


Assuntos
Anticorpos/metabolismo , Simulação por Computador , Microambiente Tumoral/imunologia , Algoritmos , Contagem de Células , Linhagem Celular Tumoral , Difusão , Fluorescência , Humanos , Neoplasias/patologia , Transporte Proteico , Processos Estocásticos
3.
Arch Toxicol ; 92(1): 411-423, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28932931

RESUMO

Prediction and management of drug-induced renal injury (DIRI) rely on the knowledge of the mechanisms of drug insult and on the availability of appropriate animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI because the larval pronephric kidney has a high homology with its human counterpart and it is fully mature at 3.5 days post-fertilization. Herein, we aimed to evaluate the usefulness of zebrafish larvae as a model of renal tubular toxicity through a comprehensive analysis of the renal alterations induced by the lethal concentrations for 10% of the larvae for gentamicin, paracetamol and tenofovir. We evaluated drug metabolic profile by mass spectrometry, renal function with the inulin clearance assay, the 3D morphology of the proximal convoluted tubule by two-photon microscopy and the ultrastructure of proximal convoluted tubule mitochondria by transmission electron microscopy. Paracetamol was metabolized by conjugation and oxidation with further detoxification with glutathione. Renal clearance was reduced with gentamicin and paracetamol. Proximal tubules were enlarged with paracetamol and tenofovir. All drugs induced mitochondrial alterations including dysmorphic shapes ("donuts", "pancakes" and "rods"), mitochondrial swelling, cristae disruption and/or loss of matrix granules. These results are in agreement with the tubular effects of gentamicin, paracetamol and tenofovir in man and demonstrate that zebrafish larvae might be a good model to assess functional and structural damage associated with DIRI.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Túbulos Renais Proximais/efeitos dos fármacos , Testes de Toxicidade/métodos , Peixe-Zebra , Acetaminofen/efeitos adversos , Acetaminofen/farmacocinética , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/patologia , Animais , Animais Geneticamente Modificados , Gentamicinas/efeitos adversos , Gentamicinas/farmacocinética , Inativação Metabólica , Testes de Função Renal , Túbulos Renais Proximais/patologia , Larva , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacocinética , Tenofovir/efeitos adversos , Tenofovir/farmacocinética , Peixe-Zebra/genética
4.
Cytometry A ; 91(2): 144-151, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28075531

RESUMO

Flow cytometry is the tool of choice for high-speed acquisition and analysis of large cell populations, with the tradeoff of lacking intracellular spatial information. Although in the last decades flow cytometry systems that can actually acquire two-dimensional spatial information were developed, some of the limitations remained though, namely constrains related to sample size and lack of depth or dynamic information. The combination of fluidics and light-sheet illumination has the potential to address these limitations. By having cells travelling with the flowing sheath one can, in a controlled fashion, force them at constant speed through the light-sheet enabling the synchronized acquisition of several optical sections, that is, three-dimensional imaging. This approach has already been used for imaging cellular spheroids, plankton, and zebra-fish embryos. In this review, we discuss the known solutions and standing challenges of performing three-dimensional high-throughput imaging of multicellular biological models using fluidics, while retaining cell and organelle-level resolution. © 2017 International Society for Advancement of Cytometry.


Assuntos
Citometria de Fluxo/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Ensaios de Triagem em Larga Escala , Plâncton/ultraestrutura , Esferoides Celulares/ultraestrutura , Peixe-Zebra
5.
Elife ; 132024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38224094

RESUMO

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.


Assuntos
Interações Hospedeiro-Patógeno , Peixe-Zebra , Animais , Macrófagos/microbiologia , Salmonella typhimurium , Fenótipo
6.
J Biol Chem ; 287(46): 38946-55, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23019323

RESUMO

The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Lectinas/química , Receptores de Superfície Celular/metabolismo , Animais , Sítios de Ligação , Biofísica/métodos , Células CHO , Membrana Celular/metabolismo , Clatrina/metabolismo , Cricetinae , Cricetulus , Citoplasma/metabolismo , Células Dendríticas/citologia , Difusão , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Propriedades de Superfície , Fatores de Tempo
7.
Micromachines (Basel) ; 14(11)2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-38004954

RESUMO

We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF4:Yb3+/Er3+) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF4:Yb3+/Er3+ nanoparticles immersed in agar. The composite materials show upconverted (UC) fluorescence bands when excited by a 980 nm near-infrared laser light-sheet. Using a single CMOS camera and a pair of interferometric optical filters to specifically image the two thermally-coupled bands (at 525 and 550 nm), the two-dimensional FIR and, hence, the temperature map can be readily obtained. The proposed method can take optically sectioned (confocal-like) images with good optical resolution over relatively large samples (up to the millimetric scale) for further 3D temperature reconstruction.

8.
Sci Rep ; 12(1): 1969, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121789

RESUMO

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.

9.
Plants (Basel) ; 11(9)2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35567165

RESUMO

Plant-parasitic nematodes are a significant cause of yield losses and food security issues. Specifically, nematodes of the genus Meloidogyne can cause significant production losses in horticultural crops around the world. Understanding the mechanisms of the ever-changing physiology of plant roots by imaging the galls induced by nematodes could provide a great insight into their control. However, infected roots are unsuitable for light microscopy investigation due to the opacity of plant tissues. Thus, samples must be cleared to visualize the interior of whole plants in order to make them transparent using clearing agents. This work aims to identify which clearing protocol and microscopy system is the most appropriate to obtain 3D images of tomato cv. Durinta and eggplant cv. Cristal samples infected with Meloidogyne incognita to visualize and study the root-nematode interaction. To that extent, two clearing solutions (BABB and ECi), combined with three different dehydration solvents (ethanol, methanol and 1-propanol), are tested. In addition, the advantages and disadvantages of alternative imaging techniques to confocal microscopy are analyzed by employing an experimental custom-made setup that combines two microscopic techniques, light sheet fluorescence microscopy and optical projection tomography, on a single instrument.

10.
Sci Rep ; 12(1): 4072, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260695

RESUMO

It is known that the organization of microtubule (MT) networks in cells is orchestrated by subcellular structures named MT organizing centers (MTOCs). In this work, we use Light Sheet Fluorescence and Confocal Microscopy to investigate how the MT network surrounding the spherical yolk is arranged in the dclk2-GFP zebrafish transgenic line. We found that during epiboly the MT network is organized by multiple aster-like MTOCS. These structures form rings around the yolk sphere. Importantly, in wt embryos, aster-like MTOCs are only found upon pharmacological or genetic induction. Using our microscopy approach, we underscore the variability in the number of such asters in the transgenic line and report on the variety of global configurations of the yolk MT network. The asters' morphology, dynamics, and their distribution in the yolk sphere are also analyzed. We propose that these features are tightly linked to epiboly timing and geometry. Key molecules are identified which support this asters role as MTOCs, where MT nucleation and growth take place. We conclude that the yolk MT network of dclk2-GFP transgenic embryos can be used as a model to organize microtubules in a spherical geometry by means of multiple MTOCs.


Assuntos
Microtúbulos , Peixe-Zebra , Animais , Citoplasma , Centro Organizador dos Microtúbulos , Morfogênese , Peixe-Zebra/genética
11.
Adv Healthc Mater ; 11(22): e2201172, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36073021

RESUMO

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.


Assuntos
Mucosa Intestinal , Organoides , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Intestinos , Intestino Delgado , Diferenciação Celular/fisiologia
13.
Biomed Opt Express ; 12(10): 6237-6254, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34745732

RESUMO

During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

14.
Biomed Opt Express ; 12(10): 6205-6227, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34745730

RESUMO

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

15.
Neuroscience ; 439: 211-229, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31689390

RESUMO

The cerebellum harbors a specialized area on the roof of the fourth ventricle that is composed of glial cells and neurons that interface with the cerebrospinal fluid. This region includes the so-called ventromedial cord (VMC), which is composed of cells that are glial fibrillary acidic protein (GFAP)-positive and nestin-positive and distributes along the midline in association with blood vessels. We hypothesized that these cells should compare to GFAP and nestin-positive cells that are known to exist in other areas of the brain, which undergo proliferation and differentiation under hypoxic conditions. Thus, we tested whether cells of the VMC would display a similar reaction to hypoxic preconditioning (HPC). Indeed, we found that the VMC does respond to HPC by reorganizing its cellular components before it gradually returns to its basal state after about a week. This response we documented by monitoring global changes in the expression of GFAP-EGFP in transgenic mice, using light-sheet fluorescence microscopy (LSFM) revealed a dramatic loss of EGFP upon HPC, and was paralleled by retraction of Bergmann glial cell processes. This EGFP loss was supported by western blot analysis, which also showed a loss in the astrocyte-markers GFAP and ALDH1L1. On the other hand, other cell-markers appeared to be upregulated in the blots (including nestin, NeuN, and Iba1). Finally, we found that HPC does not remarkably affect the incorporation of BrdU into cells on the cerebellum, but strongly augments BrdU incorporation into periventricular cells on the floor of the fourth ventricle over the adjacent medulla.


Assuntos
Quarto Ventrículo , Neuroglia , Animais , Astrócitos/metabolismo , Quarto Ventrículo/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Neuroglia/metabolismo , Neurônios/metabolismo
16.
J Exp Clin Cancer Res ; 39(1): 161, 2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807212

RESUMO

BACKGROUND: Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena. METHODS: BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-ß-estradiol and fulvestrant. RESULTS: We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling. CONCLUSIONS: The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.


Assuntos
Adenocarcinoma Mucinoso/patologia , Neoplasias da Mama/patologia , Carcinoma Lobular/patologia , Receptor alfa de Estrogênio/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Lobular/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Feminino , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral
17.
Appl Spectrosc ; 63(3): 280-5, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19281643

RESUMO

We present an appropriate methodology and results for using third-harmonic generation (THG) modality for nondestructive high resolution imaging measurements of varnished structures in model painted artifacts. Detection takes place in the reflection mode, demonstrating the ability of the technique to be applied to the evaluation of original artworks. Furthermore, multi-photon excitation fluorescence images were obtained, providing complementary information related to the identification of the chemical composition of the artifacts.

18.
Biomaterials ; 163: 185-197, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29477032

RESUMO

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.


Assuntos
Técnicas de Cultura de Células , Macrófagos/fisiologia , Microambiente Tumoral/fisiologia , Antineoplásicos/farmacologia , Apoptose , Carcinoma Pulmonar de Células não Pequenas , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Plasticidade Celular , Proliferação de Células , Sobrevivência Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Células Mieloides , Invasividade Neoplásica , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Microambiente Tumoral/efeitos dos fármacos
19.
Sci Rep ; 6: 28209, 2016 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-27312902

RESUMO

Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Proteínas de Fluorescência Verde/genética , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Transgênicos , Ratos Wistar , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo
20.
Biomaterials ; 78: 50-61, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26650685

RESUMO

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms.


Assuntos
Microambiente Tumoral , Técnicas de Cocultura , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Humanos , Células MCF-7
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