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Molecular links between breast cancer risk factors and pro-oncogenic tissue alterations are poorly understood. The goal of this study was to characterize the impact of overweight and obesity on tissue markers of risk, using normal breast biopsies, a mouse model of diet-induced obesity, and cultured breast acini. Proliferation and alteration of epithelial polarity, both necessary for tumor initiation, were quantified by immunostaining. High BMI (>30) and elevated leptin were associated with compromised epithelial polarity whereas overweight was associated with a modest increase in proliferation in human and mice mammary glands. Human serum with unfavorable adipokine levels altered epithelial polarization of cultured acini, recapitulating the effect of leptin. Weight loss in mice led to metabolic improvements and restored epithelial polarity. In acini cultures, alteration of epithelial polarity was prevented by antioxidants and could be reverted by normalizing culture conditions. This study shows that obesity and/or dietary factors modulate tissue markers of risk. It provides a framework to set target values for metabolic improvements and to assess the efficacy of interventional studies aimed at reducing breast cancer risk.
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The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.
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Microscopia , Radiobiologia , Animais , Radiação Ionizante , Raios XRESUMO
Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet-derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. SIGNIFICANCE: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3890/F1.large.jpg.
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Mama/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Animais , Carcinogênese , Feminino , Humanos , Camundongos , Microbiota , Transdução de SinaisRESUMO
Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Artefatos , Inteligência Artificial , Teorema de Bayes , Linhagem Celular Tumoral , Aprendizado Profundo , Humanos , Redes Neurais de Computação , Razão Sinal-RuídoRESUMO
P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.
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Antígenos Nucleares/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades/genética , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.
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Neoplasias da Mama/patologia , Leptina/sangue , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Proteínas Adaptadoras de Transdução de Sinal , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Animais , Índice de Massa Corporal , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Leptina/genética , Leptina/metabolismo , Glândulas Mamárias Humanas/metabolismo , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Obesidade/patologia , Lesões Pré-Cancerosas , Fuso Acromático/metabolismo , Fuso Acromático/patologiaRESUMO
Preventing cancer is vastly better than treating the disease in terms of a patient's quality of life and healthcare costs. Yet, to screen for chemopreventative drugs or evaluate interventions aimed at lowering cancer risk, quantitative readouts of risk are needed. In the breast and in other organs of epithelial origin, apical-basal polarity is key to homeostasis and is one of the first tissue characteristics lost during cancer initiation. Therefore, apical-basal polarity may be leveraged as an "architectural" determinant of cancer risk. A classic approach to quantify the localization of epithelial polarity markers is visual scoring at the microscope by trained investigators. This approach is time-intensive and limited to low throughput. To increase the speed, accuracy, and scoring volume, we developed an algorithm that essentially replaces the human eye to objectively quantify epithelial polarity in microscopy images of breast glandular units (acini). Acini in culture are identified based on a nuclear stain and the corresponding masks are divided into concentric terraces of equal width. This positional information is used to calculate radial intensity profiles (RP) of polarity markers. Profiles with a steep slope represent polarized structures, whereas more horizontal curves are indicative of non-polarized acini. To compare treatment effects, RP curves are integrated into summary values of polarity. We envision applications of this method for primary cancer prevention research with acini organoids, specifically (1) to screen for chemoprevention drugs, (2) for toxicological assessment of suspected carcinogens and pharmacological hit compounds, and (3) for personalized evaluation of cancer risk and risk-reducing interventions. The RadialProfiler algorithm developed for the MATLAB computing environment and for users without prior informatics knowledge is publicly available on the Open Science Framework (OSF).
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Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple-negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP-based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non-malignant breast epithelial cells. In contrast, TNBC cells and non-malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC-specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non-malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non-malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non-toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.
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Up to 30% of patients with metastatic breast cancer eventually develop brain metastasis, yet the pathologic mechanism behind this development remains poorly understood. Here, we profiled long noncoding RNAs in brain metastatic tumors from patients with breast cancer and found that the X-inactive-specific transcript (XIST) was significantly downregulated in these tissues. XIST expression levels inversely correlated with brain metastasis, but not with bone metastasis in patients. Silencing of XIST preferentially promoted brain metastatic growth of XISThigh cells in our xenograft models. Moreover, knockout of XIST in mice mammary glands accelerated primary tumor growth as well as metastases in the brain. Decreased expression of XIST stimulated epithelial-mesenchymal transition and activated c-Met via MSN-mediated protein stabilization, which resulted in the promotion of stemness in the tumor cells. Loss of XIST also augmented secretion of exosomal miRNA-503, which triggered M1-M2 polarization of microglia. This M1-M2 conversion upregulated immune suppressive cytokines in microglia that suppressed T-cell proliferation. Furthermore, we screened an FDA-approved drug library and identified fludarabine as a synthetic lethal drug for XISTlow breast tumor cells and found that fludarabine blocked brain metastasis in our animal model. Our results indicate that XIST plays a critical role in brain metastasis in breast cancer by affecting both tumor cells and the tumor microenvironment and that the XIST-mediated pathway may serve as an effective target for treating brain metastasis.Significance: These findings describe mechanisms of how loss of the lncRNA XIST promotes brain metastasis in breast cancer and identify fludarabine as a potential therapeutic agent that specifically eliminates XISTlow tumor cells in the brain. Cancer Res; 78(15); 4316-30. ©2018 AACR.
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Neoplasias Ósseas/genética , Exossomos/genética , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Microglia/patologia , Proteínas Proto-Oncogênicas c-met/genética , RNA Longo não Codificante/genética , Animais , Neoplasias Ósseas/patologia , Encéfalo/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Microambiente Tumoral/genética , Regulação para Cima/genéticaRESUMO
We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340 ± 30 nm, which simultaneously photoactivate a 7 × 7 matrix pattern of GFP-labeled histones, with spots 1.70 µm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.
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Cromatina/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desenho de Equipamento , Histonas/química , Humanos , Iluminação , Microscopia de Fluorescência/instrumentaçãoRESUMO
The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.
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Antígenos Nucleares/genética , Nucléolo Celular/genética , DNA Ribossômico/genética , Proteínas Associadas à Matriz Nuclear/genética , Transcrição Gênica , Antígenos Nucleares/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dactinomicina/farmacologia , Doxorrubicina/farmacologia , Humanos , Microscopia Eletrônica , Proteínas Associadas à Matriz Nuclear/metabolismo , Ligação Proteica , Interferência de RNA , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Agriculture in the United States employs youth ages ten and older in work environments with high pesticide levels. Younger children in rural areas may also be affected by indirect pesticide exposures. The long-term effects of pesticides on health and development are difficult to assess and poorly understood. Yet, epidemiologic studies suggest associations with cancer as well as cognitive deficits. We report a practical and cost-effective approach to assess environmental pesticide exposures and their biological consequences in children. Our approach combines silicone wristband personal samplers and DNA damage quantification from hair follicles, and was tested as part of a community-based participatory research (CBPR) project involving ten Latino children from farmworker households in North Carolina. Our study documents high acceptance among Latino children and their caregivers of these noninvasive sampling methods. The personal samplers detected organophosphates, organochlorines, and pyrethroids in the majority of the participants (70%, 90%, 80%, respectively). Pesticides were detected in all participant samplers, with an average of 6.2±2.4 detections/participant sampler. DNA damage in epithelial cells from the sheath and bulb of plucked hairs follicles was quantified by immunostaining 53BP1-labled DNA repair foci. This method is sensitive, as shown by dose response analyses to γ radiations where the lowest dose tested (0.1Gy) led to significant increased 53BP1 foci density. Immunolabeling of DNA repair foci has significant advantages over the comet assay in that specific regions of the follicles can be analyzed. In this cohort of child participants, significant association was found between the number of pesticide detections and DNA damage in the papilla region of the hairs. We anticipate that this monitoring approach of bioavailable pesticides and genotoxicity will enhance our knowledge of the biological effects of pesticides to guide education programs and safety policies.
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Dano ao DNA , Reparo do DNA , Exposição Ambiental , Folículo Piloso/efeitos dos fármacos , Praguicidas , Manejo de Espécimes/instrumentação , Disponibilidade Biológica , Criança , Pesquisa Participativa Baseada na Comunidade , Exposição Ambiental/efeitos adversos , Exposição Ambiental/análise , Folículo Piloso/metabolismo , Humanos , North Carolina , Praguicidas/análise , Praguicidas/farmacocinética , Praguicidas/toxicidade , Medição de RiscoRESUMO
Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.
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Antígenos Nucleares/metabolismo , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Células-Tronco Mesenquimais/citologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Adipócitos/citologia , Antígenos Nucleares/genética , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular , Núcleo Celular/ultraestrutura , Separação Celular/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Osteócitos/citologia , Análise de Célula Única/métodosRESUMO
Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.
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Cromatina/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Histonas/genética , Trifosfato de Adenosina/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Reparo do DNA , Proteínas de Fluorescência Verde/genética , Humanos , Radiação Ionizante , Raios UltravioletaRESUMO
Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks.
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Adenosina Trifosfatases/metabolismo , Antígenos Nucleares/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Associadas à Matriz Nuclear/fisiologia , Reparo de DNA por Recombinação , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , HumanosRESUMO
We present a disease-on-a-chip model in which cancer grows within phenotypically normal breast luminal epithelium on semicircular acrylic support mimicking portions of mammary ducts. The cells from tumor nodules developing within these hemichannels are morphologically distinct from their counterparts cultured on flat surfaces. Moreover, tumor nodules cocultured with the luminal epithelium in hemichannels display a different anticancer drug sensitivity compared to nodules cocultured with the luminal epithelium on a flat surface and to monocultures of tumor nodules. The mimicry of tumor development within the epithelial environment of mammary ducts provides a framework for the design and test of anticancer therapies.
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Técnicas de Cultura de Células/métodos , Glândulas Mamárias Humanas/citologia , Técnicas Analíticas Microfluídicas/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/citologia , Feminino , Humanos , Integrina alfa6/metabolismo , Glândulas Mamárias Humanas/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Preventive actions for chronic diseases hold the promise of improving lives and reducing healthcare costs. For several diseases, including breast cancer, multiple risk and protective factors have been identified by epidemiologists. The impact of most of these factors has yet to be fully understood at the organism, tissue, cellular and molecular levels. Importantly, combinations of external and internal risk and protective factors involve cooperativity thus, synergizing or antagonizing disease onset. Models are needed to mechanistically decipher cancer risks under defined cellular and microenvironmental conditions. Here, we briefly review breast cancer risk models based on 3D cell culture and propose to improve risk modeling with lab-on-a-chip approaches. We suggest epithelial tissue polarity, DNA repair and epigenetic profiles as endpoints in risk assessment models and discuss the development of 'risks-on-chips' integrating biosensors of these endpoints and of general tissue homeostasis. Risks-on-chips will help identify biomarkers of risk, serve as screening platforms for cancer preventive agents, and provide a better understanding of risk mechanisms, hence resulting in novel developments in disease prevention.
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Biomarcadores Tumorais/análise , Neoplasias da Mama/etiologia , Neoplasias da Mama/prevenção & controle , Medição de Risco/métodos , Neoplasias da Mama/genética , Técnicas de Cultura de Células , Reparo do DNA , Feminino , Humanos , Medição de Risco/normasRESUMO
Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell-cell and cell-extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions.Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer.
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Técnicas de Cultura de Células/métodos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/patologia , Fenômenos Biomecânicos , Neoplasias da Mama/patologia , Crioultramicrotomia , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/química , Imagem Molecular , FenótipoRESUMO
Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis--a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation--thus, tissue polarity and NuMA cooperate to maintain genome integrity.