TAMI-70. METABOLIC VULNERABILITY TO GPX4 INHIBITION AND FERROPTOSIS OF QUIESCENT ASTROCYTE-LIKE GLIOMA CELL POPULATIONS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi212-vi213
Author(s):  
Matei Banu ◽  
Athanassios Dovas ◽  
Michael Argenziano ◽  
Wenting Zhao ◽  
Dominique Higgins ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Glioma Cell ◽  
Ex Vivo ◽  
Tricarboxylic Acid Cycle ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Acid Oxidation ◽  
Astrocytic Tumor ◽  
Proliferating Cells ◽  
Novel Treatments

Abstract Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.

scholarly journals Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

2006 ◽  
Vol 11 (8) ◽  
pp. 922-932 ◽  
Author(s):  
Andrea Ivascu ◽  
Manfred Kubbies
Keyword(s):  
Tumor Cell ◽  
Suspension Culture ◽  
Cell Lines ◽  
Cell Function ◽  
Tumor Cell Lines ◽  
Proliferating Cells ◽  
Basement Membrane Extract ◽  
Rapid Generation ◽  
Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

scholarly journals Intestinal crypt derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus

2020 ◽  
Author(s):  
Daniel E Levin ◽  
Arabinda Mandal ◽  
Mark A Fleming ◽  
Katherine H Bae ◽  
Brielle Gerry ◽  
...  
Keyword(s):  
Myenteric Plexus ◽  
Longitudinal Muscle ◽  
Culture Media ◽  
Ex Vivo ◽  
Enteric Neurons ◽  
Intestinal Cell ◽  
Cell Populations ◽  
Complex Interactions ◽  
Proliferation And Differentiation ◽  
Intestinal Peristalsis

Abstract The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions is complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: 1) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and 2) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

scholarly journals ETMR-05. SINGLE-CELL RNA-SEQ OF ETMR REVEALS CELL PROGRAMS OF DEVELOPMENTAL HIERARCHY AND CELLULAR DIVERSITY IN THE TUMOR MICROENVIRONMENT

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii323-iii323
Author(s):  
Flavia W de Faria ◽  
Marta Interlandi ◽  
Natalia Moreno ◽  
Monika Graf ◽  
Viktoria Melcher ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Interaction Analysis ◽  
Cellular Heterogeneity ◽  
Receptor Interaction ◽  
Acid Oxidation ◽  
Bmp Receptors ◽  
New Findings ◽  
Developmental Hierarchy

Abstract Embryonal tumors with multilayered rosettes (ETMR) are deadly brain malignancies affecting young children. No standard treatment is available and the median survival is less than 12 months. Molecularly, the disease is characterized by the miRNA C19MC cluster amplification, with the expression of multiples miRNAs related to a stem cell program. The discoveries on the purely molecular mechanisms of the disease did not help to create a bridge for new treatment strategies so far and the cellular diversity of ETMR remains poorly understood. In this study, we used single-cell RNA sequencing of murine and human tumors to describe ETMR cellular heterogeneity. Our findings support that intra-tumoral heterogeneity is mainly characterized by 4 cellular programs defining a developmental hierarchy related to different metabolic states: 1) Early quiescent NSC-like cells supported by fatty-acid oxidation 2) Late NSC and NP-like proliferative cells fueled by glycolytic metabolism; 3) Post-mitotic neuroblast-like cells, relying on oxidative-phosphorylation; 4) NSC-like proliferative cells, with metabolic plasticity and capable of performing the three types of metabolism. Tumor-specific ligand-receptor interaction analysis revealed that ETMR exchange with microglia and vascular mural cells (MC) signals related to extracellular matrix (ECM) organization (Cxcl12-CxCr4), stem cell signaling (BMPs-BMP receptors), anti-apoptosis and survival (Ntf3-Ntrk), not seen in the control brain. In addition, the vascular MC showed a cancer-associated fibroblast (CAF) phenotype, with potential prognostic implications, as previously demonstrated for other tumors. This study provides new findings to build up a more robust understanding of ETMR biology and opens space for further studies in the field.

Abstract 12117: The Cardioprotection of Trimetazidine Against Ischemic Injury is Mediated by AMPK and ERK Signaling Pathway

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Zhenling Liu ◽  
Yina Ma ◽  
Michelle Kuznicki ◽  
Xingchi Chen ◽  
Wanqing Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Glucose Oxidation ◽  
Ex Vivo ◽  
Ischemic Injury ◽  
Erk Signaling ◽  
Acid Oxidation ◽  
Ampk Signaling ◽  
Heart Perfusion

Introduction: Trimetazidine (TMZ) is an anti-anginal drug that has been widely used in Europe and Asia. The TMZ can optimize energy metabolism via inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT) in the heart, with subsequent decrease in fatty acid oxidation and stimulation of glucose oxidation. However, the mechanism by which TMZ aids in cardioprotection against ischemic injury has not been characterized. Hypothesis: AMP-activated protein kinase (AMPK) is an energy sensor that control ATP supply from substrate metabolism and protect heart from energy stress. TMZ changes the cardiac AMP/ATP ratio via modulating fatty acid oxidation, thereby it may trigger AMPK signaling cascade that contribute to protection heart from ischemia/reperfusion (I/R) injury. Methods: The mouse in vivo regional ischemia and reperfusion by the ligation of the left anterior descending coronary artery (LAD) were used for determination of myocardial infarction. The infarct size was compared between C57BL/6J WT mice and AMPK kinase dead (KD) transgenic mice with or without TMZ treatment. The ex vivo working heart perfusion system was used to monitor the effect of TMZ on glucose oxidation and fatty acid oxidation in the heart. Results: TMZ treatment significantly stimulates cardiac AMPK and extracellular signal-regulated kinase (ERK) signaling pathways (p<0.05 vs. vehicle group). The administration of TMZ reduces myocardial infarction size in WT C57BL/6J hearts, the reduction of myocardial infarction size by TMZ in AMPK KD hearts was significantly impaired versus WT hearts (p<0.05). Intriguingly, the administration of ERK inhibitor, PD 98059, to AMPK KD mice abolished the cardioprotection of TMZ against I/R injury. The ex vivo working heart perfusion data demonstrated that TMZ treatment significantly activates AMPK signaling and modulating the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion, leading to reduction of oxidative stress in the I/R hearts. Conclusions: Both AMPK and ERK signaling pathways mediate the cardioprotection of TMZ against ischemic injury. The metabolic benefits of TMZ for angina patients could be due to the activation of energy sensor AMPK in the heart by TMZ administration.

TMOD-29. STANDARDIZED GENERATION OF TUMOR-ORGANOIDS AS NOVEL DRUG SCREENING MODEL IN MENINGIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi221-vi222
Author(s):  
Gerhard Jungwirth ◽  
Tao Yu ◽  
Cao Junguo ◽  
Catharina Lotsch ◽  
Andreas Unterberg ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cancer Biology ◽  
Drug Testing ◽  
Large Scale ◽  
Ex Vivo ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Drug Responses ◽  
Novel Drug ◽  
Tumor Organoids

Abstract Tumor-organoids (TOs) are novel, complex three-dimensional ex vivo tissue cultures that under optimal conditions accurately reflect genotype and phenotype of the original tissue with preserved cellular heterogeneity and morphology. They may serve as a new and exciting model for studying cancer biology and directing personalized therapies. The aim of our study was to establish TOs from meningioma (MGM) and to test their usability for large-scale drug screenings. We were capable of forming several hundred TO equal in size by controlled reaggregation of freshly prepared single cell suspension of MGM tissue samples. In total, standardized TOs from 60 patients were formed, including eight grade II and three grade III MGMs. TOs reaggregated within 3 days resulting in a reducted diameter by 50%. Thereafter, TO size remained stable throughout a 14 days observation period. TOs consisted of largely viable cells, whereas dead cells were predominantly found outside of the organoid. H&E stainings confirmed the successful establishment of dense tissue-like structures. Next, we assessed the suitability and reliability of TOs for a robust large-scale drug testing by employing nine highly potent compounds, derived from a drug screening performed on several MGM cell lines. First, we tested if drug responses depend on TO size. Interestingly, drug responses to these drugs remained identical independent of their sizes. Based on a sufficient representation of low abundance cell types such as T-cells and macrophages an overall number of 25.000 cells/TO was selected for further experiments revealing FDA-approved HDAC inhibitors as highly effective drugs in most of the TOs with a mean z-AUC score of -1.33. Taken together, we developed a protocol to generate standardized TO from MGM containing low abundant cell types of the tumor microenvironment in a representative manner. Robust and reliable drug responses suggest patient-derived TOs as a novel drug testing model in meningioma research.

scholarly journals Repurposing Cationic Amphiphilic Drugs and Derivatives to Engage Lysosomal Cell Death in Cancer Treatment

2020 ◽  
Vol 10 ◽  
Author(s):  
Michelle Hu ◽  
Kermit L. Carraway
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cellular Transformation ◽  
Therapeutic Window ◽  
Cell Populations ◽  
Necrotic Cell Death ◽  
Necrotic Cell ◽  
Membrane Rupture ◽  
Cationic Amphiphilic Drugs ◽  
Amphiphilic Drugs

A major confounding issue in the successful treatment of cancer is the existence of tumor cell populations that resist therapeutic agents and regimens. While tremendous effort has gone into understanding the biochemical mechanisms underlying resistance to each traditional and targeted therapeutic, a broader approach to the problem may emerge from the recognition that existing anti-cancer agents elicit their cytotoxic effects almost exclusively through apoptosis. Considering the myriad mechanisms cancer cells employ to subvert apoptotic death, an attractive alternative approach would leverage programmed necrotic mechanisms to side-step therapeutic resistance to apoptosis-inducing agents. Lysosomal cell death (LCD) is a programmed necrotic cell death mechanism that is engaged upon the compromise of the limiting membrane of the lysosome, a process called lysosomal membrane permeabilization (LMP). The release of lysosomal components into the cytosol upon LMP triggers biochemical cascades that lead to plasma membrane rupture and necrotic cell death. Interestingly, the process of cellular transformation appears to render the limiting lysosomal membranes of tumor cells more fragile than non-transformed cells, offering a potential therapeutic window for drug development. Here we outline the concepts of LMP and LCD, and discuss strategies for the development of agents to engage these processes. Importantly, the potential exists for existing cationic amphiphilic drugs such as antidepressants, antibiotics, antiarrhythmics, and diuretics to be repurposed to engage LCD within therapy-resistant tumor cell populations.

scholarly journals Single-Cell Sequencing: Biological Insight and Potential Clinical Implications in Pediatric Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5658
Author(s):  
Donát Alpár ◽  
Bálint Egyed ◽  
Csaba Bödör ◽  
Gábor T. Kovács
Keyword(s):  
High Resolution ◽  
Single Cell ◽  
Childhood Leukemia ◽  
Clinical Decision Making ◽  
Cellular Heterogeneity ◽  
Therapeutic Interventions ◽  
Cell Populations ◽  
Pediatric Leukemia ◽  
Single Cell Sequencing ◽  
Wide Range

Single-cell sequencing (SCS) provides high-resolution insight into the genomic, epigenomic, and transcriptomic landscape of oncohematological malignancies including pediatric leukemia, the most common type of childhood cancer. Besides broadening our biological understanding of cellular heterogeneity, sub-clonal architecture, and regulatory network of tumor cell populations, SCS can offer clinically relevant, detailed characterization of distinct compartments affected by leukemia and identify therapeutically exploitable vulnerabilities. In this review, we provide an overview of SCS studies focused on the high-resolution genomic and transcriptomic scrutiny of pediatric leukemia. Our aim is to investigate and summarize how different layers of single-cell omics approaches can expectedly support clinical decision making in the future. Although the clinical management of pediatric leukemia underwent a spectacular improvement during the past decades, resistant disease is a major cause of therapy failure. Currently, only a small proportion of childhood leukemia patients benefit from genomics-driven therapy, as 15–20% of them meet the indication criteria of on-label targeted agents, and their overall response rate falls in a relatively wide range (40–85%). The in-depth scrutiny of various cell populations influencing the development, progression, and treatment resistance of different disease subtypes can potentially uncover a wider range of driver mechanisms for innovative therapeutic interventions.

scholarly journals IFITM1 expression determines extracellular vesicle uptake in colorectal cancer

2021 ◽  
Author(s):  
Andrea Kelemen ◽  
Idan Carmi ◽  
Ádám Oszvald ◽  
Péter Lőrincz ◽  
Gábor Petővári ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transmembrane Protein ◽  
Extracellular Vesicle ◽  
Cellular Heterogeneity ◽  
Functional Difference ◽  
Proliferating Cells ◽  
Antiviral Responses ◽  
The Difference ◽  
Functional Relevance ◽  
Therapeutic Tools

AbstractThe majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which lead to the unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered potential therapeutic tools. Although CRC is a genetically heterogeneous disease, the significance of the intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induced the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1high CRC cells contained more proliferating cells and they had a markedly reduced uptake of fibroblast EVs as compared to IFITM1low/− cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 levels. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1low CRC cells. Furthermore, inactivating IFITM1 resulted in an enhanced EV uptake, highlighting the importance of this molecule in establishing the cellular difference for EV effects. Collectively, we identified CRC cells with functional difference in their EV uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.

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