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Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient‐limited cells; this translocation was mediated by a rapid post‐transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient‐limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient‐limited cells were treated with 5‐Fluorouracil (5‐FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance. [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient‐own liver‐derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient‐derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient‐derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched‐chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient‐specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process. [ABSTRACT FROM AUTHOR]

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration). [ABSTRACT FROM AUTHOR]

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing. [ABSTRACT FROM AUTHOR]