bims-cagime 2025-03-02 papers

bims-cagime

Biomed News

on Cancer, aging and metabolism

Issue of 2025–03–02
24 papers selected by
Kıvanç Görgülü, Technical University of Munich

Identification of a senescence-associated transcriptional program in skeletal muscle of cachectic pancreatic-tumor bearing mice.
TAX1BP1-dependent autophagic degradation of STING1 impairs anti-tumor immunity.
Piezo1-induced durotaxis of pancreatic stellate cells depends on TRPC1 and TRPV4 channels.
A Distinctive Form of Autophagy Induced by Oncogenic RAS.
Uncovering pre-cytokinetic block in cancer cells under shear stress using a disturbed flow-generating device.
Phase separation of p62: roles and regulations in autophagy.
A Quantitative Approach to Mapping Mitochondrial Specialization and Plasticity.
Lipid packing and local geometry influence septin curvature sensing.
Optimized Methods to Quantify Tumor Treating Fields (TTFields)-Induced Permeabilization of Glioblastoma Cell Membranes.
SQLE-catalyzed squalene metabolism promotes mitochondrial biogenesis and tumor development in K-ras-driven cancer.
Nellie: automated organelle segmentation, tracking and hierarchical feature extraction in 2D/3D live-cell microscopy.
A systems-level, semi-quantitative landscape of metabolic flux in C. elegans.
Dynamic and Biphasic Regulation of Cell Migration by Ras.
The spectrum of lysosomal stress and damage responses: from mechanosensing to inflammation.
RBM10 loss promotes metastases by aberrant splicing of cytoskeletal and extracellular matrix mRNAs.
Lipid-Directed Covalent Labeling of Plasma Membranes for Long-Term Imaging, Barcoding and Manipulation of Cells.
Pancreatic steatosis as a risk factor for pancreatic ductal adenocarcinoma: pathogenesis and clinical implications.
Autophagy induced by mechanical stress sensitizes cells to ferroptosis by the NCOA4-FTH1 axis.
Clonal Heterogeneity in Human Pancreatic Ductal Adenocarcinoma and Its Impact on Tumor Progression.
Efficient cell-wide mapping of mitochondria in electron microscopic volumes using webKnossos.
Attenuated growth factor signaling during cell death initiation sensitizes membranes towards peroxidation.
Microfluidic cell unroofing for the in situ molecular analysis of organelles without membrane permeabilization.
Ultra-rapid droplet digital PCR enables intraoperative tumor quantification.
Cooperativity in septin polymerization is tunable by ionic strength and membrane adsorption.

Am J Physiol Cell Physiol. 2025 Feb 24.

Identification of a senescence-associated transcriptional program in skeletal muscle of cachectic pancreatic-tumor bearing mice.

Jeremy B Ducharme, Madison E Carelock, Martin M Schonk, Nour M Al-Zaeed, Weizhou Zhang, Sarah M Judge, Andrew R Judge.

  Cancer cachexia is the involuntary loss of body and skeletal muscle mass, which negatively impacts physical function, quality of life, treatment tolerance, and survival. Skeletal muscles of cachectic people and mice with pancreatic tumors also exhibit skeletal muscle damage, non-resolute immune cell infiltration, and impaired regeneration. These phenotypes may be influenced by the accumulation of senescent cells, which secrete factors detrimental to skeletal muscle health. However, there is currently no comprehensive research on senescent cell accumulation in skeletal muscle of tumor-bearing hosts, with or without chemotherapy. To address this gap, we cross-referenced the SenMayo panel of 125 senescence-related genes with our RNA-seq dataset in mouse skeletal muscle during the initiation and progression of cancer cachexia, which revealed a differential expression of 39 genes at pre-cachexia, 64 genes at cachexia onset, and 72 genes when cachexia is severe. Since p16 is a canonical marker of senescence, we subsequently orthotopically injected p16-tdTomato reporter mice with murine KPC pancreatic cancer cells and treated a subset of mice with chemotherapy. At experimental endpoint, when KPC treatment-naïve mice were cachectic, we observed an increased accumulation of p16+ cells, along with increased mRNA levels of hallmark senescence markers (Cdkn1a/p21, Cdkn2a/p16, Glb1/senescent-associated-β-galactosidase), which were exacerbated by chemotherapy. Lastly, we demonstrate an increase in CDKN1A/p21 in the muscle of cachectic patients with pancreatic cancer, which associated with cachexia severity. These findings suggest that senescent cells accumulate in skeletal muscle of cachectic pancreatic tumor-bearing hosts and that chemotherapy can exacerbate this accumulation.

Keywords:  Cachexia; Cellular Senescence; Glb1; p16; p21

DOI:  https://doi.org/10.1152/ajpcell.00816.2024
Autophagy. 2025 Feb 25.

TAX1BP1-dependent autophagic degradation of STING1 impairs anti-tumor immunity.

Ruoxi Zhang, Chunhua Yu, Herbert J Zeh, Guido Kroemer, Daniel J Klionsky, Daolin Tang, Rui Kang.

  The activation of STING1 can lead to the production and secretion of cytokines, initiating antitumor immunity. Here, we screened an ion channel ligand library and identified tetrandrine, a bis-benzylisoquinoline alkaloid, as an immunological adjuvant that enhances antitumor immunity by preventing the autophagic degradation of the STING1 protein. This tetrandrine effect is independent of its known function as a calcium or potassium channel blocker. Instead, tetrandrine inhibits lysosomal function, impairing cathepsin maturation, and autophagic degradation. Proteomic analysis of lysosomes identified TAX1BP1 as a novel autophagic receptor for the proteolysis of STING1. TAX1BP1 recognizes STING1 through the physical interaction of its coiled-coil domain with the cyclic dinucleotide binding domain of STING1. Systematic mutation of lysine (K) residues revealed that K63-ubiquitination of STING1 at the K224 site ignites TAX1BP1-dependent STING1 degradation. Combined treatment with tetrandrine and STING1 agonists promotes antitumor immunity by converting "cold" pancreatic cancers into "hot" tumors. This process is associated with enhanced cytokine release and increased infiltration of cytotoxic T-cells into the tumor microenvironment. The antitumor immunity mediated by tetrandrine and STING1 agonists is limited by neutralizing antibodies to the type I interferon receptor or CD8+ T cells. Thus, these findings establish a potential immunotherapeutic strategy against pancreatic cancer by preventing the autophagic degradation of STING1.

Keywords:  Autophagy; degradation; lysosome; pancreatic cancer; tumor immunity

DOI:  https://doi.org/10.1080/15548627.2025.2471736
J Cell Sci. 2025 Feb 28. pii: jcs.263846. [Epub ahead of print]

Piezo1-induced durotaxis of pancreatic stellate cells depends on TRPC1 and TRPV4 channels.

Ilka Budde, André Schlichting, David Ing, Sandra Schimmelpfennig, Anna Kuntze, Benedikt Fels, Joelle M-J Romac, Sandip M Swain, Rodger A Liddle, Angela Stevens, Albrecht Schwab, Zoltán Pethő.

  Pancreatic stellate cells (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. However, the molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the contribution of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Our results demonstrate that optimal PSC durotaxis requires an intermediary level of ion channel activity, which we simulated via a numerically discretized mathematical model. These findings suggest that mechanosensitive Piezo1 channels detect the differential stiffness microenvironment. The resulting intracellular signals are amplified by TRPV4 and TRPC1 channels to guide efficient PSC durotaxis.

Keywords:  Mechanosensation; Mechanosignaling; Pancreatic cancer; Taxis

DOI:  https://doi.org/10.1242/jcs.263846
Autophagy. 2025 Feb 23.

A Distinctive Form of Autophagy Induced by Oncogenic RAS.

Xiaojuan Wang, Shulin Li, Min Zhang, Liang Ge.

  RAS mutations enhance macroautophagy/autophagy in tumor cells, crucial for their growth and survival, making autophagy a promising therapeutic target for RAS-mutant cancers. However, the distinction between RAS-induced autophagy and physiological autophagy is not well understood. We recently identified a unique form of autophagy, RAS-induced non-canonical autophagy via ATG8ylation (RINCAA), which differs from starvation-induced autophagy. RINCAA is regulated by different sets of autophagic factors and forms structures distinct from the double-membrane autophagosome known as RAS-induced multivesicular/multilaminar bodies of ATG8ylation (RIMMBA). A key feature of RINCAA is the phosphorylation of PI4KB by ULK1, and inhibiting this phosphorylation shows superior effects compared to general autophagy inhibitors. This work suggests a potential for specifically targeting autophagy in RAS-driven cancers as a therapeutic strategy.

Keywords:  ATG8ylation; PtdIns4KB; RAS; autophagy; cancer

DOI:  https://doi.org/10.1080/15548627.2025.2468917
Sci Rep. 2025 Feb 22. 15(1): 6457

Uncovering pre-cytokinetic block in cancer cells under shear stress using a disturbed flow-generating device.

Lucie Beresova, Jan Vitecek, Iva Protivánková, Michal Dudka, Katarina Chroma, Zdenek Skrott, Tereza Buchtova, Kamila Poláková, Jan Novotny, Ludmila Novakova, Jiri Bartek, Martin Mistrik.

  During metastasis, cancer cells navigate through harsh conditions, including various mechanical forces in the bloodstream, highlighting the need to understand the impact of mechanical and shear stresses on cancer cells. To overcome the current methodological limitations of such research, here we present a new device that replicates similar conditions by applying shear stress on cultured cells. The device provides a less complex, easily accessible alternative to traditional fluidics while generating fluid shear stress values comparable to those in human veins and capillaries. The device allows analyses of large cell numbers in standard cell culture flasks and incubators. Using this device to explore the shear stress-induced responses of various human cell lines, we discovered a previously unknown, reversible pre-cytokinetic block occurring in cells that lose anchorage during mitosis and are kept under constant shear stress. Notably, some cancer cell lines appear to bypass this unorthodox cell-cycle block, suggesting its role as a safety checkpoint to restrict the proliferation of cancer cells in the bloodstream and their overall spreading potential. These findings provide new insights into the diverse responses of normal and cancer cells to shear stress and highlight the potential of our technology for research on circulating tumor cells and metastatic spread.

Keywords:  Circulating tumor cells; Fluid shear stress; Fluidic systems; Metastasis; Mitosis

DOI:  https://doi.org/10.1038/s41598-024-83058-3
Trends Cell Biol. 2025 Feb 25. pii: S0962-8924(25)00033-9. [Epub ahead of print]

Phase separation of p62: roles and regulations in autophagy.

Xue Huang, Jinpei Zhang, Jia Yao, Na Mi, Aimin Yang.

  The phase separation of the cargo receptor sequestome-1/p62 (SQSTM1/p62) is a critical mechanism for assembling signaling complexes in autophagy. During this process, p62 undergoes phase separation upon binding to polyubiquitin chains, concentrating ubiquitinated substrates within p62 droplets. These droplets further gather membrane sources and core autophagy machineries to facilitate autophagosome formation. The dynamics of p62 droplets are finely tuned in response to autophagy signals triggered by cellular stresses. Recent studies have revealed new regulatory mechanisms that highlight the significance of p62 phase separation in regulating autophagy. This review summarizes and discusses the molecular mechanisms of p62 phase separation and its roles in autophagy, with particular emphasis on the regulation of p62 droplets and their interaction modes with autophagic membranes.

Keywords:  cargo receptor; p62; p62 droplets; phase separation; selective autophagy

DOI:  https://doi.org/10.1016/j.tcb.2025.01.010
Res Sq. 2025 Feb 14. pii: rs.3.rs-5961609. [Epub ahead of print]

A Quantitative Approach to Mapping Mitochondrial Specialization and Plasticity.

Martin Picard, Anna Monzel, Jack Devine, Darshana Kapri, Jose Enriquez, Caroline Trumpff.

Mitochondria are a diverse family of organelles that specialize to accomplish complimentary functions 1-3. All mitochondria share general features, but not all mitochondria are created equal 4.Here we develop a quantitative pipeline to define the degree of molecular specialization among different mitochondrial phenotypes - or mitotypes. By distilling hundreds of validated mitochondrial genes/proteins into 149 biologically interpretable MitoPathway scores (MitoCarta 3.0 5) the simple mitotyping pipeline allows investigators to quantify and interpret mitochondrial diversity and plasticity from transcriptomics or proteomics data across a variety of natural and experimental contexts. We show that mouse and human multi-organ mitotypes segregate along two main axes of mitochondrial specialization, contrasting anabolic (liver) and catabolic (brain) tissues. In cultured primary human fibroblasts exhibiting robust time-dependent and treatment-induced metabolic plasticity 6-8, we demonstrate how the mitotype of a given cell type recalibrates i) over time in parallel with hallmarks of aging, and ii) in response to genetic, pharmacological, and metabolic perturbations. Investigators can now use MitotypeExplorer.org and the associated code to visualize, quantify and interpret the multivariate space of mitochondrial biology.

DOI: https://doi.org/10.21203/rs.3.rs-5961609/v1
bioRxiv. 2025 Feb 16. pii: 2025.02.12.637894. [Epub ahead of print]

Lipid packing and local geometry influence septin curvature sensing.

Brandy N Curtis, Ellysa J D Vogt, Christopher Edelmaier, Amy S Gladfelter.

Septins assemble into scaffolds that direct cell growth and morphology that are often localized to the plasma membrane. While septins preferentially bind convex membranes via amphipathic helices, their assembly on varied geometries in cells suggests additional localization cues. We tested the hypothesis that lipid composition directs septin assembly through lipid packing properties. Lipid mixtures varying in lipid packing were designed by molecular dynamics simulations and incorporated onto supported lipid bilayers to measure septin adsorption in vitro. Septins strongly favor loosely-packed, disordered lipid bilayers but additional geometry cues act in conjunction with this membrane property. Introducing tighter lipid packing in cells disrupted septin structures in a curvature dependent manner, specifically limiting septin assembly and retention along flat regions of the plasma membrane. This work demonstrates that packing defects and geometry jointly regulate septin localization and highlights how multiple membrane properties are integrated to control organization of the septin cytoskeleton.
Summary: Localization of the septin cytoskeleton is controlled by regulatory factors, membrane curvature, and charge. In this study, changes to lipid composition that modulate lipid packing defects are found to impact septin assemblies in vitro and in cells.

DOI: https://doi.org/10.1101/2025.02.12.637894
Methods Protoc. 2025 Jan 22. pii: 10. [Epub ahead of print]8(1):

Optimized Methods to Quantify Tumor Treating Fields (TTFields)-Induced Permeabilization of Glioblastoma Cell Membranes.

Melisa Martinez-Paniagua, Sabbir Khan, Nikita W Henning, Sri Vaishnavi Konagalla, Chirag B Patel.

  Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5-24 h of TTFields exposure resulted in an increased uptake of FITC-dextran fluorescent probes (4-20 kDa) in human GBM cells. However, this approach, in which a fluorescence plate-based detector is used to evaluate cells attached to glass coverslips, cannot distinguish FITC-dextran uptake in live vs. dead cells. The goal of the study was to report the optimization and validation of two independent methods to quantify human GBM cell membrane permeabilization induced by TTFields exposure. First, we optimized flow cytometry by measuring mean fluorescence intensity at 72 h for 4 kDa (TTFields 6726 ± 958.0 vs. no-TTFields 5093 ± 239.7, p = 0.016) and 20 kDa (7087 ± 1137 vs. 5055 ± 897.8, p = 0.031) probes. Second, we measured the ratio of lactate dehydrogenase (LDH) to cell viability (measured using the CellTiter-Glo [CTG] viability assay); the LDH/CTG ratio was higher under TTFields (1.47 ± 0.15) than no-TTFields (1.08 ± 0.08) conditions, p < 0.0001. The findings using these two independent methods reproducibly demonstrated their utility for time-dependent evaluations. We also showed that these methods can be used to relate the cell membrane-permeabilizing effects of the non-ionizing radiation of TTFields to that of an established cell membrane permeabilizer, the non-ionic detergent Triton-X-100. Evaluating carboplatin ± TTFields, the LDH/CTG ratio was significantly higher in the TTFields vs. no-TTFields condition at each carboplatin concentration (0-30 µM), p = 0.014. We successfully optimized and validated two cost-effective methods to reproducibly quantify TTFields-induced human GBM cancer cell membrane permeabilization.

Keywords:  cell membrane permeability; flow cytometry; lactate dehydrogenase (LDH); method optimization; tumor treating fields (TTFields)

DOI:  https://doi.org/10.3390/mps8010010
Cancer Lett. 2025 Feb 25. pii: S0304-3835(25)00150-8. [Epub ahead of print] 217586

SQLE-catalyzed squalene metabolism promotes mitochondrial biogenesis and tumor development in K-ras-driven cancer.

Junchen Pan, Rui Liu, Wenhua Lu, Hongyu Peng, Jing Yang, Qianrui Zhang, Tiantian Yu, Bitao Huo, Xiaoying Wei, Haixi Liang, Lin Zhou, Yameng Sun, Yumin Hu, Shijun Wen, Jie Fu, Paul J Chiao, Xiaojun Xia, Jinyun Liu, Peng Huang.

  It is well known that activation of oncogenic K-ras alone is insufficient to drive tumor development and that additional factors are needed for full malignant transformation, but the metabolic pathways and regulatory mechanisms that facilitate K-ras-driven cancer development remain to be characterized. Here we show that SQLE, a key enzyme in cholesterol synthesis, is upregulated in K-ras-driven cancer and its high expression is correlated with poor clinical outcome. K-ras regulates SQLE expression in a biphasic manner through reactive oxygen species and MYC signaling. Surprisingly, the pro-oncogenic role of SQLE is not mediated by promoting cholesterol synthesis, but by metabolic removal of squalene and thus mitigating its suppressive effect on the PGC-1α-mediated mitochondrial biogenesis and metabolism. Genetic silencing of SQLE in pancreatic cancer cells causes an accumulation of cellular squalene, which binds to Sp1 protein and causes a formation of a tight Sp1-TFAP2E promoter DNA complex with a highly negative binding score. This aberrant squalene/Sp1/ TFAP2E promoter complex hinders the expression of TFAP2E and its downstream molecule PGC-1α, leading to suppression of mitochondrial metabolism and an almost complete inhibition of tumor formation in vivo. Importantly, administration of pharmacological squalene to mice bearing pancreatic cancer xenografts could significantly inhibit tumor growth. Our study has revealed a previously unrecognized role of SQLE in regulating gene expression and mitochondrial metabolism to facilitate K-ras-driven cancer development, and identified SQLE as a novel therapeutic target for potential treatment of pancreatic cancer.

Keywords:  K-ras; PGC-1α; SQLE; Sp1; mitochondria; pancreatic cancer; squalene

DOI:  https://doi.org/10.1016/j.canlet.2025.217586
Nat Methods. 2025 Feb 27.

Nellie: automated organelle segmentation, tracking and hierarchical feature extraction in 2D/3D live-cell microscopy.

Austin E Y T Lefebvre, Gabriel Sturm, Ting-Yu Lin, Emily Stoops, Magdalena Preciado López, Benjamin Kaufmann-Malaga, Kayley Hake.

Cellular organelles undergo constant morphological changes and dynamic interactions that are fundamental to cell homeostasis, stress responses and disease progression. Despite their importance, quantifying organelle morphology and motility remains challenging due to their complex architectures, rapid movements and the technical limitations of existing analysis tools. Here we introduce Nellie, an automated and unbiased pipeline for segmentation, tracking and feature extraction of diverse intracellular structures. Nellie adapts to image metadata and employs hierarchical segmentation to resolve sub-organellar regions, while its radius-adaptive pattern matching enables precise motion tracking. Through a user-friendly Napari-based interface, Nellie enables comprehensive organelle analysis without coding expertise. We demonstrate Nellie's versatility by unmixing multiple organelles from single-channel data, quantifying mitochondrial responses to ionomycin via graph autoencoders and characterizing endoplasmic reticulum networks across cell types and time points. This tool addresses a critical need in cell biology by providing accessible, automated analysis of organelle dynamics.

DOI: https://doi.org/10.1038/s41592-025-02612-7
Nature. 2025 Feb 26.

A systems-level, semi-quantitative landscape of metabolic flux in C. elegans.

Hefei Zhang, Xuhang Li, L Tenzin Tseyang, Gabrielle E Giese, Hui Wang, Bo Yao, Jingyan Zhang, Rachel L Neve, Elizabeth A Shank, Jessica B Spinelli, L Safak Yilmaz, Albertha J M Walhout.

Metabolic flux, or the rate of metabolic reactions, is one of the most fundamental metrics describing the status of metabolism in living organisms. However, measuring fluxes across the entire metabolic network remains nearly impossible, especially in multicellular organisms. Computational methods based on flux balance analysis have been used with genome-scale metabolic network models to predict network-level flux wiring1-6. However, such approaches have limited power because of the lack of experimental constraints. Here, we introduce a strategy that infers whole-animal metabolic flux wiring from transcriptional phenotypes in the nematode Caenorhabditis elegans. Using a large-scale Worm Perturb-Seq (WPS) dataset for roughly 900 metabolic genes7, we show that the transcriptional response to metabolic gene perturbations can be integrated with the metabolic network model to infer a highly constrained, semi-quantitative flux distribution. We discover several features of adult C. elegans metabolism, including cyclic flux through the pentose phosphate pathway, lack of de novo purine synthesis flux and the primary use of amino acids and bacterial RNA as a tricarboxylic acid cycle carbon source, all of which we validate by stable isotope tracing. Our strategy for inferring metabolic wiring based on transcriptional phenotypes should be applicable to a variety of systems, including human cells.

DOI: https://doi.org/10.1038/s41586-025-08635-6
bioRxiv. 2025 Feb 16. pii: 2025.02.13.638204. [Epub ahead of print]

Dynamic and Biphasic Regulation of Cell Migration by Ras.

Yiyan Lin, Eleana Parajón, Qinling Yuan, Siyu Ye, Guanghui Qin, Yu Deng, Jane Borleis, Ariel Koyfman, Pablo A Iglesias, Konstantinos Konstantopoulos, Douglas N Robinson, Peter N Devreotes.

Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, non-transcriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that RasGEFs generally increase cortical tension while RasGAPs decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.
Significance: This study challenges the traditional view of Ras as solely a positive regulator of cell functions by controlling of gene expression. Using optogenetics to rapidly modulate Ras activity in Dictyostelium , we demonstrate a biphasic relationship between Ras activity and migration: both excessive and insufficient Ras activity impair cell movement. Importantly, these effects occur rapidly, independent of transcriptional changes, revealing the mechanism by which Ras controls cell migration. The findings suggest that optimal Ras activity and cortical tension are crucial for efficient migration, and that targeting Ras in cancer therapy should consider the cell's initial state, aiming to push Ras activity outside the optimal range for migration. This nuanced understanding of the role of Ras in migration has significant implications for developing more effective cancer treatments, as simply inhibiting Ras might inadvertently promote metastasis in certain contexts.

DOI: https://doi.org/10.1101/2025.02.13.638204
EMBO Rep. 2025 Feb 27.

The spectrum of lysosomal stress and damage responses: from mechanosensing to inflammation.

Ori Scott, Ekambir Saran, Spencer A Freeman.

  Cells and tissues turn over their aged and damaged components in order to adapt to a changing environment and maintain homeostasis. These functions rely on lysosomes, dynamic and heterogeneous organelles that play essential roles in nutrient redistribution, metabolism, signaling, gene regulation, plasma membrane repair, and immunity. Because of metabolic fluctuations and pathogenic threats, lysosomes must adapt in the short and long term to maintain functionality. In response to such challenges, lysosomes deploy a variety of mechanisms that prevent the breaching of their membrane and escape of their contents, including pathogen-associated molecules and hydrolases. While transient permeabilization of the lysosomal membrane can have acute beneficial effects, supporting inflammation and antigen cross-presentation, sustained or repeated lysosomal perforations have adverse metabolic and transcriptional consequences and can lead to cell death. This review outlines factors contributing to lysosomal stress and damage perception, as well as remedial processes aimed at addressing lysosomal disruptions. We conclude that lysosomal stress plays widespread roles in human physiology and pathology, the understanding and manipulation of which can open the door to novel therapeutic strategies.

Keywords:  Autophagy; Glycocalyx; Host–pathogen; Phagosolysosome; Pore-forming Toxins

DOI:  https://doi.org/10.1038/s44319-025-00405-9
J Exp Med. 2025 May 05. pii: e20241029. [Epub ahead of print]222(5):

RBM10 loss promotes metastases by aberrant splicing of cytoskeletal and extracellular matrix mRNAs.

Gnana P Krishnamoorthy, Anthony R Glover, Brian R Untch, Nickole Sigcha-Coello, Bin Xu, Dina Vukel, Yi Liu, Vera Tiedje, Jose Mario Bello Pineda, Katherine Berman, Prasanna P Tamarapu, Adrian Acuña-Ruiz, Mahesh Saqcena, Elisa de Stanchina, Laura Boucai, Ronald A Ghossein, Jeffrey A Knauf, Omar Abdel-Wahab, Robert K Bradley, James A Fagin.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse HrasG12V/Rbm1OKO thyrocytes develop metastases that are reversed by RBM10 expression or by combined knockdown of VCL, CD44, and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusion in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFκB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.

DOI: https://doi.org/10.1084/jem.20241029
JACS Au. 2025 Feb 24. 5(2): 922-936

Lipid-Directed Covalent Labeling of Plasma Membranes for Long-Term Imaging, Barcoding and Manipulation of Cells.

Nathan Aknine, Remi Pelletier, Andrey S Klymchenko.

Fluorescent probes for cell plasma membranes (PM) generally exploit a noncovalent labeling mechanism, which constitutes a fundamental limitation in multiple bioimaging applications. Here, we report a concept of lipid-directed covalent labeling of PM, which exploits transient binding to the lipid membrane surface generating a high local dye concentration, thus favoring covalent ligation to random proximal membrane proteins. This concept yielded fluorescent probes for PM called MemGraft, which are built of a dye (cyanine Cy3 or Cy5) bearing a low-affinity membrane anchor and a reactive group: an activated ester or a maleimide. In contrast to specially designed control dyes and commercial Cy3-based labels of amino or thiol groups, MemGraft probes stain efficiently PM, revealing the crucial role of the membrane anchor combined with optimal reactivity of the activated ester or the maleimide. MemGraft probes overcome existing limitations of noncovalent probes, which makes them compatible with cell fixation, permeabilization, trypsinization, and the presence of serum. The latter allows long-term cell tracking and video imaging of cell PM dynamics without the signs of phototoxicity. The covalent strategy also enables staining and long-term tracking of cocultured cells labeled in different colors without exchange of probes. Moreover, the combination of MemGraft-Cy3 and MemGraft-Cy5 probes at different ratios enabled long-term cell barcoding in at least 5 color codes, important for tracking and visualizing multiple populations of cells. Ultimately, we found that the MemGraft strategy enables efficient biotinylation of the cell surface, opening the path to cell surface engineering and cell manipulation.

DOI: https://doi.org/10.1021/jacsau.4c01134
Clin Transl Gastroenterol. 2025 Feb 24.

Pancreatic steatosis as a risk factor for pancreatic ductal adenocarcinoma: pathogenesis and clinical implications.

Zoi Papalamprakopoulou, Prasenjit Dey, Rachel Frascati, Christos Fountzilas.

ABSTRACT: Pancreatic steatosis is defined as the ectopic accumulation of fat in the pancreas. While historically considered a benign incidental imaging finding, it is now recognized as a significant, and potentially reversible risk factor for pancreatic ductal adenocarcinoma (PDAC) independently of obesity. Although its epidemiology is not well characterized, meta-analysis data suggest an approximately 30% prevalence, with individual studies reporting even higher rates among patients with obesity and/or metabolic syndrome. Concurrently, PDAC incidence is rising and is projected to soon become the second leading cause of cancer-related deaths. Given the critical importance of early PDAC detection and intervention for improving survival, it is particularly timely to explore the associations between pancreatic steatosis and PDAC. This review aims to provide a comprehensive overview of the pathogenesis and clinical associations between pancreatic steatosis and PDAC, as well as to discuss future perspectives within the context of current PDAC surveillance practices.

DOI: https://doi.org/10.14309/ctg.0000000000000832
Autophagy. 2025 Feb 23.

Autophagy induced by mechanical stress sensitizes cells to ferroptosis by the NCOA4-FTH1 axis.

Chenyu Luo, Haisheng Liang, Mintao Ji, Caiyong Ye, Yiping Lin, Yuhan Guo, Zhisen Zhang, Yinyin Shu, Xiaoni Jin, Shuangshuang Lu, Wanling Lu, Yazheng Dang, Hong Zhang, Bingyan Li, Guangming Zhou, Zengli Zhang, Lei Chang.

  Ferroptosis is an iron-dependent regulated form of cell death implicated in various diseases, including cancers, with its progression influenced by iron-dependent peroxidation of phospholipids and dysregulation of the redox system. Whereas the extracellular matrix of tumors provides mechanical cues influencing tumor initiation and progression, its impact on ferroptosis and its mechanisms remains largely unexplored. In this study, we reveal that heightened mechanical tension sensitizes cells to ferroptosis, whereas decreased mechanics confers resistance. Mechanistically, reduced mechanical tension reduces intracellular free iron levels by enhancing FTH1 protein expression. Additionally, low mechanics significantly diminishes NCOA4, pivotal in mediating FTH1 phase separation-induced ferritinophagy. Targeting NCOA4 effectively rescues ferroptosis susceptibility under low mechanical tension through modulation of FTH1 phase separation-driven autophagy. In conclusion, our findings demonstrate that mechanics regulates iron metabolism via NCOA4-FTH1 phase separation-mediated autophagy, thereby influencing ferroptosis sensitivity and offering promising therapeutic avenues for future exploration.

Keywords:  Ferritinophagy; iron metabolism; mechanotransduction

DOI:  https://doi.org/10.1080/15548627.2025.2469129
bioRxiv. 2025 Feb 15. pii: 2025.02.11.637729. [Epub ahead of print]

Clonal Heterogeneity in Human Pancreatic Ductal Adenocarcinoma and Its Impact on Tumor Progression.

Despoina Kalfakakou, Daniel C Cameron, Emily A Kawaler, Motoyuki Tsuda, Lidong Wang, Xiaohong Jing, Cristina Hajdu, Dylan L Tamayo, Yoona Shim, Amanda Ackermann, Daniel Weissinger, Hayley Zimny, Rosmel Hernandez, Matthew Beier, Dacia Dimartino, Peter Meyn, Kalina Rice, Shanmugapriya Selvaraj, Cynthia Loomis, Adriana Heguy, Amanda W Lund, Rosalie C Sears, Theodore H Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M Simeone.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by profound desmoplasia and cellular heterogeneity, which cannot be fully resolved using traditional bulk sequencing approaches. To understand the contribution of this heterogeneity to PDAC biology, we analyzed a large cohort of primary human PDAC samples (n = 62), profiling 443,451 single cells and 53,236 spatial transcriptomic spots using a combined single-cell RNA sequencing and spatial transcriptomics approach. Our analysis revealed significant intratumoral heterogeneity, with multiple genetically distinct neoplastic clones co-existing within individual tumors. These clones exhibited diverse transcriptional states and subtype profiles, challenging the traditional binary classification of PDAC into basal and classical subtypes; instead, our findings support a transcriptional continuum influenced by clonal evolution and spatial organization. Additionally, these clones each interacted uniquely with surrounding cell types in the tumor microenvironment. Phylogenetic analysis uncovered a rare but consistent classical-to-basal clonal transition associated with MYC amplification and immune response depletion, which were validated experimentally, suggesting a mechanism driving the emergence of a more aggressive basal clonal phenotype. Spatial analyses further revealed dispersed clones enriched for epithelial-to-mesenchymal transition (EMT) activity and immune suppression, correlating with metastatic potential and colonization of lymph node niches. These dispersed clones tended to transition toward a basal phenotype, contributing to disease progression. Our findings highlight the critical role of clonal diversity, transcriptional plasticity, and TME interactions in shaping human PDAC biology. This work provides new insights into the molecular and spatial heterogeneity of PDAC and offers potential avenues for therapeutic intervention targeting clonal evolution and the mechanisms driving metastasis.

DOI: https://doi.org/10.1101/2025.02.11.637729
Cell Rep Methods. 2025 Feb 24. pii: S2667-2375(25)00025-6. [Epub ahead of print]5(2): 100989

Efficient cell-wide mapping of mitochondria in electron microscopic volumes using webKnossos.

Yi Jiang, Haoyu Wang, Kevin M Boergens, Norman Rzepka, Fangfang Wang, Yunfeng Hua.

  Recent technical advances in volume electron microscopy (vEM) and artificial-intelligence-assisted image processing have facilitated high-throughput quantifications of cellular structures, such as mitochondria, that are ubiquitous and morphologically diversified. A still often-overlooked computational challenge is to assign a cell identity to numerous mitochondrial instances, for which both mitochondrial and cell membrane contouring used to be required. Here, we present a vEM reconstruction procedure (called mito-SegEM) that utilizes virtual-path-based annotation to assign automatically segmented mitochondrial instances at the cellular scale, therefore bypassing the requirement of membrane contouring. The embedded toolset in webKnossos (an open-source online annotation platform) is optimized for fast annotation, visualization, and proofreading of cellular organelle networks. We demonstrate the broad applications of mito-SegEM on volumetric datasets from various tissues, including the brain, intestine, and testis, to achieve an accurate and efficient reconstruction of mitochondria in a use-dependent fashion.

Keywords:  CP: Cell biology; CP: Imaging; cell biology; image processing; mitochondrion; software; volume electron microscopy

DOI:  https://doi.org/10.1016/j.crmeth.2025.100989
Nat Commun. 2025 Feb 25. 16(1): 1774

Attenuated growth factor signaling during cell death initiation sensitizes membranes towards peroxidation.

André Gollowitzer, Helmut Pein, Zhigang Rao, Lorenz Waltl, Leonhard Bereuter, Konstantin Loeser, Tobias Meyer, Vajiheh Jafari, Finja Witt, René Winkler, Fengting Su, Silke Große, Maria Thürmer, Julia Grander, Madlen Hotze, Sönke Harder, Lilia Espada, Alexander Magnutzki, Ronald Gstir, Christina Weinigel, Silke Rummler, Günther Bonn, Johanna Pachmayr, Maria Ermolaeva, Takeshi Harayama, Hartmut Schlüter, Christian Kosan, Regine Heller, Kathrin Thedieck, Michael Schmitt, Takao Shimizu, Jürgen Popp, Hideo Shindou, Marcel Kwiatkowski, Andreas Koeberle.

Cell death programs such as apoptosis and ferroptosis are associated with aberrant redox homeostasis linked to lipid metabolism and membrane function. Evidence for cross-talk between these programs is emerging. Here, we show that cytotoxic stress channels polyunsaturated fatty acids via lysophospholipid acyltransferase 12 into phospholipids that become susceptible to peroxidation under additional redox stress. This reprogramming is associated with altered acyl-CoA synthetase isoenzyme expression and caused by a decrease in growth factor receptor tyrosine kinase (RTK)-phosphatidylinositol-3-kinase signaling, resulting in suppressed fatty acid biosynthesis, for specific stressors via impaired Akt-SREBP1 activation. The reduced availability of de novo synthesized fatty acids favors the channeling of polyunsaturated fatty acids into phospholipids. Growth factor withdrawal by serum starvation mimics this phenotype, whereas RTK ligands counteract it. We conclude that attenuated RTK signaling during cell death initiation increases cells' susceptibility to oxidative membrane damage at the interface of apoptosis and alternative cell death programs.

DOI: https://doi.org/10.1038/s41467-025-56711-2
Lab Chip. 2025 Feb 26.

Microfluidic cell unroofing for the in situ molecular analysis of organelles without membrane permeabilization.

Yuki Umeda, Shinya Yamahira, Koki Nakamura, Tomoko Takagi, Tomoko Suzuki, Kae Sato, Yusuke Hirabayashi, Akimitsu Okamoto, Satoshi Yamaguchi.

Molecular networks of organelle membranes are involved in many cell processes. However, the nature of plasma membrane as a barrier to various analytical tools, including antibodies, makes it challenging to examine intact organelle membranes without affecting their structure and functions via membrane permeabilization. Therefore, in this study, we aimed to develop a microfluidic method to unroof cells and observe the intrinsic membrane molecules in organelles. In our method, single cells were precisely arrayed on the bottom surface of microchannels in a light-guided manner using a photoactivatable cell-anchoring material. At sufficiently short cell intervals, horizontal stresses generated by the laminar flow instantly fractured the upper cell membranes, without significantly affecting some organelles inside the fractured cells. Subsequently, nucleus and other organelles in unroofed cells were observed via confocal fluorescence and scanning electron microscopy. Furthermore, distribution of the mitochondrial membrane protein, translocase of outer mitochondrial membrane 20, on the mitochondrial membrane was successfully observed via immunostaining without permeabilization. Overall, the established cell unroofing method shows great potential to examine the localization, functions, and affinities of proteins on intact organelle membranes.

DOI: https://doi.org/10.1039/d5lc00102a
Med. 2025 Feb 19. pii: S2666-6340(25)00031-5. [Epub ahead of print] 100604

Ultra-rapid droplet digital PCR enables intraoperative tumor quantification.

Zachary R Murphy, Emilia C Bianchini, Andrew Smith, Lisa I Körner, Teresa Russell, David Reinecke, Nader Maarouf, Yuxiu Wang, John G Golfinos, Alexandra M Miller, Matija Snuderl, Daniel A Orringer, Gilad D Evrony.

   BACKGROUND: The diagnosis and treatment of tumors often depend on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections.
METHODS: Here, we introduce Ultra-Rapid droplet digital PCR (UR-ddPCR), a technology that achieves the fastest measurement, to date, of mutation burdens in tissue samples, from tissue to result in 15 min. Our workflow substantially reduces the time from tissue biopsy to molecular diagnosis and provides a highly accurate means of quantifying residual tumor infiltration at surgical margins.
FINDINGS: We demonstrate UR-ddPCR assays for the IDH1 R132H and BRAF V600E clonal mutations that are present in many low-grade gliomas and melanomas, respectively, and whose intraoperative detection would shape surgical decision-making. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 22 brain tumor cases, and we further combine UR-ddPCR tumor cell percentage measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities ranging from >1,300 tumor cells/mm2 within a tumor core to <5 tumor cells/mm2 at tumor margins. UR-ddPCR measurements were virtually identical to standard ddPCR measurements performed on the same samples (R2 = 0.995).
CONCLUSIONS: The technology and workflow developed here enable intraoperative molecular-genetic assays with unprecedented speed and sensitivity. We anticipate that our method will facilitate novel point-of-care diagnostics and molecularly guided surgeries that improve clinical outcomes.
FUNDING: This study was funded by the National Institutes of Health and NYU Grossman School of Medicine institutional funds. Reagents and instruments were provided in kind by Bio-Rad.

Keywords:  BRAF; IDH1; PCR; Translation to patients; brain tumor; ddPCR; glioma; intraoperative; rapid diagnostics

DOI:  https://doi.org/10.1016/j.medj.2025.100604
bioRxiv. 2025 Feb 16. pii: 2025.02.12.637902. [Epub ahead of print]

Cooperativity in septin polymerization is tunable by ionic strength and membrane adsorption.

Ellysa Vogt, Ian Seim, Wilton Snead, Amy Gladfelter.

Cells employ cytoskeletal polymers to move, divide, and pass information inside and outside of the cell. Previous work on eukaryotic cytoskeletal elements such as actin, microtubules, and intermediate filaments investigating the mechanisms of polymerization have been critical to understand how cells control the assembly of the cytoskeleton. Most biophysical analyses have considered cooperative versus isodesmic modes of polymerization; this framework is useful for specifying functions of regulatory proteins that control nucleation and understanding how cells regulate elongation in time and space. The septins are considered a fourth component of the eukaryotic cytoskeleton, but they are poorly understood in many ways despite their conserved roles in membrane dynamics, cytokinesis, and cell shape, and in their links to a myriad of human diseases. Because septin function is intimately linked to their assembled state, we set out to investigate the mechanisms by which septin polymers elongate under different conditions. We used simulations, in vitro reconstitution of purified septin complexes, and quantitative microscopy to directly interrogate septin polymerization behaviors in solution and on synthetic lipid bilayers of different geometries. We first used reactive Brownian dynamics simulations to determine if the presence of a membrane induces cooperativity to septin polymerization. We then used fluorescence correlation spectroscopy (FCS) to assess septins ability to form filaments in solution at different salt conditions. Finally, we investigated septin membrane adsorption and polymerization on planar and curved supported lipid bilayers. Septins clearly show signs of salt-dependent cooperative assembly in solution, but cooperativity is limited by binding a membrane. Thus, septin assembly is dramatically influenced by extrinsic conditions and substrate properties and can show properties of both isodesmic and cooperative polymers. This versatility in assembly modes may explain the extensive array of assembly types, functions, and subcellular locations in which septins act.

DOI: https://doi.org/10.1101/2025.02.12.637902