RESUMEN
Achieving complete tumor resection is challenging and can be improved by real-time fluorescence-guided surgery with molecular-targeted probes. However, pre-clinical identification and validation of probes presents a lengthy process that is traditionally performed in animal models and further hampered by inter- and intra-tumoral heterogeneity in target expression. To screen multiple probes at patient scale, we developed a multispectral real-time 3D imaging platform that implements organoid technology to effectively model patient tumor heterogeneity and, importantly, healthy human tissue binding.
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Imagenología Tridimensional , Organoides , Humanos , Imagenología Tridimensional/métodos , Cirugía Asistida por Computador/métodos , Imagen Óptica/métodos , Animales , Neoplasias/cirugía , Colorantes Fluorescentes/químicaRESUMEN
Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.
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Imagenología Tridimensional , Neoplasias , Linfocitos T , Humanos , Linfocitos T/inmunología , Imagenología Tridimensional/métodos , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Inmunoterapia/métodos , Técnicas de Cocultivo/métodosRESUMEN
Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.
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Vesículas Extracelulares , Neoplasias , Humanos , Linfocitos T , Taxoides/farmacología , Apoptosis , Células Epiteliales , Neoplasias/tratamiento farmacológicoRESUMEN
Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.
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Neoplasias , Linfocitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Inmunoterapia/métodos , Organoides/patologíaRESUMEN
Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Transformación Celular Neoplásica/genética , Genes Supresores de Tumor , Humanos , Ratones , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Neoplasias/diagnóstico por imagen , Biomarcadores de Tumor/metabolismo , Aprendizaje Profundo , Colorantes Fluorescentes , Humanos , Riñón/diagnóstico por imagen , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Programas InformáticosRESUMEN
Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
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Neoplasias de la Mama/patología , Mama/patología , Técnicas de Cultivo de Célula/métodos , Técnicas Genéticas , Organoides/patología , Trasplante Heterólogo , Animales , Bancos de Muestras Biológicas , Células Clonales , Femenino , Humanos , Ratones , Factores de TiempoRESUMEN
This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019).
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Bioensayo/métodos , Colforsina/farmacología , Colon , Fibrosis Quística/metabolismo , Organoides , Células Cultivadas , Colon/efectos de los fármacos , Colon/metabolismo , Edema , Humanos , Organoides/efectos de los fármacos , Organoides/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Neuroblastoma resection represents a major challenge in pediatric surgery, because of the high risk of complications. Fluorescence-guided surgery (FGS) could lower this risk by facilitating discrimination of tumor from normal tissue and is gaining momentum in adult oncology. Here, we provide the first molecular-targeted fluorescent agent for FGS in pediatric oncology, by developing and preclinically evaluating a GD2-specific tracer consisting of the immunotherapeutic antibody dinutuximab-beta, recently approved for neuroblastoma treatment, conjugated to near-infrared (NIR) fluorescent dye IRDye800CW. We demonstrated specific binding of anti-GD2-IRDye800CW to human neuroblastoma cells in vitro and in vivo using xenograft mouse models. Furthermore, we defined an optimal dose of 1 nmol, an imaging time window of 4 days after administration and show that neoadjuvant treatment with anti-GD2 immunotherapy does not interfere with fluorescence imaging. Importantly, as we observed universal, yet heterogeneous expression of GD2 on neuroblastoma tissue of a wide range of patients, we implemented a xenograft model of patient-derived neuroblastoma organoids with differential GD2 expression and show that even low GD2 expressing tumors still provide an adequate real-time fluorescence signal. Hence, the imaging advancement presented in this study offers an opportunity for improving surgery and potentially survival of a broad group of children with neuroblastoma.
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Bencenosulfonatos/uso terapéutico , Neoplasias Encefálicas/cirugía , Colorantes Fluorescentes/uso terapéutico , Gangliósidos/metabolismo , Indoles/uso terapéutico , Neuroblastoma/cirugía , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales , Neuroblastoma/metabolismo , Análisis de Matrices TisularesRESUMEN
Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.
Asunto(s)
Imagenología Tridimensional/métodos , Organoides/diagnóstico por imagen , Animales , Humanos , Ratones , Organoides/crecimiento & desarrolloRESUMEN
PURPOSE: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. EXPERIMENTAL DESIGN: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. RESULTS: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. CONCLUSIONS: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/terapia , Terapia Neoadyuvante/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Adulto , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Mama/patología , Mama/cirugía , Neoplasias de la Mama/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Quimioterapia Adyuvante/métodos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Fulvestrant/farmacología , Fulvestrant/uso terapéutico , Humanos , Mastectomía , Ratones , Persona de Mediana Edad , Organoides , Piperazinas/farmacología , Piperazinas/uso terapéutico , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Piridinas/uso terapéutico , Receptores de Estrógenos/análisis , Receptores de Estrógenos/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.
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Neoplasias de la Mama/genética , Mama/fisiología , Animales , Mama/citología , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Femenino , Edición Génica/métodos , Técnicas de Inactivación de Genes , Genes p53 , Xenoinjertos , Humanos , Ratones Endogámicos NOD , Ratones SCID , Organoides , Fosfohidrolasa PTEN/genética , Proteínas de Unión a Retinoblastoma/genética , Ingeniería de Tejidos/métodos , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: CFTR function measurements in intestinal organoids may help to better characterise individual disease expression in F508del homozygous people. Our objective was to study correlations between CFTR function as measured with forskolin-induced swelling in rectal organoids with clinical parameters in adult patients with homozygous F508del mutations. METHODS: Multicentre observational study. Thirty-four adults underwent rectal biopsy, pulmonary function tests (FEV1 and FVC), chest X-ray and chest CT. Body-mass index (BMI) was assessed at study visit and exacerbation rate was determined during five years prior to study visit. Organoids were cultured and measured after stimulation with 5⯵m forskolin for three hours to quantitate CFTR residual function. FINDINGS: FIS was positively correlated with FEV1 (râ¯=â¯0.36, 95% CI 0.02-0.62, pâ¯=â¯0.04) and BMI (râ¯=â¯0.42, 95% CI 0.09-0.66, pâ¯=â¯0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = -0.37, 95% CI -0.62- -0.03, pâ¯=â¯0.049). We found no significant correlation between FIS and chest radiography score for CF (r = -0.16, 95% CI -0.48-0.20, pâ¯=â¯0.44). We observed a trend between higher FIS and a lower mean number of exacerbations over the last 5 years of observation, but this was not statistically significant (Poisson regression, pâ¯=â¯0.089). INTERPRETATION: FIS of intestinal organoids varied between subjects with homozygous F508del and correlated with pulmonary and nutritional parameters. These findings suggest that differences at low CFTR residual function may contribute to clinical heterogeneity in F508del homozygous patients and small changes in CFTR residual function might impact long-term disease expression.
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Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Organoides , Recto , Adyuvantes Inmunológicos/farmacología , Adulto , Biopsia/métodos , Correlación de Datos , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Humanos , Masculino , Mutación , Estado Nutricional , Organoides/efectos de los fármacos , Organoides/metabolismo , Organoides/patología , Recto/metabolismo , Recto/patología , Pruebas de Función Respiratoria , Índice de Severidad de la EnfermedadRESUMEN
In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Organoides/ultraestructura , Fijación del Tejido/métodos , Animales , Mama/ultraestructura , Colon/ultraestructura , Femenino , Humanos , Inmunohistoquímica/métodos , Riñón/ultraestructura , Hígado/ultraestructura , RatonesRESUMEN
Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.
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Linaje de la Célula/genética , Células Enteroendocrinas/metabolismo , Perfilación de la Expresión Génica/métodos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Células Enteroendocrinas/fisiología , Colorantes Fluorescentes , Proteínas de Homeodominio/genética , Mucosa Intestinal/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Imagen Óptica/métodos , Organoides , Fenotipo , Análisis de la Célula Individual/métodos , Células Madre , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.
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Fibrosis Quística/terapia , Organoides/patología , Recto/patología , Fibrosis Quística/patología , Femenino , Humanos , MasculinoRESUMEN
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Fibrosis Quística/patología , Células Epiteliales/patología , Técnicas de Cultivo de Órganos/métodos , Organoides/patología , Infecciones por Virus Sincitial Respiratorio/patología , Sistema Respiratorio/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células Cultivadas , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Células Epiteliales/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Organoides/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/aislamiento & purificación , Sistema Respiratorio/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r=â-0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/diagnóstico , Insuficiencia Pancreática Exocrina/diagnóstico , Organoides/patología , Biomarcadores/metabolismo , Cloruros/metabolismo , Fibrosis Quística/complicaciones , Femenino , Humanos , Lactante , Transporte Iónico , Modelos Lineales , Masculino , Prueba de Estudio Conceptual , Índice de Severidad de la EnfermedadRESUMEN
Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.