Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 706
Filtrar
1.
Nat Biomed Eng ; 4(4): 446-462, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32284552

RESUMO

Environmental factors are the largest contributors to cardiovascular disease. Here we show that cardiac organoids that incorporate an oxygen-diffusion gradient and that are stimulated with the neurotransmitter noradrenaline model the structure of the human heart after myocardial infarction (by mimicking the infarcted, border and remote zones), and recapitulate hallmarks of myocardial infarction (in particular, pathological metabolic shifts, fibrosis and calcium handling) at the transcriptomic, structural and functional levels. We also show that the organoids can model hypoxia-enhanced doxorubicin cardiotoxicity. Human organoids that model diseases with non-genetic pathological factors could help with drug screening and development.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Coração/efeitos dos fármacos , Modelos Cardiovasculares , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Organoides/efeitos dos fármacos , Cardiotoxicidade/metabolismo , Cardiotoxicidade/patologia , Desenvolvimento de Medicamentos , Humanos , Infarto do Miocárdio/induzido quimicamente , Infarto do Miocárdio/genética , Organoides/metabolismo , Organoides/patologia , Oxigênio/metabolismo
2.
PLoS One ; 15(2): e0229283, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084202

RESUMO

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother's expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.


Assuntos
Bifidobacterium longum subspecies infantis/fisiologia , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Ácidos Graxos Voláteis/biossíntese , Ácidos Graxos Voláteis/farmacologia , Intestinos/microbiologia , Leite Humano/microbiologia , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Enterócitos/metabolismo , Indução Enzimática/efeitos dos fármacos , Feto/microbiologia , Histona Desacetilases/genética , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Intestinos/citologia , Camundongos , Mutagênese Insercional/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
3.
PLoS One ; 15(2): e0229407, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32097436

RESUMO

Therapeutic resistance plagues cancer outcomes, challenging treatment particularly in aggressive disease. A unique method to decipher drug interactions with their targets and inform therapy is to employ fluorescence-based screening tools; however, to implement productive screening assays, adequate model systems must be developed. Patient-derived pancreatic cancer models (e.g., cell culture, patient-derived xenograft mouse models, and organoids) have been traditionally utilized to predict personalized therapeutic response. However, cost, long read out times and the inability to fully recapitulate the tumor microenvironment have rendered most models incompatible with clinical decision making for pancreatic ductal adenocarcinoma (PDAC) patients. Tumor explant cultures, where patient tissue can be kept viable for up to weeks, have garnered interest as a platform for delivering personalized therapeutic prediction on a clinically relevant timeline. To fully explore this ex vivo platform, a series of studies were completed to quantitatively compare in vivo models with tumor explants, examining gemcitabine therapeutic efficacy, small molecule uptake and drug-target engagement using a novel fluorescently-labeled gemcitabine conjugate. This initial work shows promise for patient-specific therapeutic selection, where tumor explant drug distribution and response recapitulated the in vivo behavior and could provide a valuable platform for understanding mechanisms of therapeutic response and resistance.


Assuntos
Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Modelos Animais de Doenças , Corantes Fluorescentes/farmacocinética , Organoides/patologia , Neoplasias Pancreáticas/patologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Desoxicitidina/química , Desoxicitidina/farmacologia , Feminino , Corantes Fluorescentes/química , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Organoides/efeitos dos fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Cell Biol ; 22(2): 167-174, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029896

RESUMO

Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.


Assuntos
Adenocarcinoma/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transaminases/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Transaminases/metabolismo
5.
PLoS One ; 15(1): e0227116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31951625

RESUMO

In this study we aimed to explore the potential biological effect of ethanol exposure on healthy colon epithelial cells using normal human colon 3D organoid "mini-gut" cultures. In numerous published studies ethanol use has been shown to be an environmental risk factor for colorectal cancer (CRC) development; however, the influence of ethanol exposure on normal colon epithelial cell biology remains poorly understood. We investigated the potential molecular effects of ethanol exposure in normal colon 3D organoids in a small pilot study (n = 3) using RNA-seq and ATAC-seq. We identify 1965 differentially expressed genes and 2217 differentially accessible regions of chromatin in response to ethanol treatment. Further, by cross-referencing our results with previously published analysis in colorectal cancer cell lines, we have not only validated a number of reported differentially expressed genes, but also identified several novel candidates for future investigation. In summary, our data highlights the potential importance for the use of normal colon 3D organoid models as a novel tool for the investigation of the relationship between the effects of environmental risk factors associated with colorectal cancer and the molecular mechanisms through which they confer this risk.


Assuntos
Montagem e Desmontagem da Cromatina , Colo/efeitos dos fármacos , Etanol/farmacologia , Organoides/efeitos dos fármacos , Transcriptoma , Adulto , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Colo/citologia , Colo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Organoides/metabolismo
6.
Cancer Sci ; 111(1): 137-147, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31724799

RESUMO

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3-K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase-inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium-induced colitis, the kinase-inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin-1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Intestinos/patologia , Organoides/metabolismo , Organoides/patologia , Receptor ErbB-3/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinogênese/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Organoides/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Pólipos/tratamento farmacológico , Pólipos/patologia , Inibidores de Proteínas Quinases/farmacologia
7.
Nat Med ; 25(10): 1607-1614, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31591597

RESUMO

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.


Assuntos
Quimiorradioterapia , Organoides/patologia , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/radioterapia , Animais , Fluoruracila/farmacologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica , Organoides/efeitos dos fármacos , Organoides/efeitos da radiação , Neoplasias Retais/patologia
8.
PLoS Biol ; 17(10): e3000498, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613879

RESUMO

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.


Assuntos
Proteína Morfogenética Óssea 4/genética , Gastrulação/genética , Mesoderma/metabolismo , Proteína Nodal/genética , Transdução de Sinais , Proteínas Wnt/genética , Benzotiazóis/farmacologia , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Gástrula/citologia , Gástrula/efeitos dos fármacos , Gástrula/metabolismo , Gastrulação/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Modelos Biológicos , Modelos Estatísticos , Proteína Nodal/deficiência , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteínas Wnt/metabolismo
9.
Nat Commun ; 10(1): 4407, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562298

RESUMO

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.


Assuntos
Diferenciação Celular/genética , Integrina alfa6/genética , Organoides/metabolismo , Receptores Notch/metabolismo , Células-Tronco/metabolismo , Urotélio/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Integrina alfa6/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Organoides/citologia , Organoides/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Urotélio/citologia
10.
Nihon Yakurigaku Zasshi ; 154(2): 50-55, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31406042

RESUMO

Colorectal cancer is a disease with high unmet medical needs. An increase in the number of cancer patients who are resistant to anti-cancer drugs is one of factors that increase the number of fatalities. Since there was no suitable experimental model to recapitulate the tumor environment in which various cells in the tissues exist, it was extremely difficult to develop a medicine that overcomes the anti-cancer drug resistance in each colorectal cancer patient. In this review, we describe the current status and problems of drug therapy for colorectal cancer patients, and introduce our study to develop the new targeting drugs using human colon tissue-derived air liquid interface organoid culture method.


Assuntos
Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Organoides/efeitos dos fármacos , Humanos , Técnicas de Cultura de Tecidos
11.
Nutrients ; 11(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426533

RESUMO

Growth hormone (GH) and glutamine (Gln) stimulate the growth of the intestinal mucosa. GH activates the proliferation of intestinal stem cells (ISCs), enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and drives the differentiation of ISCs into Paneth cells and enterocytes. Gln enhances the proliferation of ISCs and increases crypt organoid formation; however, it mainly acts on the post-proliferation activity of ISCs to maintain the stability of crypt organoids and the intestinal mucosa, as well as to stimulate the differentiation of ISCs into goblet cells and possibly Paneth cells and enteroendocrine cells. Since GH and Gln have differential effects on ISCs. Their use in combination may have synergistic effects on ISCs. In this review, we summarize the evidence of the actions of GH and/or Gln on crypt cells and ISCs in the literature. Overall, most studies demonstrated that GH and Gln in combination exerted synergistic effects to activate the proliferation of crypt cells and ISCs and enhance crypt organoid formation and mucosal growth. This treatment influenced the proliferation of ISCs to a similar degree as GH treatment alone and the differentiation of ISCs to a similar degree as Gln treatment alone.


Assuntos
Glutamina/farmacologia , Hormônio do Crescimento/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células , Sinergismo Farmacológico , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco/fisiologia
12.
World J Gastroenterol ; 25(30): 4125-4147, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31435168

RESUMO

The introduction of biologics such as anti-tumor necrosis factor (TNF) monoclonal antibodies followed by anti-integrins has dramatically changed the therapeutic paradigm of inflammatory bowel diseases (IBD). Furthermore, a newly developed anti-p40 subunit of interleukin (IL)-12 and IL-23 (ustekinumab) has been recently approved in the United States for patients with moderate to severe Crohn's disease who have failed treatment with anti-TNFs. However, these immunosuppressive therapeutics which focus on anti-inflammatory mechanisms or immune cells still fail to achieve long-term remission in a significant percentage of patients. This strongly underlines the need to identify novel treatment targets beyond immune suppression to treat IBD. Recent studies have revealed the critical role of intestinal epithelial cells (IECs) in the pathogenesis of IBD. Physical, biochemical and immunologic driven barrier dysfunctions of epithelial cells contribute to the development of IBD. In addition, the recent establishment of adult stem cell-derived intestinal enteroid/organoid culture technology has allowed an exciting opportunity to study human IECs comprising all normal epithelial cells. This long-term epithelial culture model can be generated from endoscopic biopsies or surgical resections and recapitulates the tissue of origin, representing a promising platform for novel drug discovery in IBD. This review describes the advantages of intestinal enteroids/organoids as a research tool for intestinal diseases, introduces studies with these models in IBD, and gives a description of the current status of therapeutic approaches in IBD. Finally, we provide an overview of the current endeavors to identify a novel drug target for IBD therapy based on studies with human enteroids/organoids and describe the challenges in using enteroids/organoids as an IBD model.


Assuntos
Descoberta de Drogas/métodos , Imunossupressores/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Organoides/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Biópsia , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais , Humanos , Imunossupressores/uso terapêutico , Células-Tronco Pluripotentes Induzidas , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Organoides/imunologia , Cultura Primária de Células/métodos
13.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452509

RESUMO

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.


Assuntos
Anticorpos/metabolismo , Receptores Frizzled/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Camundongos , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Ligação Proteica
14.
Nat Cell Biol ; 21(8): 1041-1051, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31371824

RESUMO

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Neoplasias do Endométrio/patologia , Organoides/patologia , Doenças Uterinas/patologia , Técnicas de Cultura de Células/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Doenças Uterinas/tratamento farmacológico , Doenças Uterinas/metabolismo
15.
PLoS One ; 14(7): e0219697, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361756

RESUMO

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFß) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFß-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFß-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFß has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFß has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFß actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Proteoglicanas/metabolismo , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Peso Molecular , Invasividade Neoplásica , Organoides/efeitos dos fármacos , Organoides/patologia , Proteínas Recombinantes/farmacologia , Proteínas Smad/metabolismo
16.
PLoS Comput Biol ; 15(7): e1007214, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31310602

RESUMO

The dynamics of tumor progression is driven by multiple factors, which can be exogenous to the tumor (microenvironment) or intrinsic (genetic, epigenetic or due to intercellular interactions). While tumor heterogeneity has been extensively studied on the level of cell genetic profiles or cellular composition, tumor morphological diversity has not been given as much attention. The limited analysis of tumor morphophenotypes may be attributed to the lack of accurate models, both experimental and computational, capable of capturing changes in tumor morphology with fine levels of spatial detail. Using a three-dimensional, agent-based, lattice-free computational model, we generated a library of multicellular tumor organoids, the experimental analogues of in vivo tumors. By varying three biologically relevant parameters-cell radius, cell division age and cell sensitivity to contact inhibition, we showed that tumor organoids with similar growth dynamics can express distinct morphologies and possess diverse cellular compositions. Taking advantage of the high-resolution of computational modeling, we applied the quantitative measures of compactness and accessible surface area, concepts that originated from the structural biology of proteins. Based on these analyses, we demonstrated that tumor organoids with similar sizes may differ in features associated with drug effectiveness, such as potential exposure to the drug or the extent of drug penetration. Both these characteristics might lead to major differences in tumor organoid's response to therapy. This indicates that therapeutic protocols should not be based solely on tumor size, but take into account additional tumor features, such as their morphology or cellular packing density.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/patologia , Organoides/efeitos dos fármacos , Organoides/patologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Biologia Computacional , Simulação por Computador , Progressão da Doença , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imageamento Tridimensional , Modelos Biológicos , Neoplasias/metabolismo , Organoides/metabolismo , Fenótipo , Propriedades de Superfície , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia
17.
PLoS One ; 14(7): e0213114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31295264

RESUMO

BACKGROUND: 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. METHODS AND RESULTS: Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. CONCLUSIONS: COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.


Assuntos
Adenosina/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Adenosina/análogos & derivados , Linhagem Celular , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/metabolismo
18.
PLoS One ; 14(7): e0219944, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339921

RESUMO

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent "Warburg effect" blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.


Assuntos
Neoplasias Colorretais/metabolismo , Metabolismo dos Lipídeos , Organoides/metabolismo , Animais , Antineoplásicos/farmacologia , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Fluoruracila/farmacologia , Glicólise , Hipoglicemiantes/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Metformina/farmacologia , Camundongos , Organoides/efeitos dos fármacos
19.
Cancer Sci ; 110(9): 2806-2821, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31254429

RESUMO

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.


Assuntos
Técnicas de Cultura de Células/métodos , Doenças do Cão/patologia , Organoides/patologia , Neoplasias da Bexiga Urinária/veterinária , Bexiga Urinária/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doenças do Cão/tratamento farmacológico , Doenças do Cão/genética , Doenças do Cão/urina , Cães , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Masculino , Organoides/efeitos dos fármacos , Organoides/metabolismo , Análise de Sequência de RNA , Regulação para Cima , Bexiga Urinária/citologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina , Urina/citologia , Urotélio/citologia
20.
Curr Med Sci ; 39(3): 403-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209810

RESUMO

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Ácido Láctico/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Simportadores/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Via de Sinalização Wnt/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA