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1.
Cell ; 176(5): 1158-1173.e16, 2019 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-30712869

RESUMEN

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.


Asunto(s)
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/genética
2.
EMBO J ; 38(4)2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30643021

RESUMEN

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.


Asunto(s)
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 Xenoinjerto
3.
PLoS Biol ; 16(8): e2004986, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30080881

RESUMEN

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.


Asunto(s)
Glándulas Mamarias Animales/embriología , Complejo Represivo Polycomb 2/fisiología , Animales , Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/fisiología , Femenino , Heterocromatina/metabolismo , Código de Histonas , Histonas/metabolismo , Lisina/metabolismo , Glándulas Mamarias Animales/metabolismo , Metilación , Ratones , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb , Cultivo Primario de Células , Procesamiento Proteico-Postraduccional
4.
Development ; 144(6): 1065-1071, 2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-27993977

RESUMEN

Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation in vitro Here, we have built upon previously described culture assays to define culture conditions that enable the efficient generation of clonal organoid structures from single sorted basal mammary epithelial cells (MECs). Analysis of Confetti-reporter mice revealed the formation of uni-colored structures and thus the clonal nature of these organoids. High-resolution 3D imaging demonstrated that basal cell-derived complex organoids comprised an inner compartment of polarized luminal cells with milk-producing capacity and an outer network of elongated myoepithelial cells. Conversely, structures generated from luminal MECs rarely contained basal/myoepithelial cells. Moreover, flow cytometry and 3D microscopy of organoids generated from lineage-specific reporter mice established the bipotent capacity of basal cells and the restricted potential of luminal cells. In summary, we describe optimized in vitro conditions for the efficient generation of mouse mammary organoids that recapitulate features of mammary tissue architecture and function, and can be applied to understand tissue dynamics and cell-fate decisions.


Asunto(s)
Glándulas Mamarias Animales/crecimiento & desarrollo , Organoides/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Linaje de la Célula , Células Clonales , Células Epiteliales/citología , Femenino , Citometría de Flujo , Genes Reporteros , Imagenología Tridimensional , Glándulas Mamarias Animales/citología , Ratones , Microscopía Confocal
5.
Eur Respir J ; 52(3)2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30166324

RESUMEN

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= -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.


Asunto(s)
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 Enfermedad
6.
Thorax ; 72(2): 137-147, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27852956

RESUMEN

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.


Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/terapia , Terapia Genética/métodos , Lentivirus/genética , Animales , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones , Factor 1 de Elongación Peptídica , Regiones Promotoras Genéticas
7.
PLoS Biol ; 12(11): e1001998, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25406061

RESUMEN

Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer's disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure-function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/etiología , Proteínas de Unión al ADN/genética , Deficiencias en la Proteostasis/genética , Factores de Transcripción/genética , Animales , Antineoplásicos Alquilantes/uso terapéutico , Caenorhabditis elegans , Línea Celular , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/metabolismo , Diterpenos/uso terapéutico , Evaluación Preclínica de Medicamentos , Compuestos Epoxi/uso terapéutico , Silenciador del Gen , Factores de Transcripción del Choque Térmico , Humanos , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Ratones Transgénicos , Organoides , Fenantrenos/uso terapéutico , Prostaglandina-E Sintasas , Pliegue de Proteína , Mucosa Respiratoria/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo
8.
Am J Respir Crit Care Med ; 193(3): 288-98, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26509335

RESUMEN

RATIONALE: Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. OBJECTIVES: We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. METHODS: We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. MEASUREMENTS AND MAIN RESULTS: rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. CONCLUSIONS: Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.


Asunto(s)
Fibrosis Quística/terapia , Dependovirus , Terapia Genética/métodos , Vectores Genéticos , Intestinos , Organoides , Animales , Líquidos Corporales/metabolismo , Canales de Cloruro/genética , Cloruros/metabolismo , Colforsina/farmacología , Fibrosis Quística/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Genotipo , Células HeLa , Humanos , Ratones , Organoides/metabolismo , Transducción Genética
9.
Eur Respir J ; 48(2): 451-8, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27103391

RESUMEN

Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Mutación , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Biopsia , Genotipo , Homocigoto , Humanos , Organoides , Pliegue de Proteína , Transporte de Proteínas , Recto/patología , Tiazoles/química , Resultado del Tratamiento
10.
Eur Respir J ; 48(3): 768-79, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27471203

RESUMEN

We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified ß2-adrenergic receptor agonists as the most potent inducers of CFTR function.ß2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled ß2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.


Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Administración Oral , Albuterol/administración & dosificación , Bioensayo , Bronquios/patología , Línea Celular , Células Cultivadas , Cloruros/química , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Evaluación Preclínica de Medicamentos , Células Epiteliales/metabolismo , Epitelio/metabolismo , Humanos , Mutación , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Organoides , Proyectos Piloto , Sistema Respiratorio/metabolismo , Transducción de Señal
11.
Nat Chem Biol ; 9(7): 444-54, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23666117

RESUMEN

The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.


Asunto(s)
Aminopiridinas/farmacología , Benzodioxoles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Animales , Sitios de Unión , Bronquios/citología , Membrana Celular/metabolismo , Cricetinae , Fibrosis Quística/genética , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Retículo Endoplásmico/metabolismo , Células Epiteliales/metabolismo , Glicosilación , Humanos , Mutación , Nucleótidos/química , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química
12.
Cell Microbiol ; 15(12): 1955-68, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23869880

RESUMEN

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.


Asunto(s)
Pared Celular/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Bacterias Grampositivas/inmunología , Sitios de Unión , Complemento C3b/inmunología , Humanos , Peptidoglicano/inmunología , Unión Proteica/inmunología
13.
Nat Protoc ; 19(7): 2052-2084, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38504137

RESUMEN

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.


Asunto(s)
Imagenología Tridimensional , Neoplasias , Linfocitos T , Humanos , Linfocitos T/inmunología , Imagenología Tridimensional/métodos , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Inmunoterapia/métodos , Técnicas de Cocultivo/métodos
14.
EMBO Mol Med ; 16(7): 1495-1514, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38831131

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.


Asunto(s)
Imagenología Tridimensional , Organoides , Humanos , Imagenología Tridimensional/métodos , Cirugía Asistida por Computador/métodos , Imagen Óptica/métodos , Animales , Neoplasias/cirugía , Colorantes Fluorescentes/química
15.
Cancer Cell ; 41(6): 1170-1185.e12, 2023 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-37311414

RESUMEN

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.


Asunto(s)
Vesículas Extracelulares , Neoplasias , Humanos , Linfocitos T , Taxoides/farmacología , Apoptosis , Células Epiteliales , Neoplasias/tratamiento farmacológico
16.
Nat Biotechnol ; 41(1): 60-69, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-35879361

RESUMEN

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.


Asunto(s)
Neoplasias , Linfocitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Inmunoterapia/métodos , Organoides/patología
17.
Mol Oncol ; 16(5): 1119-1131, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35000262

RESUMEN

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.


Asunto(s)
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/metabolismo
18.
Nat Protoc ; 16(4): 1936-1965, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33692550

RESUMEN

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.


Asunto(s)
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 Tiempo
19.
Nat Biotechnol ; 39(10): 1239-1245, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34083793

RESUMEN

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.


Asunto(s)
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 Informáticos
20.
J Vis Exp ; (160)2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32568249

RESUMEN

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 & desarrollo
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