bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2024‒01‒14
29 papers selected by
Kıvanç Görgülü, Technical University of Munich

  1. Proc Natl Acad Sci U S A. 2024 Jan 16. 121(3): e2314093121
      Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.
    Keywords:  lipid droplet turnover; lipid transport protein; membrane dynamics
  2. Nat Cancer. 2024 Jan 09.
      Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.
  3. Clin Lab. 2024 Jan 01. 70(1):
      BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor originating from the epithelium of the pancreatic duct. Neoadjuvant chemotherapy FOLFIRINOX (a combination of oxaliplatin, irinotecan, and 5-fluorouracil/leucovorin) is considered to be the most effective regimen for patients with resected pancreatic cancer.METHODS: This article reports a case of a pancreatic ductal adenocarcinoma patient who exhibited regular periodic fluctuations in the serum iron level during FOLFIRINOX.
    RESULTS: It indicates that an unexplained increase in serum iron levels after each cycle of FOLFIRINOX is non-cell destructive and due to a reduction in iron consumption, after ruling out other potential causes.
    CONCLUSIONS: FOLFIRINOX in pancreatic cancer patients may cause an elevation of serum iron levels.
  4. BMC Cancer. 2024 Jan 12. 24(1): 67
      BACKGROUND: Despite some therapeutic advances, improvement in survival rates of unresectable and/or metastatic pancreatic ductal adenocarcinoma (PDAC) has been minimal over recent decade. We aimed to evaluate the impact of different treatment sequences on clinical outcomes of advanced PDAC at our academic institution.METHODS: In this single institution retrospective analysis, we assessed characteristics and survival rates of unresectable and/or metastatic pancreatic PDAC patients who started a systemic treatment between 01/2015 and 12/2021. Survival analyses were performed by Kaplan-Meier and Cox proportional hazards model.
    RESULTS: The number of 285 patients received at least two lines of treatment, but only 137 patients were suitable for third-line treatment. Subgroup analysis showed that thirty-seven patients received A line (gemcitabine/nab-paclitaxel or nab-paclitaxel combined therapy to FOLFIRINOX) therapy, 37 patients received B line (nab-paclitaxel combined therapy to gemcitabine combined therapy to FOLFIRINOX) therapy, 21 patients received C line (nab-paclitaxel combined therapy to gemcitabine combined therapy to oxaliplatin or irinotecan combined therapy) therapy. Survival rates for different treatment lines were significantly different and median overall survival (OS) was 14.00, 18.00, and 14.00 months, respectively (p<0.05).
    CONCLUSION: Our study provides real-world evidence for the effectiveness of different treatment sequences and underscores the treatment sequences on survival outcome when considering the entire management in advanced PDAC.
    Keywords:  FOLFIRINOX; Irinotecan; Nab-paclitaxel; New chemotherapy regimens; Pancreatic ductal adenocarcinoma; Treatment landscape; Treatment patterns; Treatment sequences
  5. Autophagy Rep. 2023 Dec 31. pii: 27694127.2023.2188523. [Epub ahead of print]2(1):
      The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.
    Keywords:  AIM; Atg8; Autophagy; LDS; LIR; UDS; phosphorylation; protein-protein interaction
  6. J Clin Invest. 2024 Jan 09. pii: e171788. [Epub ahead of print]
      Choline deficiency causes disorders including hepatic abnormalities and is associated with an increased risk of multiple types of cancer(1, 2). Here, by choline free diet-associated RNA-seq analyses, we found that the tumor suppressor p53 drives the Kennedy pathway via PCYT1B to control the growth of lipid droplets (LDs) and their fueling role in tumorigenesis. Mechanistically, through upregulation of PCYT1B, p53 channeled depleted choline stores to phosphatidylcholine (PC) biosynthesis during choline starvation, thus preventing LD coalescence. Cells lacking p53 failed to complete this response to choline depletion, leading to hepatic steatosis and tumorigenesis, and these effects could be reversed by enforcing PCYT1B expression or restoring PC abundance. Furthermore, loss of p53 or defects in the Kennedy pathway increased surface localization of hormone-sensitive lipase (HSL) on LDs to release specific fatty acids that fueled tumor cells in vivo and in vitro. Thus, p53 loss leads to dysregulation of choline metabolism and LD growth, and couples perturbed LD homeostasis to tumorigenesis.
    Keywords:  Cancer; Cell Biology; Cholesterol; Tumor suppressors
  7. Aging Cell. 2024 Jan 09. e14083
      Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.
    Keywords:  3D genome organization; SASP; cellular ageing; chromatin; senescence; single cell genomics
  8. bioRxiv. 2023 Dec 23. pii: 2023.12.22.572956. [Epub ahead of print]
      Here, we examine the impact of mechanosensitive ion channels on the durotaxis of pancreatic stellate cells (PSCs). PSCs are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. The molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional linear stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the role of the ion channels Piezo1, TRPC1, and TRPV4 in durotaxis of primary murine PSCs. We found that PSCs migrate towards a stiffer substrate, which is abolished by clamping Piezo1 activity to zero (knockout) or to its maximal value (Piezo1 activator Yoda1). Hence, PSC durotaxis is optimal with an intermediary, dynamically changing level of Piezo1 channel activity. Based on these findings we developed and numerically discretized a mathematical model of partial differential equations to simulate PSC durotaxis, raising the possibility that PSC accumulation during PDAC progression is partly due to durotaxis. We extended our biological model to TRPV4 and TRPC1, key sensors, and signal transducers for mechanical forces in PSCs. Disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. These findings suggest that mechanosensitive ion channels, particularly Piezo1, detect the mechanical microenvironment to guide PSC migration. Yet, Piezo1 relies on TRPC1 and TRPV4 to orchestrate durotaxis.
    Keywords:  Biological Sciences/Physiology; Physical Sciences/Applied Mathematics; mechanosensation; mechanotransduction; pancreatic cancer; taxis
  9. Curr Protoc. 2024 Jan;4(1): e950
      Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.
    Keywords:  autophagy; density purification; lysosomes; organelle isolation
  10. Nat Commun. 2024 Jan 09. 15(1): 375
      Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.
  11. J Cell Sci. 2024 Jan 01. pii: jcs260986. [Epub ahead of print]137(1):
      Mitochondria are multifunctional organelles of key importance for cell homeostasis. The outer mitochondrial membrane (OMM) envelops the organelle, and the inner mitochondrial membrane (IMM) is folded into invaginations called cristae. As cristae composition and functions depend on the cell type and stress conditions, they recently started to be considered as a dynamic compartment. A number of proteins are known to play a role in cristae architecture, such as OPA1, MIC60, LETM1, the prohibitin (PHB) complex and the F1FO ATP synthase. Furthermore, phospholipids are involved in the maintenance of cristae ultrastructure and dynamics. The use of new technologies, including super-resolution microscopy to visualize cristae dynamics with superior spatiotemporal resolution, as well as high-content techniques and datasets have not only allowed the identification of new cristae proteins but also helped to explore cristae plasticity. However, a number of open questions remain in the field, such as whether cristae-resident proteins are capable of changing localization within mitochondria, or whether mitochondrial proteins can exit mitochondria through export. In this Review, we present the current view on cristae morphology, stability and composition, and address important outstanding issues that might pave the way to future discoveries.
    Keywords:  Cristae; Cristae dynamics; High-content approaches; Mitochondria; Quantitative microscopy
  12. Nat Aging. 2024 Jan 10.
      Epigenetic 'clocks' based on DNA methylation have emerged as the most robust and widely used aging biomarkers, but conventional methods for applying them are expensive and laborious. Here we develop tagmentation-based indexing for methylation sequencing (TIME-seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-seq, we applied multi-tissue and tissue-specific epigenetic clocks in over 1,800 mouse DNA samples from eight tissue and cell types. We show that TIME-seq clocks are accurate and robust, enriched for polycomb repressive complex 2-regulated loci, and benchmark favorably against conventional methods despite being up to 100-fold less expensive. Using dietary treatments and gene therapy, we find that TIME-seq clocks reflect diverse interventions in multiple tissues. Finally, we develop an economical human blood clock (R > 0.96, median error = 3.39 years) in 1,056 demographically representative individuals. These methods will enable more efficient epigenetic clock measurement in larger-scale human and animal studies.
  13. Trends Endocrinol Metab. 2024 Jan 10. pii: S1043-2760(23)00250-3. [Epub ahead of print]
      Tumours are heterogeneous tissues containing diverse populations of cells and an abundant extracellular matrix (ECM). This tumour microenvironment prompts cancer cells to adapt their metabolism to survive and grow. Besides epigenetic factors, the metabolism of cancer cells is shaped by crosstalk with stromal cells and extracellular components. To date, most experimental models neglect the complexity of the tumour microenvironment and its relevance in regulating the dynamics of the metabolism in cancer. We discuss emerging strategies to model cellular and extracellular aspects of cancer metabolism. We highlight cancer models based on bioengineering, animal, and mathematical approaches to recreate cell-cell and cell-matrix interactions and patient-specific metabolism. Combining these approaches will improve our understanding of cancer metabolism and support the development of metabolism-targeting therapies.
    Keywords:  cancer metabolism; cancer models; mathematical models; tumour microenvironment
  14. Am J Physiol Cell Physiol. 2024 Jan 08.
      Immune cell driven pathways are linked to cancer cachexia. Tumor presence is associated with immune cell infiltration whereas cytotoxic chemotherapies reduce immune cell counts. Despite these paradoxical effects, both cancer and chemotherapy can cause cachexia; however, our understanding of immune responses in the cachexia condition with cancer and chemotherapy is largely unknown. We sought to advance our understanding of the immunology underlying cancer and cancer with chemotherapy induced cachexia. CD2F1 mice were given 106 C26 cells, followed by 5 doses of 5-fluorouracil (5FU; 30mg/kg LM, i.p.) or PBS. Indices of cachexia and tumor (TUM), skeletal muscle (SKM), and adipose tissue (AT) immune cell populations were examined using high-parameter flow cytometry. While 5FU was able to stunt tumor growth, % body weight loss and muscle mass were not different between C26 and C26+5FU. C26 increased CD11b+Ly6g+ and CD11b+Ly6cInt inflammatory myeloid cells in SKM and AT, however both populations were reduced with C26+5FU. tSNE analysis revealed 24 SKM macrophage subsets wherein 8 were changed with C26 or C26+5FU. C26+5FU increased SKM CD11b-CD11c+ dendritic cells, CD11b-NK1.1+ NK-cells, and CD11b-B220+ B-cells, and reduced Ly6cHiCX3CR1+CD206+CD163IntCD11c-MHCII- infiltrated macrophages and other CD11b+Ly6cHi myeloid cells compared to C26. Both C26 and C26+5FU had elevated CD11b+F480+CD206+MHCII- or more specifically Ly6cLoCX3CR1+CD206+CD163IntCD11c-MHCII- pro-fibrotic macrophages. 5FU suppressed tumor growth and decreased SKM and AT inflammatory immune cells without protecting against cachexia suggesting that these cells are not required for wasting. However, pro-fibrotic cells and muscle inflammatory/atrophic signaling appears consistent with cancer- and cancer with chemotherapy-induced wasting and remain potential therapeutic targets.
    Keywords:  Adipose tissue; Cancer; Chemotherapy; Macrophages; Skeletal muscle
  15. bioRxiv. 2023 Dec 18. pii: 2023.12.17.572088. [Epub ahead of print]
      Aging is accompanied by multiple molecular changes that contribute to aging-associated pathologies, such as accumulation of cellular damage and mitochondrial dysfunction. Tissue metabolism can also change with age, in part because mitochondria are central to cellular metabolism. Moreover, the co-factor NAD+, which is reported to decline across multiple tissue types during aging, plays a central role in metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the oxidative synthesis of nucleotides, amino acids, and lipids. To further characterize how tissue metabolism changes with age, we intravenously infused [U-13C]-glucose into young and old C57BL/6J, WSB/EiJ, and Diversity Outbred mice to trace glucose fate into downstream metabolites within plasma, liver, gastrocnemius muscle, and brain tissues. We found that glucose incorporation into central carbon and amino acid metabolism was robust during healthy aging across these different strains of mice. We also observed that levels of NAD+, NADH, and the NAD+/NADH ratio were unchanged in these tissues with healthy aging. However, aging tissues, particularly brain, exhibited evidence of up-regulated fatty acid and sphingolipid metabolism reactions that regenerate NAD+ from NADH. Because mitochondrial respiration, a major source of NAD+ regeneration, is reported to decline with age, our data supports a model where NAD+-generating lipid metabolism reactions may buffer against changes in NAD+/NADH during healthy aging.
  16. Nat Cancer. 2024 Jan 09.
      The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.
  17. ESMO Open. 2024 Jan 08. pii: S2059-7029(23)01460-6. [Epub ahead of print]9(1): 102219
      BACKGROUND: Despite the prognostic relevance of cachexia in pancreatic cancer, individual body composition has not been routinely integrated into treatment planning. In this multicenter study, we investigated the prognostic value of sarcopenia and myosteatosis automatically extracted from routine computed tomography (CT) scans of patients with advanced pancreatic ductal adenocarcinoma (PDAC).PATIENTS AND METHODS: We retrospectively analyzed clinical imaging data of 601 patients from three German cancer centers. We applied a deep learning approach to assess sarcopenia by the abdominal muscle-to-bone ratio (MBR) and myosteatosis by the ratio of abdominal inter- and intramuscular fat to muscle volume. In the pooled cohort, univariable and multivariable analyses were carried out to analyze the association between body composition markers and overall survival (OS). We analyzed the relationship between body composition markers and laboratory values during the first year of therapy in a subgroup using linear regression analysis adjusted for age, sex, and American Joint Committee on Cancer (AJCC) stage.
    RESULTS: Deep learning-derived MBR [hazard ratio (HR) 0.60, 95% confidence interval (CI) 0.47-0.77, P < 0.005] and myosteatosis (HR 3.73, 95% CI 1.66-8.39, P < 0.005) were significantly associated with OS in univariable analysis. In multivariable analysis, MBR (P = 0.019) and myosteatosis (P = 0.02) were associated with OS independent of age, sex, and AJCC stage. In a subgroup, MBR and myosteatosis were associated with albumin and C-reactive protein levels after initiation of therapy. Additionally, MBR was also associated with hemoglobin and total protein levels.
    CONCLUSIONS: Our work demonstrates that deep learning can be applied across cancer centers to automatically assess sarcopenia and myosteatosis from routine CT scans. We highlight the prognostic role of our proposed markers and show a strong relationship with protein levels, inflammation, and anemia. In clinical practice, automated body composition analysis holds the potential to further personalize cancer treatment.
    Keywords:  body composition; computed tomography; deep learning; pancreatic cancer; prognosis
  18. Pancreas. 2024 Jan 09.
      ABSTRACT: Pancreatic cancer remains one of the deadliest of all cancer types with a 5-year overall survival rate of just 12%. Preclinical models available for understanding the disease pathophysiology have evolved significantly in recent years. Traditionally, commercially available 2-dimensional cell lines were developed to investigate mechanisms underlying tumorigenesis, metastasis, and drug resistance. However, these cells grow as monolayer cultures that lack heterogeneity and do not effectively represent tumor biology. Developing patient-derived xenografts and genetically engineered mouse models led to increased cellular heterogeneity, molecular diversity, and tissues that histologically represent the original patient tumors. However, these models are relatively expensive and very timing consuming. More recently, the advancement of fast and inexpensive in vitro models that better mimic disease conditions in vivo are on the rise. Three-dimensional cultures like organoids and spheroids have gained popularity and are considered to recapitulate complex disease characteristics. In addition, computational genomics, transcriptomics, and metabolomic models are being developed to simulate pancreatic cancer progression and predict better treatment strategies. Herein, we review the challenges associated with pancreatic cancer research and available analytical models. We suggest that an integrated approach toward using these models may allow for developing new strategies for pancreatic cancer precision medicine.
  19. Mol Cell. 2024 Jan 04. pii: S1097-2765(23)01035-3. [Epub ahead of print]
      Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.
    Keywords:  FA; Fe-S cluster; Friedreich’s ataxia; METTL17; frataxin; mitochondria; mitoribosome
  20. Cold Spring Harb Perspect Med. 2024 Jan 08. pii: a041411. [Epub ahead of print]
      The tumor microenvironment (TME) is a complex ecosystem of both cellular and noncellular components that functions to impact the evolution of cancer. Various aspects of the TME have been targeted for the control of cancer; however, TME composition is dynamic, with the overall abundance of immune cells, endothelial cells (ECs), fibroblasts, and extracellular matrix (ECM) as well as subsets of TME components changing at different stages of progression and in response to therapy. To effectively treat cancer, an understanding of the functional role of the TME is needed. Genetically engineered mouse models have enabled comprehensive insight into the complex interactions within the TME ecosystem that regulate disease progression. Here, we review recent advances in mouse models that have been employed to understand how the TME regulates cancer initiation, progression, metastasis, and response to therapy.
  21. Lab Chip. 2024 Jan 11.
      Enzymatically isolated pancreatic islets are the most commonly used ex vivo testbeds for diabetes research. Recently, precision-cut living slices of human pancreas are emerging as an exciting alternative because they maintain the complex architecture of the endocrine and exocrine tissues, and do not suffer from the mechanical and chemical stress of enzymatic isolation. We report a fluidic pancreatic SliceChip platform with dynamic environmental controls that generates a warm, oxygenated, and bubble-free fluidic pathway across singular immobilized slices with continuous deliver of fresh media and the ability to perform repeat serial perfusion assessments. A degasser ensures the system remains bubble-free while systemic pressurization with compressed oxygen ensures slice medium remains adequately oxygenated. Computational modeling of perfusion and oxygen dynamics within SliceChip guide the system's physiomimetic culture conditions. Maintenance of the physiological glucose dependent insulin secretion profile across repeat perfusion assessments of individual pancreatic slices kept under physiological oxygen levels demonstrated the culture capacity of our platform. Fluorescent images acquired every 4 hours of transgenic murine pancreatic slices were reliably stable and recoverable over a 5 day period due to the inclusion of a 3D-printed bioinert metallic anchor that maintained slice position within the SliceChip. Our slice on a chip platform has the potential to expand the useability of human pancreatic slices for diabetes pathogenesis and the development of new therapeutic approaches, while also enabling organotypic culture and assessment of other tissue slices such as brain and patient tumors.
  22. Bio Protoc. 2024 Jan 05. 14(1): e4917
      Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12-ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout. Key features • Preparation of ATG7 E1 from insect cells (Sf9 cells). • Preparation of ATG3 E2 from bacteria (E. coli). • Preparation of LC3B S3C from bacteria (E. coli). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.
    Keywords:  Autophagy; Fluorescence spectroscopy; In vitro ATG8 lipidation; Liposomes; NBD; Real-time ATG8 lipidation assay; Site-directed fluorescence; Ubiquitin-like conjugation
  23. Lab Chip. 2024 Jan 09.
      Cancer metastasis, the leading cause of cancer-related deaths, remains a complex challenge in medical science. Stephen Paget's "seed and soil theory" introduced the concept of organotropism, suggesting that metastatic success depends on specific organ microenvironments. Understanding organotropism not only offers potential for curbing metastasis but also novel treatment strategies. Microphysiological systems (MPS), especially organ-on-a-chip models, have emerged as transformative tools in this quest. These systems, blending microfluidics, biology, and engineering, grant precise control over cell interactions within organ-specific microenvironments. MPS enable real-time monitoring, morphological analysis, and protein quantification, enhancing our comprehension of cancer dynamics, including tumor migration, vascularization, and pre-metastatic niches. In this review, we explore innovative applications of MPS in investigating cancer metastasis, particularly focusing on organotropism. This interdisciplinary approach converges the field of science, engineering, and medicine, thereby illuminating a path toward groundbreaking discoveries in cancer research.
  24. Adv Mater. 2024 Jan 12. e2311176
      In biology, membranes are the key structures to separate and spatially organize cellular reaction systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries many of which operate through energy dissipation. Likewise, man-made molecular rotary motors powered by light have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here we systematically investigate the impact of rotary molecular motors on well-defined biological membrane systems, focusing on supported lipid bilayers (SLBs) and giant unilamellar vesicles (GUVs). Notably, we observe dramatic mechanical transformations of these systems upon motor irradiation, indicative of motor-induced membrane expansion. We systematically explore the influence of several factors on this phenomenon, such as motor concentration and membrane composition, and find that in particular, membrane fluidity plays a crucial role in motor-induced deformations. At the same time, only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, we find that membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of how molecular motors interact with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology. This article is protected by copyright. All rights reserved.
    Keywords:  GUV shape transitions; membrane area expansion; membrane biophysics; membrane deformation; molecular motors
  25. EMBO J. 2024 Jan 11.
      Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.
    Keywords:  Brown Adipose Tissue; Coenzyme Q; FGF21; Mitochondrial Unfolded Protein Response; Mitohormesis
  26. J Immunother Cancer. 2024 Jan 08. pii: e008086. [Epub ahead of print]12(1):
      BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor. Prognosis is poor and survival is low in patients diagnosed with this disease, with a survival rate of ~12% at 5 years. Immunotherapy, including adoptive T cell transfer therapy, has not impacted the outcomes in patients with PDAC, due in part to the hostile tumor microenvironment (TME) which limits T cell trafficking and persistence. We posit that murine models serve as useful tools to study the fate of T cell therapy. Currently, genetically engineered mouse models (GEMMs) for PDAC are considered a "gold-standard" as they recapitulate many aspects of human disease. However, these models have limitations, including marked tumor variability across individual mice and the cost of colony maintenance.METHODS: Using flow cytometry and immunohistochemistry, we characterized the immunological features and trafficking patterns of adoptively transferred T cells in orthotopic PDAC (C57BL/6) models using two mouse cell lines, KPC-Luc and MT-5, isolated from C57BL/6 KPC-GEMM (KrasLSL-G12D/+p53-/- and KrasLSL-G12D/+p53LSL-R172H/+, respectively).
    RESULTS: The MT-5 orthotopic model best recapitulates the cellular and stromal features of the TME in the PDAC GEMM. In contrast, far more host immune cells infiltrate the KPC-Luc tumors, which have less stroma, although CD4+ and CD8+ T cells were similarly detected in the MT-5 tumors compared with KPC-GEMM in mice. Interestingly, we found that chimeric antigen receptor (CAR) T cells redirected to recognize mesothelin on these tumors that signal via CD3ζ and 41BB (Meso-41BBζ-CAR T cells) infiltrated the tumors of mice bearing stroma-devoid KPC-Luc orthotopic tumors, but not MT-5 tumors.
    CONCLUSIONS: Our data establish for the first time a reproducible and realistic clinical system useful for modeling stroma-rich and stroma-devoid PDAC tumors. These models shall serve an indepth study of how to overcome barriers that limit antitumor activity of adoptively transferred T cells.
    Keywords:  CD4-CD8 ratio; T-lymphocytes; receptors, chimeric antigen; receptors, immunologic; tumor microenvironment
  27. Cell Metab. 2023 Dec 29. pii: S1550-4131(23)00459-X. [Epub ahead of print]
      The risk associated with multiple cancers, cardiovascular disease, diabetes, and all-cause mortality is decreased in individuals who meet the current recommendations for physical activity. Therefore, regular exercise remains a cornerstone in the prevention and treatment of non-communicable diseases. An acute bout of exercise results in the coordinated interaction between multiple tissues to meet the increased energy demand of exercise. Over time, the associated metabolic stress of each individual exercise bout provides the basis for long-term adaptations across tissues, including the cardiovascular system, skeletal muscle, adipose tissue, liver, pancreas, gut, and brain. Therefore, regular exercise is associated with a plethora of benefits throughout the whole body, including improved cardiorespiratory fitness, physical function, and glycemic control. Overall, we summarize the exercise-induced adaptations that occur within multiple tissues and how they converge to ultimately improve cardiometabolic health.
    Keywords:  adaptations; cardiometabolic health; exercise; exercise physiology; exercise signaling; metabolism; multi-tissue
  28. Nat Immunol. 2024 Jan 08.
      Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the molecular and cellular impact of sepsis across organs remains rudimentary. Here, we characterize the pathogenesis of sepsis by measuring dynamic changes in gene expression across organs. To pinpoint molecules controlling organ states in sepsis, we compare the effects of sepsis on organ gene expression to those of 6 singles and 15 pairs of recombinant cytokines. Strikingly, we find that the pairwise effects of tumor necrosis factor plus interleukin (IL)-18, interferon-gamma or IL-1β suffice to mirror the impact of sepsis across tissues. Mechanistically, we map the cellular effects of sepsis and cytokines by computing changes in the abundance of 195 cell types across 9 organs, which we validate by whole-mouse spatial profiling. Our work decodes the cytokine cacophony in sepsis into a pairwise cytokine message capturing the gene, cell and tissue responses of the host to the disease.
  29. bioRxiv. 2023 Dec 23. pii: 2023.12.22.573123. [Epub ahead of print]
      Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated β-galactosidase activity (SA-β-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-β-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-β-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that exacerbate or mitigate senescence burden (i.e. senescence inducers and senolytics, respectively). This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.
    Keywords:  Cellular senescence; aging; high-content image analysis; high-throughput screening; senescence associated-β-galactosidase; senescence-associated markers