The steady-state morphology of the mitochondrial network is maintained by a balance of constitutive fission and fusion reactions. Disruption of this steady-state morphology results in either a ...fragmented or elongated network, both of which are associated with altered metabolic states and disease. How the processes of fission and fusion are balanced by the cell is unclear. Here we show that mitochondrial fission and fusion are spatially coordinated at ER membrane contact sites (MCSs). Multiple measures indicate that the mitochondrial fusion machinery, Mitofusins, accumulate at ER MCSs where fusion occurs. Furthermore, fission and fusion machineries colocalize to form hotspots for membrane dynamics at ER MCSs that can persist through sequential events. Because these hotspots can undergo fission and fusion, they have the potential to quickly respond to metabolic cues. Indeed, we discover that ER MCSs define the interface between polarized and depolarized segments of mitochondria and can rescue the membrane potential of damaged mitochondria by ER-associated fusion.
Abstract
MicroRNAs (miRNAs) are noncoding RNAs with 18–26 nucleotides; they pair with target mRNAs to regulate gene expression and produce significant changes in various physiological and ...pathological processes. In recent years, the interaction between miRNAs and their target genes has become one of the mainstream directions for drug development. As a large-scale biological database that mainly provides miRNA–target interactions (MTIs) verified by biological experiments, miRTarBase has undergone five revisions and enhancements. The database has accumulated >2 200 449 verified MTIs from 13 389 manually curated articles and CLIP-seq data. An optimized scoring system is adopted to enhance this update’s critical recognition of MTI-related articles and corresponding disease information. In addition, single-nucleotide polymorphisms and disease-related variants related to the binding efficiency of miRNA and target were characterized in miRNAs and gene 3′ untranslated regions. miRNA expression profiles across extracellular vesicles, blood and different tissues, including exosomal miRNAs and tissue-specific miRNAs, were integrated to explore miRNA functions and biomarkers. For the user interface, we have classified attributes, including RNA expression, specific interaction, protein expression and biological function, for various validation experiments related to the role of miRNA. We also used seed sequence information to evaluate the binding sites of miRNA. In summary, these enhancements render miRTarBase as one of the most research-amicable MTI databases that contain comprehensive and experimentally verified annotations. The newly updated version of miRTarBase is now available at https://miRTarBase.cuhk.edu.cn/.
Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA ...expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA
cells in a tumor.
RM1 cells expressing different levels of PSMA (PSMA
, PSMA
, PSMA
, PSMA
; study 1) or a mix of PSMA
and PSMA
RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received
Lu- (studies 1-3) or
Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified.
Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with
Lu-PSMA617 uptake and the degree of DNA damage. Compared with
Lu-PSMA617,
Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups.
In the current models, both the degree of PSMA expression and the fraction of PSMA
cells correlate with
Lu-/
Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.
.
Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. ...However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.
Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for ...the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), homogeneous cultures that do not take into account tumor heterogeneity, three-dimensional (3D) cell-cell contacts between tumor and/or host cells or interactions with extracellular matrix proteins. Here we describe a 3D tumor spheroid-based migration assay which more accurately reflects the solid tumor microenvironment and can accommodate both extracellular matrix and host cell interactions. It is a rapid and highly reproducible 96-well plate-based technique and we demonstrate its utility for the evaluation of therapeutic agents/drugs with anti-migratory properties.
Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour ...microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.
Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumour cell population ...including mature non-stem-like cell and immature stem-like cells within the tumour. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumour growth in mice. Here, we report the critical role of PRMT5 in GBM differentiated cells (GBMDC) grown in serum and GBM neurospheres (GBMNS) grown as neurospheres in vitro. Our results uncover a very significant role for PRMT5 in GBMNS self-renewal capacity and proliferation. PRMT5 knockdown in GBMDC led to apoptosis, knockdown in GBMNS led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of retinoblastoma protein, leading to senescence. Comparison of impact of PRMT5 on cellular signalling by the Human Phospho-Kinase Array and chromatin immunoprecipitation-PCR revealed that unlike GBMDC, PRMT5 regulates PTEN expression and controls Akt and ERk activity in GBMNS. In vivo transient depletion of PRMT5 decreased intracranial tumour size and growth rate in mice implanted with both primary tumour-derived GBMNS and GBMDC. This is the first study to identify PTEN as a potential downstream target of PRMT5 and PRMT5 is vital to support both mature and immature GBM tumour cell populations.
This paper discusses "bioink", bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to ...fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader.
The amino acid antiporter system Xc
is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ...ferroptosis). While the activity of system Xc
often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc
activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc
is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc
, depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc
and promote ferroptosis repressed by this antiporter.
High fatality in ovarian cancer is attributed to metastasis, propagated by the release of multi-cellular aggregates/spheroids into the peritoneal cavity and their subsequent mesothelial invasion of ...peritoneal organs. Spheroids are therefore a common and clinically relevant in vitro model for ovarian cancer research. Spheroids in patients vary significantly in size and shape and display enhanced resistance to anti-cancer drugs compared to monolayers. However, there is no consensus on how spheroid size and shape affect drug resistance. Moreover, existing data regarding the influence of epithelial-to-mesenchymal transition (EMT) profile on spheroid shape and migration is inconclusive.
We formed spheroids with OVCAR-3 and OVCAR-8 cells, chosen for their established genetic similarity to the patient tumor samples. We monitored their morphology using confocal microscope with dipping objective and fluorescent microscope. We characterized important EMT biomarkers; E-cadherin, Vimentin and Slug through western blotting in monolayers and spheroids. We treated these spheroids with Taxol and Cisplatin and investigated their migratory profile based on their morphology.
We report two distinct multicellular structures: loose aggregates (OVCAR-3) and compact spheroids (OVCAR-8). We attribute these different morphologies to the expression of the EMT biomarkers, and their changes upon spheroid formation. Importantly, we did not observe a difference in resistance to the anti-cancer drugs as a function of spheroid size and shape. However, migration capacity of compact spheroid (OVCAR-8) was 15-fold higher compared to that of loose aggregates (OVCAR-3).
These results highlight the importance of spheroid size and shape on anti-cancer drug resistance and migration profiles. The results of this study can, therefore, help to elucidate general rules for ovarian cancer studies based on 3D samples.
Formation of loose aggregates and compact spheroids using OVCAR-3 and OVCAR-8 cells, respectively. Display omitted
•High-throughput formation of ovarian cancer spheroids in different sizes.•Observation of EMT biomarkers (E-cadherin, Vimentin, Slug) in different spheroid morphologies.•Observation of the effect of spheroid size and shape on anti-cancer drug resistance.•Influence of the shape of the spheroids on their migratory profile.
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