RESUMEN
Tumor organoids are three-dimensional in vitro models which can recapitulate the complex mutational landscape and tissue architecture observed in cancer patients, providing a realistic model for testing novel therapies, including immunotherapies. A significant challenge in organoid research in oncology lies in developing efficient and reliable methods for segmenting organoid images, quantifying organoid growth, regression and response to treatments, as well as predicting the behavior of organoid systems. Up to now, a curated dataset of organoids co-cultured with immune cells is not available. To address this gap, we present a new public dataset, comprising both phase-contrast images of murine and patient-derived tumor organoids of one of the deadliest cancer types, the Pancreatic Ductal Adenocarcinoma, co-cultured with immune cells, and state-of-the-art algorithms for object detection and segmentation. Our dataset, OrganoIDNetData, encompassing 180 images with 33906 organoids, can be a potential common benchmark for different organoids segmentation protocols, moving beyond the current practice of training and testing these algorithms on isolated datasets.
Asunto(s)
Algoritmos , Organoides , Neoplasias Pancreáticas , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Humanos , Ratones , Animales , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Inteligencia ArtificialRESUMEN
Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.
Asunto(s)
Proteínas de Drosophila , Vesículas Extracelulares , Animales , Secreciones Corporales , Drosophila melanogaster , Endosomas , Cinesinas , Transducción de Señal , Proteínas de Unión al GTP rabRESUMEN
During endosome maturation, neutral sphingomyelinase 2 (nSMase2, encoded by SMPD3) is involved in budding of intraluminal vesicles (ILVs) into late endosomes or multivesicular bodies (MVBs). Fusion of these with the plasma membrane results in secretion of exosomes or small extracellular vesicles (sEVs). Here, we report that nSMase2 activity controls sEV secretion through modulation of vacuolar H+-ATPase (V-ATPase) activity. Specifically, we show that nSMase2 inhibition induces V-ATPase complex assembly that drives MVB lumen acidification and consequently reduces sEV secretion. Conversely, we further demonstrate that stimulating nSMase2 activity with the inflammatory cytokine TNFα (also known as TNF) decreases acidification and increases sEV secretion. Thus, we find that nSMase2 activity affects MVB membrane lipid composition to counteract V-ATPase-mediated endosome acidification, thereby shifting MVB fate towards sEV secretion. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Exosomas , ATPasas de Translocación de Protón Vacuolares , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Exosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cuerpos Multivesiculares/metabolismo , Transporte de Proteínas , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fusión de Membrana/fisiología , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Drosophila , Células HCT116 , Células HEK293 , Humanos , Fosforilación/fisiología , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Ageing and obesity are two major risk factors for the development of type 2 diabetes (T2D). A chronic, low-grade, sterile inflammation contributes to insulin resistance and ß-cell failure. Toll-like receptor-4 (TLR4) is a major pro-inflammatory pathway; its ligands as well as downstream signals are increased systemically in patients with T2D and at-risk individuals. In the present study we investigated the combined effects of high fat/high sucrose diet (HFD) feeding, ageing and TLR4-deficiency on tissue inflammation, insulin resistance and ß-cell failure. In young mice, a short-term HFD resulted in a mildly impaired glucose tolerance and reduced insulin secretion, together with a ß-cell mass compensation. In older mice, HFD further deteriorated insulin secretion and induced a significantly impaired glucose tolerance and augmented tissue inflammation in adipose, liver and pancreatic islets, all of which was attenuated by TLR4 deficiency. Our results show that ageing exacerbates HFD-induced impairment of glucose homeostasis and pancreatic ß-cell function and survival, and deteriorates HFD-induced induction of mRNA expression of inflammatory cytokines and pro-inflammatory macrophage markers. TLR4-deficiency protects against these combined deleterious effects of a high fat diet and ageing through a reduced expression of inflammatory products in both insulin sensitive tissues and pancreatic islets.