bims-cagime 2025-03-16 papers

bims-cagime

Biomed News

on Cancer, aging and metabolism

Issue of 2025–03–16
43 papers selected by
Kıvanç Görgülü, Technical University of Munich

STAT3 Sustains Tumorigenicity Following Mutant KRAS Ablation.
MYC ecDNA promotes intratumour heterogeneity and plasticity in PDAC.
Propionyl-CoA carboxylase subunit B regulates anti-tumor T cells in a pancreatic cancer mouse model.
Mechanisms and functions of lysosomal lipid homeostasis.
CYP51A1 drives resistance to pH-dependent cell death in pancreatic cancer.
The Biology in the Pattern: Metastatic organotropism and clinical outcome depend on DNA damage response and immune interactions in pancreatic cancer.
Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors.
Integrating model systems and genomic insights to decipher mechanisms of cancer metastasis.
Oncogenic RAS induces a distinctive form of non-canonical autophagy mediated by the P38-ULK1-PI4KB axis.
Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits.
Reply to: Overcoming Barriers to Cancer-Associated Cachexia Diagnosis and Management.
Autophagy Suppresses CCL2 to Preserve Appetite and Prevent Lethal Cachexia.
Toward universal models for collective interactions in biomolecular condensates.
Structural biology at the plasma membrane.
Selective Sorting of Senescent Cell Subpopulations Compatible with Downstream Genomics Applications.
T-cell dependency of tumor regressions and complete responses with RAS(ON) multi-selective inhibition in preclinical models of PDAC.
Cholesterol inhibits assembly and oncogenic activation of the EphA2 receptor.
Cellular homeostatic responses to lysosomal damage.
Lipid Dynamics at Membrane Contact Sites.
Oncological and Survival Endpoints in Cancer Cachexia Clinical Trials: Systematic Review 6 of the Cachexia Endpoints Series.
A functional single-cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumours.
Highly multiplexed digital PCR assay for simultaneous quantification of variant allele frequencies and copy number alterations of KRAS and GNAS in pancreatic cancer precursors.
LivecellX: A Deep-learning-based, Single-Cell Object-Oriented Framework for Quantitative Analysis in Live-Cell Imaging.
ADT-1004: a first-in-class, oral pan-RAS inhibitor with robust antitumor activity in preclinical models of pancreatic ductal adenocarcinoma.
Epithelial-mesenchymal Transition Promotes Metabolic Reprogramming to Suppress Ferroptosis.
A dual-purification system to isolate mitochondrial subpopulations.
The TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis: a novel regulatory mechanism for Lysophagy.
Measuring and manipulating mechanical forces during development.
Muscle loss in cancer cachexia: what is the basis for nutritional support?
When tumour cells get excited.
Detection and Characterization of Senescent Cells with Flow Cytometry.
Detecting Senescence in T Cells by Flow Cytometry Using the SA-β-Galactosidase Assay.
Chemical Probe-Enabled Lipid Droplet Proteomics.
Dynamics of Microsphere Inclusions within Biomimetic Membranes Reveal Membrane Heterogeneity.
Aneuploidy as a cancer vulnerability.
Direct sensing of dietary ω-6 linoleic acid through FABP5-mTORC1 signaling.
Ultrasound-mediated mechanical forces activate selective tumor cell apoptosis.
Tissue stiffness mapping by light sheet elastography.
Glycogen drives tumour initiation and progression in lung adenocarcinoma.
ATG9A vesicles are a subtype of intracellular nanovesicle.
Unlocking the potential of digital pathology: Novel baselines for compression.
Novel orthotopic patient-derived xenograft model using human pancreatic cancer tissue fragments to recapitulate distant metastasis and cancer-related hypercoagulability.
Phosphatidylcholine synthesis and remodeling in brain endothelial cells.

bioRxiv. 2025 Feb 27. pii: 2025.02.21.639533. [Epub ahead of print]

STAT3 Sustains Tumorigenicity Following Mutant KRAS Ablation.

Stephen D'Amico, Varvara Kirillov, Jingxuan Liu, Zhijuan Qiu, Xinyuan Lei, Hong Qin, Brian S Sheridan, Nancy C Reich.

Oncogenic KRAS mutations underlie some of the deadliest human cancers. Genetic or pharmacological inactivation of mutant KRAS is not sufficient for long-term control of advanced tumors. Using a conceptual framework of pancreatic ductal adenocarcinoma, we find that CRISPR-mediated ablation of mutant KRAS can terminate tumor progression contingent on the concomitant inactivation of STAT3. STAT3 inactivation is needed to ensure that KRAS-ablated tumor cells lose their malignant identity. Mechanistically, the combined loss of mutant KRAS and STAT3 disrupts a core transcriptional program of cancer cells critical to oncogenic competence. This in turn impairs tumor growth in mice and enhances immune rejection, leading to tumor clearance. We propose that the STAT3 transcriptional program operating in cancer cells enforces their malignant identity, rather than providing classical features of transformation, and shapes cancer persistence following KRAS inactivation. Our findings establish STAT3 as a critical enforcer of oncogenic identity in KRAS-ablated tumors, revealing a key vulnerability that could be exploited for combination therapies.
Significance: The limited clinical success of KRAS inhibitors points to the need to identify means by which tumor cells maintain stemness and immune evasion. We make an unprecedented finding that the STAT3 transcription factor can sustain tumorigenicity of pancreatic cancer cells following depletion of the KRAS oncogenic driver. The results have important implications for successful therapeutic intervention.

DOI: https://doi.org/10.1101/2025.02.21.639533
Nature. 2025 Mar 12.

MYC ecDNA promotes intratumour heterogeneity and plasticity in PDAC.

Elena Fiorini, Antonia Malinova, Daniel Schreyer, Davide Pasini, Michele Bevere, Giorgia Alessio, Diego Rosa, Sabrina D'Agosto, Luca Azzolin, Salvatore Milite, Silvia Andreani, Francesca Lupo, Lisa Veghini, Sonia Grimaldi, Serena Pedron, Monica Castellucci, Craig Nourse, Roberto Salvia, Giuseppe Malleo, Andrea Ruzzenente, Alfredo Guglielmi, Michele Milella, Rita T Lawlor, Claudio Luchini, Antonio Agostini, Carmine Carbone, Christian Pilarsky, Andrea Sottoriva, Aldo Scarpa, David A Tuveson, Peter Bailey, Vincenzo Corbo.

Intratumour heterogeneity and phenotypic plasticity drive tumour progression and therapy resistance1,2. Oncogene dosage variation contributes to cell-state transitions and phenotypic heterogeneity3, thereby providing a substrate for somatic evolution. Nonetheless, the genetic mechanisms underlying phenotypic heterogeneity are still poorly understood. Here we show that extrachromosomal DNA (ecDNA) is a major source of high-level focal amplification in key oncogenes and a major contributor of MYC heterogeneity in pancreatic ductal adenocarcinoma (PDAC). We demonstrate that ecDNAs drive varying levels of MYC dosage, depending on their regulatory landscape, enabling cancer cells to rapidly and reversibly adapt to microenvironmental changes. In the absence of selective pressure, a high ecDNA copy number imposes a substantial fitness cost on PDAC cells. We also show that MYC dosage affects cell morphology and dependence of cancer cells on stromal niche factors. Our work provides a detailed analysis of ecDNAs in PDAC and describes a new genetic mechanism driving MYC heterogeneity in PDAC.

DOI: https://doi.org/10.1038/s41586-025-08721-9
Elife. 2025 Mar 11. pii: RP96925. [Epub ahead of print]13

Propionyl-CoA carboxylase subunit B regulates anti-tumor T cells in a pancreatic cancer mouse model.

Han V Han, Richard Efem, Barbara Rosati, Kevin Lu, Sara Maimouni, Ya-Ping Jiang, Valeria Montoya, Ando Van Der Velden, Wei-Xing Zong, Richard Z Lin.

  Most human pancreatic ductal adenocarcinoma (PDAC) are not infiltrated with cytotoxic T cells and are highly resistant to immunotherapy. Over 90% of PDAC have oncogenic KRAS mutations, and phosphoinositide 3-kinases (PI3Ks) are direct effectors of KRAS. Our previous study demonstrated that ablation of Pik3ca in KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cells induced host T cells to infiltrate and completely eliminate the tumors in a syngeneic orthotopic implantation mouse model. Now, we show that implantation of Pik3ca-/- KPC (named αKO) cancer cells induces clonal enrichment of cytotoxic T cells infiltrating the pancreatic tumors. To identify potential molecules that can regulate the activity of these anti-tumor T cells, we conducted an in vivo genome-wide gene-deletion screen using αKO cells implanted in the mouse pancreas. The result shows that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (named p-αKO) leads to immune evasion, tumor progression, and death of host mice. Surprisingly, p-αKO tumors are still infiltrated with clonally enriched CD8+ T cells but they are inactive against tumor cells. However, blockade of PD-L1/PD1 interaction reactivated these clonally enriched T cells infiltrating p-αKO tumors, leading to slower tumor progression and improve survival of host mice. These results indicate that Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers and this understanding may lead to improvement in immunotherapy for this difficult-to-treat cancer.

Keywords:  CD8 T cells; KPC cells; PD1; cancer biology; mouse; pancreatic cancer

DOI:  https://doi.org/10.7554/eLife.96925
Cell Chem Biol. 2025 Feb 28. pii: S2451-9456(25)00035-2. [Epub ahead of print]

Mechanisms and functions of lysosomal lipid homeostasis.

Michael Ebner, Florian Fröhlich, Volker Haucke.

  Lysosomes are the central degradative organelle of mammalian cells and have emerged as major intersections of cellular metabolite flux. Macromolecules derived from dietary and intracellular sources are delivered to the acidic lysosomal lumen where they are subjected to degradation by acid hydrolases. Lipids derived from lipoproteins, autophagy cargo, or autophagosomal membranes themselves constitute major lysosomal substrates. Dysregulation of lysosomal lipid processing, defective export of lipid catabolites, and lysosomal membrane permeabilization underly diseases ranging from neurodegeneration to metabolic syndromes and lysosomal storage disorders. Mammalian cells are equipped with sophisticated homeostatic control mechanisms that protect the lysosomal limiting membrane from excessive damage, prevent the spillage of luminal hydrolases into the cytoplasm, and preserve the lysosomal membrane composition in the face of constant fusion with heterotypic organelles such as endosomes and autophagosomes. In this review we discuss the molecular mechanisms that govern lysosomal lipid homeostasis and, thereby, lysosome function in health and disease.

Keywords:  contact sites; lipids; lysosomes; membrane homeostasis; phosphoinositides; signalling

DOI:  https://doi.org/10.1016/j.chembiol.2025.02.003
Nat Commun. 2025 Mar 07. 16(1): 2278

CYP51A1 drives resistance to pH-dependent cell death in pancreatic cancer.

Fangquan Chen, Hu Tang, Changfeng Li, Rui Kang, Daolin Tang, Jiao Liu.

Disrupted pH homeostasis can precipitate cell death and represents a viable therapeutic target in oncological interventions. Here, we utilize mass spectrometry-based drug analysis, transcriptomic screens, and lipid metabolomics to explore the metabolic mechanisms underlying pH-dependent cell death. We reveal CYP51A1, a gene involved in cholesterol synthesis, as a key suppressor of alkalization-induced cell death in pancreatic cancer cells. Inducing intracellular alkalization by the small molecule JTC801 leads to a decrease in endoplasmic reticulum cholesterol levels, subsequently activating SREBF2, a transcription factor responsible for controlling the expression of genes involved in cholesterol biosynthesis. Specifically, SREBF2-driven upregulation of CYP51A1 prevents cholesterol accumulation within lysosomes, leading to TMEM175-dependent lysosomal proton efflux, ultimately resulting in the inhibition of cell death. In animal models, including xenografts, syngeneic orthotopic, and patient-derived models, the genetic or pharmacological inhibition of CYP51A1 enhances the effectiveness of JTC801 in suppressing pancreatic tumors. These findings demonstrate a role of the CYP51A1-dependent lysosomal pathway in inhibiting alkalization-induced cell death and highlight its potential as a targetable vulnerability in pancreatic cancer.

DOI: https://doi.org/10.1038/s41467-025-57583-2
Cancer Res. 2025 Mar 11.

The Biology in the Pattern: Metastatic organotropism and clinical outcome depend on DNA damage response and immune interactions in pancreatic cancer.

Aristeidis Papargyriou, Maximilian Reichert.

Metastatic cancer remains a leading cause of cancer-related mortality, with a 5-year survival rate of just 8% for pancreatic ductal adenocarcinoma (PDAC). Among patients with metastatic PDAC, those with liver metastases experience significantly worse outcomes compared to the rare cases of isolated lung metastases. Recent findings by Link and colleagues reveal that these distinct metastatic patterns reflect underlying biological differences beyond established molecular subtypes. Specifically, the authors curated a primary organotropism (pORG) gene signature that is enriched in the liver cohorts. In detail they found that high-pORG/liver-avid tumors are characterized by high replication stress, enriched DNA repair pathways, and an immunosuppressive microenvironment, whereas low-pORG/lung-avid tumors display stronger immune infiltration, higher T cell density and reduce the richness of TCR repertoire, and better survival outcomes. These insights suggest that the clinical pattern of metastasis provides meaningful information about tumor biology and prognosis, complementing current subtype classifications in PDAC.

DOI: https://doi.org/10.1158/0008-5472.CAN-25-1085
EMBO Mol Med. 2025 Mar 07.

Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors.

Xun Zhou, Eva Berenger, Yong Shi, Vera Shirokova, Elena Kochetkova, Tina Becirovic, Boxi Zhang, Vitaliy O Kaminskyy, Yashar Esmaeilian, Kayoko Hosaka, Cecilia Lindskog, Per Hydbring, Simon Ekman, Yihai Cao, Maria Genander, Marcin Iwanicki, Erik Norberg, Helin Vakifahmetoglu-Norberg.

  Tumors often recapitulate programs to acquire invasive and dissemination abilities, during which pro-metastatic proteins are distinctively stabilized in cancer cells to drive further progression. Whether failed protein degradation affects the metastatic programs of cancer remains unknown. Here, we show that the human cancer cell-specific knockout (KO) of LAMP-2A, a limiting protein for chaperone-mediated autophagy (CMA), promotes the aggressiveness of mesenchymal tumors. Deficient CMA resulted in widespread tumor cell dissemination, invasion into the vasculature and cancer metastasis. In clinical samples, metastatic lesions showed suppressed LAMP-2A expression compared to primary tumors from the same cancer patients. Mechanistically, while stimulating TGFβ signaling dampens LAMP-2A levels, genetic suppression of CMA aggravated TGFβ signaling in cancer cells and tumors. Conversely, pharmacological inhibition of TGFβ signaling repressed the growth of LAMP-2A KO-driven tumors. Furthermore, we found that multiple EMT-driving proteins, such as TGFβR2, are degraded by CMA. Our study demonstrates that the tumor suppressive function of CMA involves negative regulation of TGFβ-driven EMT and uncovers a mechanistic link between CMA and a major feature of metastatic invasiveness.

Keywords:  Cancer; Chaperone-mediated Autophagy; EMT; Metastasis; TGFβ

DOI:  https://doi.org/10.1038/s44321-025-00210-w
Nat Rev Genet. 2025 Mar 10.

Integrating model systems and genomic insights to decipher mechanisms of cancer metastasis.

Michelle M Leung, Charles Swanton, Nicholas McGranahan.

Deciphering metastatic processes is crucial for understanding cancer progression and potential treatment options. Genetic studies of model systems engineered to mimic metastatic disease, including organoids, genetically engineered mice and human cell lines, have had an important role in shaping our understanding of the metastatic cascade and how it can be manipulated. More recently, advances in high-throughput sequencing have enabled human metastases to be studied at single-cell and single-nucleotide resolution, providing insights into metastatic evolution and phenotypes of both cancer cells and immune cells. However, human tissue studies are often correlative and descriptive, whereas experimental models are reductionistic by nature, meaning that individual results should be interpreted with caution. Crucially, these seemingly disparate branches of metastasis research can and should complement each other to strengthen and validate findings. Here we explore the synergies between model systems and sequencing studies and outline key areas that must be explored to improve our understanding of the metastatic process.

DOI: https://doi.org/10.1038/s41576-025-00825-2
Cell Res. 2025 Mar 07.

Oncogenic RAS induces a distinctive form of non-canonical autophagy mediated by the P38-ULK1-PI4KB axis.

Xiaojuan Wang, Shulin Li, Shiyin Lin, Yaping Han, Tong Zhan, Zhiying Huang, Juanjuan Wang, Ying Li, Haiteng Deng, Min Zhang, Du Feng, Liang Ge.

Cancer cells with RAS mutations exhibit enhanced autophagy, essential for their proliferation and survival, making it a potential target for therapeutic intervention. However, the regulatory differences between RAS-induced autophagy and physiological autophagy remain poorly understood, complicating the development of cancer-specific anti-autophagy treatments. In this study, we identified a form of non-canonical autophagy induced by oncogenic KRAS expression, termed RAS-induced non-canonical autophagy via ATG8ylation (RINCAA). RINCAA involves distinct autophagic factors compared to those in starvation-induced autophagy and incorporates non-autophagic components, resulting in the formation of non-canonical autophagosomes with multivesicular/multilaminar structures labeled by ATG8 family proteins (e.g., LC3 and GABARAP). We have designated these structures as RAS-induced multivesicular/multilaminar bodies of ATG8ylation (RIMMBA). A notable feature of RINCAA is the substitution of the class III PI3K in canonical autophagy with PI4KB in RINCAA. We identified a regulatory P38-ULK1-PI4KB-WIPI2 signaling cascade governing this process, where ULK1 triggers PI4KB phosphorylation at S256 and T263, initiating PI4P production, ATG8ylation, and non-canonical autophagy. Importantly, elevated PI4KB phosphorylation at S256 and T263 was observed in RAS-mutated cancer cells and colorectal cancer specimens. Inhibition of PI4KB S256 and T263 phosphorylation led to a reduction in RINCAA activity and tumor growth in both xenograft and KPC models of pancreatic cancer, suggesting that targeting ULK1-mediated PI4KB phosphorylation could represent a promising therapeutic strategy for RAS-mutated cancers.

DOI: https://doi.org/10.1038/s41422-025-01085-9
Cell. 2025 Mar 10. pii: S0092-8674(25)00197-7. [Epub ahead of print]

Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits.

Loϊc Binan, Aiping Jiang, Serwah A Danquah, Vera Valakh, Brooke Simonton, Jon Bezney, Robert T Manguso, Kathleen B Yates, Ralda Nehme, Brian Cleary, Samouil L Farhi.

  Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

Keywords:  multimodal screening; pooled CRISPR screen; pooled optical profiling; single-cell perturbations; spatial transcriptomics

DOI:  https://doi.org/10.1016/j.cell.2025.02.012
JCO Oncol Pract. 2025 Mar 13. OP2500108

Reply to: Overcoming Barriers to Cancer-Associated Cachexia Diagnosis and Management.

Marcus D Goncalves, Tobias Janowitz.

DOI: https://doi.org/10.1200/OP-25-00108
bioRxiv. 2025 Feb 24. pii: 2025.02.20.638910. [Epub ahead of print]

Autophagy Suppresses CCL2 to Preserve Appetite and Prevent Lethal Cachexia.

Maria Ibrahim, Maria Gomez-Jenkins, Adina Scheinfeld, Zhengqiao Zhao, Eduardo Cararo Lopes, Akshada Sawant, Zhixian Hu, Aditya Dharani, Michael Sun, Sarah Siddiqui, Emily T Mirek, Johan Abram-Saliba, Edmund C Lattime, Xiaoyang Su, Tobias Janowitz, Marcus D Goncalves, Steven M Dunn, Yuri Pritykin, Tracy G Anthony, Joshua D Rabinowitz, Eileen White.

Macroautophagy (autophagy hereafter) captures intracellular components and delivers them to lysosomes for degradation and recycling 1 . In adult mice, autophagy sustains metabolism to prevent wasting by cachexia and to survive fasting, and also suppresses inflammation, liver steatosis, neurodegeneration, and lethality 2,3 . Defects in autophagy contribute to metabolic, inflammatory and degenerative diseases, however, the specific mechanisms involved were unclear 4 . Here we profiled metabolism and inflammation in adult mice with conditional, whole-body deficiency in an essential autophagy gene and found that autophagy deficiency altered fuel usage, and reduced ambulatory activity, energy expenditure, and food intake, and elevated circulating GDF15, CXCL10, and CCL2. While deletion of Gdf15 or Cxcl10 provided no or mild benefit, deletion of Ccl2 restored food intake, suppressed cachexia and rescued lethality of autophagy-deficient mice. To test if appetite suppression by CCL2 was responsible for lethal cachexia we performed single nucleus RNA sequencing of the hypothalamus, the center of appetite control in the brain. Notably, we found that autophagy deficiency was specifically toxic to PMCH and HCRT neurons that produce orexigenic neuropeptides that promote food intake, which was rescued by deficiency in CCL2. Finally, the restoration of food intake via leptin deficiency prevented lethal cachexia in autophagy-deficient mice. Our findings demonstrate a novel mechanism where autophagy prevents induction of a cachexia factor, CCL2, which damages neurons that maintain appetite, the destruction of which may be central to degenerative wasting conditions.
Key points of paper: 1) Autophagy-deficient mice have reduced food intake, systemic inflammation, and cachexia2) CCL2, but not GDF15 or CXCL10, induces lethal cachexia caused by autophagy defect3) Autophagy-deficient mice have CCL2-dependent destruction of appetite-promoting neurons in the hypothalamus4) Leptin deficiency restores appetite and rescues lethal cachexia in autophagy-deficient mice5) Autophagy-deficient mice die from cachexia mediated by appetite loss6) Degenerative conditions due to impaired autophagy are caused by the inflammatory response to the damage7) Targeting CCL2 may be a viable approach to prevent degenerative wasting disorders.

DOI: https://doi.org/10.1101/2025.02.20.638910
Biophys Rev (Melville). 2025 Mar;6(1): 011401

Toward universal models for collective interactions in biomolecular condensates.

Edoardo Milanetti, Karan K H Manjunatha, GianCarlo Ruocco, Amos Maritan, Monika Fuxreiter.

A wide range of higher-order structures, including dense, liquid-like assemblies, serve as key components of cellular matter. The molecular language of how protein sequences encode the formation and biophysical properties of biomolecular condensates, however, is not completely understood. Recent notion on the scale invariance of the cluster sizes below the critical concentration for phase separation suggests a universal mechanism, which can operate from oligomers to non-stoichiometric assemblies. Here, we propose a model for collective interactions in condensates, based on context-dependent variable interactions. We provide the mathematical formalism, which is capable of describing growing dynamic clusters as well as changes in their material properties. Furthermore, we discuss the consequences of the model to maximize sensitivity to the environmental signals and to increase correlation lengths.

DOI: https://doi.org/10.1063/5.0244227
Nat Methods. 2025 Mar;22(3): 453

Structural biology at the plasma membrane.

Rita Strack.

DOI: https://doi.org/10.1038/s41592-025-02640-3
Methods Mol Biol. 2025 ;2906 45-55

Selective Sorting of Senescent Cell Subpopulations Compatible with Downstream Genomics Applications.

Athanasia Mizi, Markos Tsitsianopoulos, Argyris Papantonis.

  The dynamic character of senescence renders detection and selection of senescent cells challenging. One key feature of senescence is the alteration of chromatin features, and many methods for studying chromatin require only mild fixation of cells. The recent development of GLF16 compound allows for the selection of senescent cells in a population via fluorescence-activated cell sorting. Here, we detail two versions of a modified protocol that uses GLF16 to selectively sort senescent cells so as to be used in downstream genomics applications that require special fixation and permeabilization conditions. As proof of principle, we sort a subpopulation of senescent fetal lung fibroblasts and subject it to standard transcriptomics analysis, while the same procedure could potentially be coupled to other assays like CUT&RUN, CUT&Tag, ATAC-seq, or Hi-C/Micro-C.

Keywords:  Chromatin; Epigenomics; FACS; GLF16; Primary cells; RNA-seq; Senescence

DOI:  https://doi.org/10.1007/978-1-0716-4426-3_3
Cancer Discov. 2025 Mar 07.

T-cell dependency of tumor regressions and complete responses with RAS(ON) multi-selective inhibition in preclinical models of PDAC.

Margo I Orlen, William P Vostrejs, Rina Sor, Jayne C McDevitt, Samantha B Kemp, Il-Kyu Kim, Adam B Kramer, Nataliya Tovbis Shifrin, Nune Markosyan, Cynthia Clendenin, Mallika Singh, Elsa Quintana, Marie Menard, Robert H Vonderheide, Ben Z Stanger.

Activating mutations in KRAS drive tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), promoting tumor cell proliferation and contributing to an immunosuppressive tumor microenvironment (TME) rendering PDAC tumors insensitive to immunotherapy. RAS(ON) multi-selective inhibitors, such as daraxonrasib (RMC-6236) and RMC-7977, target the active state of RAS, with potent anti-tumor activity in PDAC murine models. Here, we report that RAS(ON) multi-selective inhibition led to rapid and profound PDAC regressions in immunocompetent mice, decreasing myeloid cells and increasing T cells and macrophages in the TME. The depth and duration of tumor regression depended on T cells and conventional dendritic cells. Moreover, the combination of RAS(ON) multi-selective inhibitors with immunotherapy conferred deeper and more durable tumor regressions, including complete responses not seen with either treatment alone. In summary, concurrent inhibition of mutant and wild-type RAS is active in concert with T cell immunotherapy, revealing RAS(ON) multi-selective inhibitors as a potential therapeutic immuno-sensitizing strategy in PDAC.

DOI: https://doi.org/10.1158/2159-8290.CD-24-1475
Commun Biol. 2025 Mar 11. 8(1): 411

Cholesterol inhibits assembly and oncogenic activation of the EphA2 receptor.

Ryan J Schuck, Alyssa E Ward, Amita R Sahoo, Jennifer A Rybak, Robert J Pyron, Thomas N Trybala, Timothy B Simmons, Joshua A Baccile, Ioannis Sgouralis, Matthias Buck, Rajan Lamichhane, Francisco N Barrera.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, we have a poor understanding regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments reveal that a reduction of plasma membrane cholesterol strongly promotes EphA2 self-assembly. Indeed, low cholesterol levels cause a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines reveal that low cholesterol increased phospho-serine levels in EphA2, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but it also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

DOI: https://doi.org/10.1038/s42003-025-07786-6
Trends Cell Biol. 2025 Mar 10. pii: S0962-8924(25)00041-8. [Epub ahead of print]

Cellular homeostatic responses to lysosomal damage.

Jingyue Jia, Suttinee Poolsup, Jay E Salinas.

  Lysosomes are essential membrane-bound organelles that control cellular homeostasis by integrating intracellular functions with external signals. Their critical roles make lysosomal membranes vulnerable to rupture under various stressors, leading to cellular dysfunction. However, the mechanisms by which cells respond to lysosomal damage have only recently begun to be explored. In this review, we summarize the cellular mechanisms activated by lysosomal damage, emphasizing those that restore lysosomal integrity and sustain homeostasis, including recognition, repair, removal, replacement, and remodeling. Drawing on our expertise, we provide an in-depth focus on the remodeling process involved in these responses, including metabolic signaling and stress granule formation. Finally, we discuss the implications of lysosomal damage in human diseases, underscoring potential therapeutic strategies to preserve lysosomal function and alleviate related disorders.

Keywords:  damaged lysosomes; recognition; remodeling; removal; repair; replacement

DOI:  https://doi.org/10.1016/j.tcb.2025.02.007
Annu Rev Biochem. 2025 Mar 11.

Lipid Dynamics at Membrane Contact Sites.

Karin M Reinisch, Pietro De Camilli, Thomas J Melia.

In eukaryotes, lipid building blocks for cellular membranes are made largely in the endoplasmic reticulum and then redistributed to other organelles. Lipids are transported between organelles by vesicular trafficking or else by proteins located primarily at sites where different organelles are closely apposed. Here we discuss transport at organelle contact sites mediated by shuttle-like proteins that carry single lipids between membranes to fine-tune their composition and by the more recently discovered bridge-like proteins that tether two organelles and provide a path for bulk lipid movement. Protein-mediated lipid transport is assisted by integral membrane proteins that have roles in (a) lowering the energy barrier for lipid transfer between the membrane and the lipid transfer protein, a key parameter determining the transfer rate, and (b) scrambling lipids to counteract the bilayer asymmetry that would result from such transfer. Advances in this field are shedding new light on a variety of physiological mechanisms.

DOI: https://doi.org/10.1146/annurev-biochem-083024-122821
J Cachexia Sarcopenia Muscle. 2025 Apr;16(2): e13756

Oncological and Survival Endpoints in Cancer Cachexia Clinical Trials: Systematic Review 6 of the Cachexia Endpoints Series.

Cancer Cachexia Endpoints Working Group

   BACKGROUND: In patients receiving anti-cancer treatment, cachexia results in poorer oncological outcomes. However, there is limited understanding and no systematic review of oncological endpoints in cancer cachexia (CC) trials. This review examines oncological endpoints in CC clinical trials.
METHODS: An electronic literature search of MEDLINE, Embase and Cochrane databases (1990-2023) was performed. Eligibility criteria comprised participants ≥ 18 years old; controlled design; ≥ 40 participants; and a cachexia intervention for > 14 days. Trials reporting at least one oncological endpoint were selected for analysis. Data extraction was performed using Covidence and followed PRISMA guidelines and the review was registered (PROSPERO CRD42022276710).
RESULTS: Fifty-seven trials were eligible, totalling 9743 patients (median: 107, IQR: 173). Twenty-six (46%) trials focussed on a single tumour site: eight in lung, six in pancreatic, six in head and neck and six in GI cancers. Forty-two (74%) studies included patients with Stage III/IV disease, and 41 (70%) included patients receiving palliative anti-cancer treatment. Ten studies (18%) involved patients on curative treatment. Twenty-eight (49%) studies used pharmacological interventions, 29 (50%) used oral nutrition, and two (4%) used enteral or parenteral nutrition. Reported oncological endpoints included overall survival (OS, n = 46 trials), progression-free survival (PFS, n = 7), duration of response (DR, n = 1), response rate (RR, n = 9), completion of treatment (TC, n = 11) and toxicity/adverse events (AE, n = 42). Median OS differed widely from 60 to 3468 days. Of the 46 studies, only three reported a significant positive effect on survival. Seven trials showed a difference in AE, four in TC, one in PFS and one in RR. Reported significances were unreliable due to missing adjustments for extensive multiple testing. Only three of the six trials using OS as the primary endpoint reported pre-trial sample size calculations, but only one recruited the planned number of patients.
CONCLUSION: In CC trials, oncological endpoints were mostly secondary and only few significant findings have been reported. Due to heterogeneity in oncological settings, nutritional and metabolic status and interventions, firm conclusions about CC treatment are not possible. OS and AE are relevant endpoints, but future trials targeting clinically meaningful hazard ratios will required more homogeneous patient cohorts, adequate pre-trial power analyses and adherence to statistical testing standards.

Keywords:  adverse events; cachexia; cancer; clinical trials; survival

DOI:  https://doi.org/10.1002/jcsm.13756
Nat Metab. 2025 Mar 10.

A functional single-cell metabolic survey identifies Elovl1 as a target to enhance CD8+ T cell fitness in solid tumours.

Samantha Pretto, Qian Yu, Pierre Bourdely, Sarah Trusso Cafarello, Heleen H Van Acker, Joren Verelst, Elena Richiardone, Lotte Vanheer, Amir Roshanzadeh, Franziska Schneppenheim, Charlotte Cresens, Maria Livia Sassano, Jonas Dehairs, Martin Carion, Shehab Ismail, Patrizia Agostinis, Susana Rocha, Tobias Bald, Johan Swinnen, Cyril Corbet, Sophia Y Lunt, Bernard Thienpont, Mario Di Matteo, Massimiliano Mazzone.

Reprogramming T cell metabolism can improve intratumoural fitness. By performing a CRISPR/Cas9 metabolic survey in CD8+ T cells, we identified 83 targets and we applied single-cell RNA sequencing to disclose transcriptome changes associated with each metabolic perturbation in the context of pancreatic cancer. This revealed elongation of very long-chain fatty acids protein 1 (Elovl1) as a metabolic target to sustain effector functions and memory phenotypes in CD8+ T cells. Accordingly, Elovl1 inactivation in adoptively transferred T cells combined with anti-PD-1 showed therapeutic efficacy in resistant pancreatic and melanoma tumours. The accumulation of saturated long-chain fatty acids in Elovl1-deficient T cells destabilized INSIG1, leading to SREBP2 activation, increased plasma membrane cholesterol and stronger T cell receptor signalling. Elovl1-deficient T cells increased mitochondrial fitness and fatty acid oxidation, thus withstanding the metabolic stress imposed by the tumour microenvironment. Finally, ELOVL1 in CD8+ T cells correlated with anti-PD-1 response in patients with melanoma. Altogether, Elovl1 targeting synergizes with anti-PD-1 to promote effective T cell responses.

DOI: https://doi.org/10.1038/s42255-025-01233-w
Mol Oncol. 2025 Mar 12.

Highly multiplexed digital PCR assay for simultaneous quantification of variant allele frequencies and copy number alterations of KRAS and GNAS in pancreatic cancer precursors.

Junko Tanaka, Tatsuo Nakagawa, Yusuke Ono, Yoshio Kamura, Takeshi Ishida, Hidemasa Kawabata, Kenji Takahashi, Hiroki Sato, Andrew S Liss, Yusuke Mizukami, Takahide Yokoi.

  Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursor lesions. Detecting these precursors and monitoring their progression are crucial for early PDAC diagnosis. Digital PCR (dPCR) is a highly sensitive nucleic acid quantification technique and offers a cost-effective option for patient follow-up. However, the clinical utility of conventional dPCR is restricted by multiplexing constraints, particularly due to the challenge of simultaneously quantifying multiple mutations and amplifications. In this study, we applied highly multiplexed dPCR and melting curve analysis to simultaneously measure single nucleotide mutations and amplifications of KRAS and GNAS. The developed 14-plex assay included both wild-type and mutant KRAS, a common driver gene in both PanIN and IPMN, and GNAS, which is specifically mutated in IPMN, along with RPP30, a reference gene for copy number alterations (CNAs). This multiplex dPCR method detected all target mutations with a limit of detection below 0.2% while quantifying CNAs. Additionally, the assay accurately quantified variant allele frequencies in liquid biopsy and tissue samples from both pancreatic neoplasm precursor and PDAC patients, indicating its potential for use in comprehensive patient follow-up.

Keywords:  copy number alterations; digital PCR; melting curve analysis; multiplex; pancreatic cancer; variant allele frequency

DOI:  https://doi.org/10.1002/1878-0261.70011
bioRxiv. 2025 Feb 27. pii: 2025.02.23.639532. [Epub ahead of print]

LivecellX: A Deep-learning-based, Single-Cell Object-Oriented Framework for Quantitative Analysis in Live-Cell Imaging.

Ke Ni, Gaohan Yu, Zhiqian Zheng, Yong Lu, Dante Poe, Shiman Zhou, Weikang Wang, Jianhua Xing.

Analyzing single-cell dynamics is crucial for understanding developmental biology, cancer biology, and other complex biological processes. This analysis depends on accurately detecting and tracking individual cells across both spatial and temporal scales, with live-cell imaging serving as a key tool. However, extracting reliable dynamic information from live-cell imaging data remains a significant challenge. The task involves constructing long single-cell trajectories and representing dynamic behaviors through multi-dimensional features. Despite recent advances in deep learning-driven segmentation, pre-trained and fine-tuned models often fail to achieve perfect segmentation in live-cell imaging scenarios. The extended duration of live-cell imaging further amplifies segmentation errors, complicating the maintenance of precise and consistent segmentation. To address these challenges, we introduce LivecellX, a comprehensive framework for live-cell imaging data analysis. LivecellX provides an integrated solution for segmentation, tracking, and dynamic analysis by adopting a single-cell, object-oriented architecture. This architecture not only enhances segmentation and tracking accuracy but also simplifies the extraction of trajectory dynamics, making it easier for users to analyze complex biological processes. Central to Live-cellX is the Correct Segmentation Network (CSN), a context-aware, multi-scale machine learning architecture designed to correct segmentation inaccuracies. To effectively apply CSN to large datasets, we developed trajectory-level algorithms that systematically address specific segmentation issues. To ensure robustness and user accessibility, we developed an asynchronous graphical user interface (GUI) based on Napari, allowing seamless interaction with the data both interactively and programmatically at any stage of the analysis. By combining automated methods with interactive correction capabilities, Live-cellX provides a comprehensive solution for precise, large-scale live-cell imaging analysis, empowering researchers to obtain more accurate biological insights.

DOI: https://doi.org/10.1101/2025.02.23.639532
Mol Cancer. 2025 Mar 13. 24(1): 76

ADT-1004: a first-in-class, oral pan-RAS inhibitor with robust antitumor activity in preclinical models of pancreatic ductal adenocarcinoma.

Dhana Sekhar Reddy Bandi, Ganji Purnachandra Nagaraju, Sujith Sarvesh, Julienne L Carstens, Jeremy B Foote, Emily C Graff, Yu-Hua D Fang, Adam B Keeton, Xi Chen, Jacob Valiyaveettil, Kristy L Berry, Sejong Bae, Mehmet Akce, Greg Gorman, Karina J Yoon, Upender Manne, Michael R Boyd, Donald J Buchsbaum, Asfar S Azmi, Yulia Y Maxuitenko, Gary A Piazza, Bassel F El-Rayes.

   BACKGROUND: Oncogenic KRAS mutations occur in nearly, 90% of patients with pancreatic ductal adenocarcinoma (PDAC). Targeting KRAS has been complicated by mutational heterogeneity and rapid resistance. We developed a novel pan-RAS inhibitor, ADT-1004 (an oral prodrug of ADT-007) and evaluated antitumor activity in murine and human PDAC models.
METHODOLOGY: Murine PDAC cells with KRASG12D mutation (KPC-luc or 2838c3-luc) were orthotopically implanted into the pancreas of C57BL/6J mice, and four PDX PDAC tumors with KRAS mutations were implanted subcutaneously in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental and resistant MIA PaCa-2 PDAC cells (KRASG12C mutation) were implanted subcutaneously. Subcutaneously implanted RASWT BxPC-3 cells were used to assess the selectivity of ADT-1004.
RESULTS: ADT-1004 potently blocked tumor growth and RAS activation in mouse PDAC models without discernable toxicity with target engagement and reduced activated RAS and ERK phosphorylation. In addition, ADT-1004 suppressed tumor growth in PDX PDAC models with KRASG12D, KRASG12V, KRASG12C, or KRASG13Q mutations and increased CD4+ and CD8+ T cells in the TME consistent with exhaustion and increased MHCII+ M1 macrophage and dendritic cells. ADT-1004 demonstrated superior efficacy over sotorasib and adagrasib in tumor models resistant to these KRASG12C inhibitors and MRTX1133 resistant KRASG12D mutant cells. As evidence of selectivity for tumors with mutant KRAS, ADT-1004 did not impact the growth of tumors from RASWT PDAC cells.
CONCLUSION/SIGNIFICANCE: ADT-1004 has strong antitumor activity in aggressive and clinically relevant PDAC models with unique selectivity to block RAS-mediated signaling in RAS mutant cells. As a pan-RAS inhibitor, ADT-1004 has broad activity and potential efficacy advantages over allele-specific KRAS inhibitors. These findings support clinical trials of ADT-1004 for KRAS mutant PDAC.

Keywords:  KRAS; Pancreatic ductal adenocarcinoma; RAS-driven malignancies; Tumor immune microenvironment; pan-RAS inhibitor

DOI:  https://doi.org/10.1186/s12943-025-02288-9
Semin Cancer Biol. 2025 Mar 07. pii: S1044-579X(25)00041-0. [Epub ahead of print]

Epithelial-mesenchymal Transition Promotes Metabolic Reprogramming to Suppress Ferroptosis.

Wenzheng Guo, Zhibing Duan, Jingjing Wu, Binhua P Zhou.

  Epithelial-mesenchymal transition (EMT) is a cellular de-differentiation process that provides cells with the increased plasticity and stem cell-like traits required during embryonic development, tissue remodeling, wound healing and metastasis. Morphologically, EMT confers tumor cells with fibroblast-like properties that lead to the rearrangement of cytoskeleton (loss of stiffness) and decrease of membrane rigidity by incorporating high level of poly-unsaturated fatty acids (PUFA) in their phospholipid membrane. Although large amounts of PUFA in membrane reduces rigidity and offers capabilities for tumor cells with the unbridled ability to stretch, bend and twist in metastasis, these PUFA are highly susceptible to lipid peroxidation, which leads to the breakdown of membrane integrity and, ultimately results in ferroptosis. To escape the ferroptotic risk, EMT also triggers the rewiring of metabolic program, particularly in lipid metabolism, to enforce the epigenetic regulation of EMT and mitigate the potential damages from ferroptosis. Thus, the interplay among EMT, lipid metabolism, and ferroptosis highlights a new layer of intricated regulation in cancer biology and metastasis. Here we summarize the latest findings and discuss these mutual interactions. Finally, we provide perspectives of how these interplays contribute to cellular plasticity and ferroptosis resistance in metastatic tumor cells that can be explored for innovative therapeutic interventions.

Keywords:  EMT; Ferroptosis; Lipid metabolism; Membrane plasticity; Metastasis

DOI:  https://doi.org/10.1016/j.semcancer.2025.02.013
J Cell Sci. 2025 Mar 13. pii: jcs.263693. [Epub ahead of print]

A dual-purification system to isolate mitochondrial subpopulations.

Corey N Cunningham, Jonathan G Van Vranken, Jakeline Larios, Katarina Heyden, Steven P Gygi, Jared Rutter.

  Mitochondria perform diverse functions, such as producing ATP through oxidative phosphorylation, synthesizing macromolecule precursors, maintaining redox balance, and many others. Given this diversity of functions, we and others have hypothesized that cells maintain specialized subpopulations of mitochondria. To begin addressing this hypothesis, we developed a new dual-purification system to isolate subpopulations of mitochondria for chemical and biochemical analyses. We used APEX2 proximity labeling such that mitochondria were biotinylated based on proximity to another organelle. All mitochondria were isolated by an elutable MitoTag-based affinity precipitation system. Biotinylated mitochondria were then purified using immobilized avidin. We used this system to compare the proteomes of endosome- and lipid droplet-associated mitochondria in U-2 OS cells, which demonstrated that these subpopulations were indistinguishable from one another but were distinct from the global mitochondria proteome. Our results suggest that this purification system could aid in describing subpopulations that contribute to intracellular mitochondrial heterogeneity, and that this heterogeneity might be more substantial than previously imagined.

Keywords:  Biochemistry; Mitochondria; Proximity Labeling; Purification

DOI:  https://doi.org/10.1242/jcs.263693
Autophagy. 2025 Mar 13.

The TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis: a novel regulatory mechanism for Lysophagy.

Na Yeon Park, Dong-Hyung Cho.

  Lysophagy, the selective macroautophagic/autophagic clearance of damaged lysosomes, is a critical mechanism for maintaining cellular homeostasis. Our recent study identified a novel regulatory axis involving TBK1, SCFFBXO3, TMEM192, and TAX1BP1 that orchestrates lysophagic flux following lysosomal damage. We demonstrated that TBK1-dependent phosphorylation of FBXO3 facilitates its interaction with TMEM192, promoting its ubiquitination and subsequent recognition by the autophagy receptor TAX1BP1. Perturbing this pathway significantly reduces lysophagic flux and results in accumulation of damaged lysosomes. These findings establish a previously unrecognized mechanistic link between ubiquitination, receptor recruitment, and lysophagic degradation, broadening our understanding of lysosomal quality control.

Keywords:  FBXO3; TAX1BP1; TBK1; TMEM192; lysophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2025.2479669
Nat Cell Biol. 2025 Mar 10.

Measuring and manipulating mechanical forces during development.

Clémentine Villeneuve, Kaitlin P McCreery, Sara A Wickström.

Tissue deformations are a central feature of development, from early embryogenesis, growth and building the body plan to the establishment of functional organs. These deformations often result from active contractile forces generated by cells and cell collectives, and are mediated by changes in their mechanical properties. Mechanical forces drive the formation of functional organ architectures, but they also coordinate cell behaviour and fate transitions, ensuring robustness of development. Advances in microscopy, genetics and chemistry have enabled increasingly powerful tools for measuring, generating and perturbing mechanical forces. Here we discuss approaches to measure and manipulate mechanical forces with a focus on developmental processes, ranging from quantification of molecular interactions to mapping the mechanical properties of tissues. We focus on contemporary methods, and discuss the biological discoveries that these approaches have enabled. We conclude with an outlook to methodologies at the interface of physics, chemistry and biology to build an integrated understanding of tissue morphodynamics.

DOI: https://doi.org/10.1038/s41556-025-01632-x
Front Pharmacol. 2025 ;16 1519278

Muscle loss in cancer cachexia: what is the basis for nutritional support?

Jaline Faiad, Márcia Fábia Andrade, Gabriela de Castro, Joyce de Resende, Marina Coêlho, Giovana Aquino, Marilia Seelaender.

  Cancer cachexia (CC) is characterized by significant skeletal muscle wasting, and contributes to diminished quality of life, while being associated with poorer response to treatment and with reduced survival. Chronic inflammation plays a central role in driving CC progression, within a complex interplay favoring catabolism. Although cachexia cannot be fully reversed by conventional nutritional support, nutritional intervention shows promise for the prevention and treatment of the syndrome. Of special interest are nutrients with antioxidant and anti-inflammatory potential and those that activate pathways involved in muscle mass synthesis and/or in the inhibition of muscle wasting. Extensive research has been carried out on novel nutritional supplements' power to mitigate CC impact, while the mechanisms through which some nutrients or bioactive compounds exert beneficial effects on muscle mass are still not totally clear. Here, we discuss the most studied supplements and nutritional strategies for dealing with muscle loss in CC.

Keywords:  cancer cachexia; chronic inflammation; muscle wasting; nutritional supplementation; protein synthesis

DOI:  https://doi.org/10.3389/fphar.2025.1519278
Nat Rev Cancer. 2025 Mar 07.

When tumour cells get excited.

Daniela Senft.

DOI: https://doi.org/10.1038/s41568-025-00808-9
Methods Mol Biol. 2025 ;2906 57-71

Detection and Characterization of Senescent Cells with Flow Cytometry.

Panagiotis Bakouros, Ariadni Archavli, Ioannis V Kostopoulos, Evangelos Terpos, Efstathios Kastritis, Meletios A Dimopoulos, Ourania Tsitsilonis, Pantelis Rousakis.

  Cellular senescence, a stable cell cycle arrest induced by various stressors or DNA damage, plays a critical role in various aspects of human physiology, including embryonic development, wound healing, and age-related diseases like cancer. Despite its discovery nearly 50 years ago, a universal marker for senescent cells remains elusive. Current identification methods, such as the detection of senescence-associated β-galactosidase activity (SA-β-Gal), immunohistochemistry, and qPCR, have limitations, including subjective assessment of the results and complex prerequisites. Flow cytometry (FC) has emerged as a reliable technique for detecting senescent phenotypes at single-cell level, traditionally based on proteins like Ki-67 and p16INK4a and, more recently, via the detection of lipofuscin with a novel Sudan Black-B analog, namely, GLF16. Here, we present detailed protocols using GLF16 in FC, to detect senescent cells and specifically, of the human myeloma cell lines NCI-H929 and L363. In the protocols, we describe the induction of cellular senescence using H2O2, the staining process, and the gating strategy to accurately identify senescent cells. Additionally, we provide a comprehensive method for detecting senescent cells in bone marrow clinical samples from patients with multiple myeloma, following red blood cell lysis and leukocyte staining. These techniques broaden the potential of FC applications in the field of senescence and could be utilized as surrogate tests in clinical diagnostics.

Keywords:  Flow cytometry; GLF16; Multiple myeloma; Oxidative stress; Plasma cells; Senescence

DOI:  https://doi.org/10.1007/978-1-0716-4426-3_4
Methods Mol Biol. 2025 ;2906 73-81

Detecting Senescence in T Cells by Flow Cytometry Using the SA-β-Galactosidase Assay.

Luca Pangrazzi, Ines Martic, Maria Cavinato, Birgit Weinberger.

  Cellular senescence is a biological process where somatic cells undergo irreversible growth arrest. One key marker of cellular senescence is senescence-associated β-galactosidase (SA-β-gal), an enzyme whose activity can be detected at pH 6 in senescent cells. Traditional methods for SA-β-gal detection often rely on the substrate X-Gal, which requires fixed cells and offers limited sensitivity for quantitative analysis. This study introduces a refined method using the SPiDER β-gal substrate from the Cellular Senescence Detection Kit (Dojindo), designed to improve cell permeability and enable quantification through flow cytometry in both live and fixed cells. We employed this novel protocol to detect SA-β-gal in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors of different ages. The results revealed a progressive increase in SA-β-gal+ CD8+ T cells with advancing age, suggesting that the accumulation of senescent immune cells is a feature of aging. To further validate this method, UVB-treated fibroblasts were analyzed, a well-established model for studying senescence. Overall, this method provides a powerful tool for studying cellular senescence across different cell types and experimental contexts, with broad applications for research on aging, immune function, and cancer biology.

Keywords:  Aging; Flow cytometry; Senescence; T cells; β-galactosidase

DOI:  https://doi.org/10.1007/978-1-0716-4426-3_5
J Am Chem Soc. 2025 Mar 11.

Chemical Probe-Enabled Lipid Droplet Proteomics.

Jing Xiang, Tao Li, Junzhe Zhang, Wenxian Wu, Guangyu Xu, Jiaqian Yan, Hao Wang, Suyuan Chen, Shao Q Yao, Miaomiao Wang, Fan Yi, Jigang Wang, Yusheng Xie.

Emerging evidence indicates that lipid droplets (LDs) play important roles in lipid metabolism, energy homeostasis, and cell stress management. Notably, dysregulation of LDs is tightly linked to numerous diseases, including lipodystrophies, cancer, obesity, atherosclerosis, and others. The pivotal physiological roles of LDs have led to an exploration of research in recent years. The functions of LDs are inherently connected to the composition of their proteome. Current methods for profiling LD proteins mostly utilize LD fractionation, including those based on proximity-based labeling techniques. Global profiling of the LD proteome in live cells without the isolation of LDs is still challenging. Herein, we disclose two small-molecule chemical probes, termed LDF and LDPL. Both LDF/LDPL are small in size and could freely and specifically migrate within the lipid context of LDs. Consequently, they were successfully used for live-cell fluorescence imaging of LDs and from animal tissues. We further showed that LDPL was capable of large-scale profiling of the LD proteome without the need of LD isolation. By using LDPL, 1584 high-confidence proteins, most of which could be annotated to prominent LD functions, were next identified. Importantly, further validation studies by using representative "hit" proteins revealed that CHMP6 and PRDX4 could act as the lipophagy receptor and lipolysis suppressor, respectively. Our results thus confirmed for the first time that LDPL is a powerful chemical tool for in situ profiling of LD proteomes. With the ability to provide a deeper understanding of LD proteomics from the native cellular environments, our newly developed strategy may be used in future to decipher the dynamics and molecular mechanism of LDs in various diseases.

DOI: https://doi.org/10.1021/jacs.5c01710
Langmuir. 2025 Mar 13.

Dynamics of Microsphere Inclusions within Biomimetic Membranes Reveal Membrane Heterogeneity.

Shefali Srivastava, Prerna Sharma.

Extracellular macromolecules and particles often bind and diffuse over cell membranes during transport processes like endocytosis, exocytosis, drug imbibition, etc. However, imaging the real-time dynamics of these phenomena at nanometer scale resolution is highly challenging. Here, we use a model system of polystyrene microspheres diffusing over the surface of colloidal membranes as 3 orders of magnitude scaled up analogue of this process. Colloidal membranes are freely fluctuating two-dimensional smectic A monolayers of one micrometer long rod-shaped viruses that bind to polystyrene microspheres through the generic mechanism of depletion attraction. We find that the microsphere causes local deformation in the membrane, behaving as an inclusion that diffuses freely in the bulk of the membrane but surprisingly gets radially trapped near the edge of the membrane. We identify the critical particle size and membrane composition required to observe trapping of particles at the membrane edge. A quantitative analysis of the motion of the particle in the bulk enabled us to determine the local membrane interfacial viscosity of the colloidal membranes. Overall, our study shows that the complex interplay of membrane fluctuations and particle characteristics can lead to a rich phenomenology even in highly simplified few component model systems.

DOI: https://doi.org/10.1021/acs.langmuir.4c05247
Curr Opin Cell Biol. 2025 Mar 06. pii: S0955-0674(25)00028-6. [Epub ahead of print]94 102490

Aneuploidy as a cancer vulnerability.

Jinghui Cao, Cai Liang, Hongtao Yu.

Aneuploidy is prevalent in cancer and has complicated roles in tumorigenesis. Paradoxically, artificially engineered aneuploidy in normal cells reduces cellular fitness by inducing proteotoxic and genotoxic stresses. A better molecular understanding of the multifaceted roles of aneuploidy in cancer evolution offers promising avenues for future cancer therapies. Here, we discuss the patterns and consequences of aneuploidy in human cancer. We highlight recent efforts to explore aneuploidy as a cancer vulnerability and new interventions that exploit this vulnerability for cancer treatment.

DOI: https://doi.org/10.1016/j.ceb.2025.102490
Science. 2025 Mar 14. 387(6739): eadm9805

Direct sensing of dietary ω-6 linoleic acid through FABP5-mTORC1 signaling.

Nikos Koundouros, Michal J Nagiec, Nayah Bullen, Evan K Noch, Guillermo Burgos-Barragan, Zhongchi Li, Long He, Sungyun Cho, Bobak Parang, Dominique Leone, Eleni Andreopoulou, John Blenis.

Diet influences macronutrient availability to cells, and although mechanisms of sensing dietary glucose and amino acids are well characterized, less is known about sensing lipids. We defined a nutrient signaling mechanism involving fatty acid-binding protein 5 (FABP5) and mechanistic target of rapamycin complex 1 (mTORC1) that is activated by the essential polyunsaturated fatty acid (PUFA) ω-6 linoleic acid (LA). FABP5 directly bound to the regulatory-associated protein of mTOR (Raptor) to enhance formation of functional mTORC1 and substrate binding, ultimately converging on increased mTOR signaling and proliferation. The amounts of FABP5 protein were increased in tumors and serum from triple-negative compared with those from receptor-positive breast cancer patients, which highlights its potential role as a biomarker that mediates cellular responses to ω-6 LA intake in this disease subtype.

DOI: https://doi.org/10.1126/science.adm9805
Bioeng Transl Med. 2025 Mar;10(2): e10737

Ultrasound-mediated mechanical forces activate selective tumor cell apoptosis.

Ajay Tijore, Felix Margadant, Nehal Dwivedi, Leslie Morgan, Mingxi Yao, Anushya Hariharan, Claire Alexandra Zhen Chew, Simon Powell, Glenn Kunnath Bonney, Michael Sheetz.

  Recent studies show that tumor cells undergo apoptosis after mechanical stretching, which promotes normal cell growth. Since ultrasound can produce similar sub-cellular mechanical stresses on the nanoscale, here we test the effect of ultrasound-mediated mechanical forces on tumors and normal cell survival. Surprisingly, tumor cells undergo apoptosis through a calpain-dependent mitochondrial pathway that relies upon calcium entry through the mechanosensitive Piezo1 channels. This is a general property of all tumor cell lines tested irrespective of tissue origin, but normal cells are unaffected. In vivo, ultrasound treatment promotes tumor cell killing in a mouse model with invasive CT26 cancer cell subcutaneous tumors and in the chick chorioallantoic membrane (CAM) model with relatively minor damage to chick embryos. Further, patient-derived pancreatic tumor organoids are killed by ultrasound treatment. Because ultrasound-mediated mechanical forces cause apoptosis of tumor cells from many different tissues in different microenvironments, it may offer a safe, non-invasive approach to augment tumor treatments.

Keywords:  Piezo1; apoptosis; cancer treatment; mechanical forces; ultrasound

DOI:  https://doi.org/10.1002/btm2.10737
Sci Adv. 2025 Mar 14. 11(11): eadt7274

Tissue stiffness mapping by light sheet elastography.

Min Zhu, Kaiwen Zhang, Evan C Thomas, Ran Xu, Brian Ciruna, Sevan Hopyan, Yu Sun.

Tissue stiffness plays a crucial role in regulating morphogenesis. The ability to measure and monitor the dynamic progression of tissue stiffness is important for generating and testing mechanistic hypotheses. Methods to measure tissue properties in vivo have been emerging but present challenges with spatial and temporal resolution especially in 3D, by their reliance on highly specialized equipment, and/or due to their invasive nature. Here, we introduce light sheet elastography, a noninvasive method that couples low-frequency shear waves with light sheet fluorescence microscopy by adapting commercially available instruments. With this method, we achieved in toto stiffness mapping of organ-stage mouse and zebrafish embryos at cellular resolution. Versatility of the method enabled time-lapse stiffness mapping during tissue remodeling and of the beating embryonic heart. This method expands the spectrum of tools available to biologists and presents opportunities for uncovering the mechanical basis of morphogenesis.

DOI: https://doi.org/10.1126/sciadv.adt7274
Nat Metab. 2025 Mar 11.

Glycogen drives tumour initiation and progression in lung adenocarcinoma.

Harrison A Clarke, Tara R Hawkinson, Cameron J Shedlock, Terrymar Medina, Roberto A Ribas, Lei Wu, Zizhen Liu, Xin Ma, Yi Xia, Yu Huang, Xing He, Josephine E Chang, Lyndsay E A Young, Jelena A Juras, Michael D Buoncristiani, Alexis N James, Anna Rushin, Matthew E Merritt, Annette Mestas, Jessica F Lamb, Elena C Manauis, Grant L Austin, Li Chen, Pankaj K Singh, Jiang Bian, Craig W Vander Kooi, B Mark Evers, Christine F Brainson, Derek B Allison, Matthew S Gentry, Ramon C Sun.

Lung adenocarcinoma (LUAD) is an aggressive cancer defined by oncogenic drivers and metabolic reprogramming. Here we leverage next-generation spatial screens to identify glycogen as a critical and previously underexplored oncogenic metabolite. High-throughput spatial analysis of human LUAD samples revealed that glycogen accumulation correlates with increased tumour grade and poor survival. Furthermore, we assessed the effect of increasing glycogen levels on LUAD via dietary intervention or via a genetic model. Approaches that increased glycogen levels provided compelling evidence that elevated glycogen substantially accelerates tumour progression, driving the formation of higher-grade tumours, while the genetic ablation of glycogen synthase effectively suppressed tumour growth. To further establish the connection between glycogen and cellular metabolism, we developed a multiplexed spatial technique to simultaneously assess glycogen and cellular metabolites, uncovering a direct relationship between glycogen levels and elevated central carbon metabolites essential for tumour growth. Our findings support the conclusion that glycogen accumulation drives LUAD cancer progression and provide a framework for integrating spatial metabolomics with translational models to uncover metabolic drivers of cancer.

DOI: https://doi.org/10.1038/s42255-025-01243-8
J Cell Sci. 2025 Mar 07. pii: jcs.263852. [Epub ahead of print]

ATG9A vesicles are a subtype of intracellular nanovesicle.

Mary Fesenko, Daniel J Moore, Peyton Ewbank, Elizabeth Courthold, Stephen J Royle.

  Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprises of multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous, and enriched for transmembrane cargo molecules including integrins, transporters, and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in 'ATG9A vesicles': small vesicles that contribute to autophagosome formation. Using in-cell vesicle capture assays we found that ATG9A, as well as other ATG9A vesicle cargos, were in INVs. Quantitative analysis showed that virtually all ATG9A vesicles are INVs, but that only ∼20% of INVs are ATG9A vesicles, suggesting that ATG9A vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impaired the autophagy response induced by starvation.

Keywords:  Autophagy; Cargo; Membrane traffic; Proteomics; Transport vesicle

DOI:  https://doi.org/10.1242/jcs.263852
J Pathol Inform. 2025 Apr;17 100421

Unlocking the potential of digital pathology: Novel baselines for compression.

Maximilian Fischer, Peter Neher, Peter Schüffler, Sebastian Ziegler, Shuhan Xiao, Robin Peretzke, David Clunie, Constantin Ulrich, Michael Baumgartner, Alexander Muckenhuber, Silvia Dias Almeida, Michael Gőtz, Jens Kleesiek, Marco Nolden, Rickmer Braren, Klaus Maier-Hein.

  Digital pathology offers a groundbreaking opportunity to transform clinical practice in histopathological image analysis, yet faces a significant hurdle: the substantial file sizes of pathological whole slide images (WSIs). Whereas current digital pathology solutions rely on lossy JPEG compression to address this issue, lossy compression can introduce color and texture disparities, potentially impacting clinical decision-making. Whereas prior research addresses perceptual image quality and downstream performance independently of each other, we jointly evaluate compression schemes for perceptual and downstream task quality on four different datasets. In addition, we collect an initially uncompressed dataset for an unbiased perceptual evaluation of compression schemes. Our results show that deep learning models fine-tuned for perceptual quality outperform conventional compression schemes like JPEG-XL or WebP for further compression of WSI. However, they exhibit a significant bias towards the compression artifacts present in the training data and struggle to generalize across various compression schemes. We introduce a novel evaluation metric based on feature similarity between original files and compressed files that aligns very well with the actual downstream performance on the compressed WSI. Our metric allows for a general and standardized evaluation of lossy compression schemes and mitigates the requirement to independently assess different downstream tasks. Our study provides novel insights for the assessment of lossy compression schemes for WSI and encourages a unified evaluation of lossy compression schemes to accelerate the clinical uptake of digital pathology.

Keywords:  Feature similarity; Lossy compression; Whole slide images

DOI:  https://doi.org/10.1016/j.jpi.2025.100421
Med Mol Morphol. 2025 Mar 10.

Novel orthotopic patient-derived xenograft model using human pancreatic cancer tissue fragments to recapitulate distant metastasis and cancer-related hypercoagulability.

Takuma Miura, Arisa Watanabe, Mutsumi Miyake, Sayaka Suga, Makoto Miyoshi, Kumiko Miyashita, Shohei Komatsu, Noriyuki Nishimura, Kazuya Shimizu, Yuichi Hori.

  Pancreatic cancer is a malignant tumor that metastasizes to distant organs, such as the liver and lungs from an early stage. Few animal models can reproduce early metastasis. In addition, no model has been reported that reproduces cancer-related hypercoagulability, which is characteristic of pancreatic ductal adenocarcinoma and other adenocarcinomas. We hypothesized that the reason why the commonly used orthotopic cell xenograft model cannot reproduce the disease is inadequate construction of the cancer microenvironment. We developed an orthotopic tissue fragment xenograft model in which tumor tissue was transplanted into the pancreas of mice while preserving the microenvironment. Briefly, we injected single cancer cells subcutaneously to form a tumor, which was then cut with a scalpel into tumor fragments. A fragment was then sutured and fixed to the surface of the pancreatic tail. In this study, we evaluated the superiority of this model over a conventional orthotopic cell xenograft model. As a result, the novel orthotopic tissue xenograft model reproduced early distant metastasis to the liver and lung, nerve invasion, and cancer-related hypercoagulability of human pancreatic cancer, and showed greater similarity to clinical cases than the control orthotopic cell xenograft model.

Keywords:  Metastasis; Orthotopic transplantation; Pancreatic cancer; Patient-derived xenograft (PDX); Trousseau’s syndrome

DOI:  https://doi.org/10.1007/s00795-025-00425-3
J Lipid Res. 2025 Mar 10. pii: S0022-2275(25)00033-1. [Epub ahead of print] 100773

Phosphatidylcholine synthesis and remodeling in brain endothelial cells.

Mohamed H Yaghmour, Theja Sajeevan, Christoph Thiele, Lars Kuerschner.

  Mammalian cells synthesize hundreds of different variants of their prominent membrane lipid phosphatidylcholine (PC), all differing in the side chain composition. This batch is constantly remodeled by the Lands cycle, a metabolic pathway replacing one chain at the time. Using the alkyne lipid lyso-phosphatidylpropargylcholine (LpPC), a precursor and intermediate in PC synthesis and remodeling, we study both processes in brain endothelial bEND3 cells. A novel method for multiplexed sample analysis by mass spectrometry is developed that offers high throughput and molecular species resolution of the propargyl-labeled PC lipids. Their time resolved profiles and kinetic parameters of metabolism demonstrate the plasticity of the PC pool and the acute handling of lipid influx in endothelial cells differs from that in hepatocytes. Side chain remodeling as a form of lipid cycling adapts the PC pool to the cells need and maintains lipid homeostasis. We estimate that endothelial cells possess the theoretical capacity to remodel up to 99% of their PC pool within 3.5 h using the Lands cycle. However, PC species are not subjected stochastically to this remodeling pathway as different species containing duplets of saturated, omega-3 and omega-6 side chains show different decay kinetics. Our findings emphasize the essential function of Lands cycling for monitoring and adapting the side chain composition of PC in endothelial cells.

Keywords:  alkyne tracer; click; lipid cycling; lipidomics; lysophosphatdiylcholine; lysophospholipids; phospholipase; propargyl-PC

DOI:  https://doi.org/10.1016/j.jlr.2025.100773