bims-cagime 2024-08-11 papers

bims-cagime

Biomed News

on Cancer, aging and metabolism

Issue of 2024–08–11
23 papers selected by
Kıvanç Görgülü, Technical University of Munich

Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.
Glycosphingolipid synthesis mediates immune evasion in KRAS-driven cancer.
Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.
Proximity labeling reveals dynamic changes in the SQSTM1 protein network.
Titration of RAS alters senescent state and influences tumour initiation.
Acvr1b loss increases formation of pancreatic precancerous lesions from acinar and ductal cells of origin.
INPP4B promotes PDAC aggressiveness via PIKfyve and TRPML-1-mediated lysosomal exocytosis.
Single-cell sensor analyses reveal signaling programs enabling Ras-G12C drug resistance.
p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells.
PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.
Spermidine is essential for fasting-mediated autophagy and longevity.
SLC25A48 controls mitochondrial choline import and metabolism.
A Phase 1b/2 Randomized Clinical Trial of Oleclumab with or without Durvalumab plus Chemotherapy in Patients with Metastatic Pancreatic Ductal Adenocarcinoma.
Protocol for establishing spontaneous metastasis in mice using a subcutaneous tumor model.
Membrane prewetting by condensates promotes tight-junction belt formation.
Distance-depending transcriptome changes of pancreatic stellate cells in paracrine pancreatic ductal adenocarcinoma co-culture models.
Precision Cancer Medicine 2.0-Oncology in the postgenomic era.
A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.
The Chorioallantoic Membrane (CAM) Assay for the Analysis of Starvation-Induced Autophagy.
Hepatic BMAL1 and HIF1α regulate a time-dependent hypoxic response and prevent hepatopulmonary-like syndrome.
Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.
Thermodynamic Characterization of LC3/GABARAP:Ligand Interactions by Isothermal Titration Calorimetry.
GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise.

Methods Mol Biol. 2024 ;2845 191-196

Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.

Shuhei Takada, Masaaki Komatsu, Hideaki Morishita.

  p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents. In this chapter, we describe a method for purifying p62 bodies using fluorescence-activated particle sorting. This method contributes to the identification of novel components of p62 bodies under various physiological and stress conditions.

Keywords:  Autophagy; Cell sorter; Fluorescence-activated particle sorting; Liquid–liquid phase separation; Selective autophagy; Ubiquitinated proteins; p62/SQSTM1 body

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_15
Nature. 2024 Aug 07.

Glycosphingolipid synthesis mediates immune evasion in KRAS-driven cancer.

Mariluz Soula, Gokhan Unlu, Rachel Welch, Aleksey Chudnovskiy, Beste Uygur, Vyom Shah, Hanan Alwaseem, Paul Bunk, Vishvak Subramanyam, Hsi-Wen Yeh, Artem Khan, Søren Heissel, Hani Goodarzi, Gabriel D Victora, Semir Beyaz, Kıvanç Birsoy.

Cancer cells frequently alter their lipids to grow and adapt to their environment1-3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.

DOI: https://doi.org/10.1038/s41586-024-07787-1
Cancer Prev Res (Phila). 2024 Aug 05.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Lincoln N Strickland, Wendao Liu, Usama Hussein, Nicolette Mardik, Xian Chen, Tingting Mills, Lana A Vornik, Michelle I Savage, Shizuko Sei, John Clifford, Holger K Eltzschig, Powel H Brown, Zhongming Zhao, Florencia McAllister, Jennifer M Bailey-Lundberg.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate (AMP) to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse (GEM) model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery three days/week at 10mg/kg, beginning when the mice were two months old and lasting three months. We euthanized the mice at five months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single Cell RNA sequencing (scRNA-seq) of pancreata of AB-680 treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment.

DOI: https://doi.org/10.1158/1940-6207.CAPR-24-0200
J Biol Chem. 2024 Aug 02. pii: S0021-9258(24)02122-7. [Epub ahead of print] 107621

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Alejandro N Rondón Ortiz, Lushuang Zhang, Peter E A Ash, Avik Basu, Sambhavi Puri, Sophie J F van der Spek, Zihan Wang, Luke Dorrian, Andrew Emili, Benjamin Wolozin.

  Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and of the relationship of the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

Keywords:  MAPT; SQSTM1; TurboID; autophagy; p62; protein aggregation; proximity labeling; tau

DOI:  https://doi.org/10.1016/j.jbc.2024.107621
Nature. 2024 Aug 07.

Titration of RAS alters senescent state and influences tumour initiation.

Adelyne S L Chan, Haoran Zhu, Masako Narita, Liam D Cassidy, Andrew R J Young, Camino Bermejo-Rodriguez, Aleksandra T Janowska, Hung-Chang Chen, Sarah Gough, Naoki Oshimori, Lars Zender, Sarah J Aitken, Matthew Hoare, Masashi Narita.

Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with premalignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS dose-escalation models in vitro and in vivo, revealing a RAS dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRAS(G12V) does not induce tumours, in part owing to OIS-driven immune clearance3. Single-cell RNA sequencing analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high and intermediate levels of NRAS(G12V), respectively. When titred down, NRAS(G12V)-expressing hepatocytes become immune resistant and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma. The molecular signature of each mouse tumour type is associated with different progenitor features and enriched in distinct human hepatocellular carcinoma subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.

DOI: https://doi.org/10.1038/s41586-024-07797-z
Cell Mol Gastroenterol Hepatol. 2024 Aug 05. pii: S2352-345X(24)00142-5. [Epub ahead of print] 101387

Acvr1b loss increases formation of pancreatic precancerous lesions from acinar and ductal cells of origin.

Kiyoshi Saeki, Ian S Wood, Wei Chuan K Wang, Shilpa Patil, Yanping Sun, David F Schaeffer, Gloria H Su, Janel L Kopp.

   BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) can develop from precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN). Previous studies indicated that loss of Acvr1b accelerates the Kras-mediated development of papillary IPMN in the mouse pancreas, however, the cell type predominantly affected by these genetic changes remains unclear.
METHODS: We investigated the contribution of cellular origin by inducing IPMN associated mutations- KRASG12D expression and Acvr1b loss - specifically in acinar (Ptf1aCreER;KrasLSL-G12D;Acvr1bfl/fl mice) or ductal (Sox9CreER;KrasLSL-G12D;Acvr1bfl/fl mice) cells in mice. We then performed MRI imaging and a thorough histopathological analysis of their pancreatic tissues.
RESULTS: The loss of Acvr1b increased the development of PanIN and IPMN-like lesions when either acinar and ductal cells expressed a Kras mutation. MRI, immunohistochemistry and histology revealed large IPMN-like lesions in these mice that exhibited features of flat, gastric epithelium. In addition, cyst formation in both mouse models was accompanied by chronic pancreatitis. Experimental acute pancreatitis accelerated the development of large mucinous cysts and PanIN when acinar, but not ductal, cells expressed mutant Kras and lost Acvr1b.
CONCLUSION: These findings indicate that loss of Acvr1b in the presence of the Kras oncogene promotes the development of large and small precancerous lesions from both ductal and acinar cells. However, the IPMN-like phenotype was not equivalent to that observed when these mutations were made in all pancreatic cells during development. Our study underscores the significance of the cellular context in the initiation and progression of precursor lesions from exocrine cells.

Keywords:  carcinogenesis; cellular origin; intraductal papillary mucinous neoplasm (IPMN); pancreatic intraepithelial neoplasia (PanIN)

DOI:  https://doi.org/10.1016/j.jcmgh.2024.101387
J Cell Biol. 2024 Nov 04. pii: e202401012. [Epub ahead of print]223(11):

INPP4B promotes PDAC aggressiveness via PIKfyve and TRPML-1-mediated lysosomal exocytosis.

Golam T Saffi, Lydia To, Nicholas Kleine, Ché M P Melo, Keyue Chen, Gizem Genc, K C Daniel Lee, Jonathan Tak-Sum Chow, Gun Ho Jang, Steven Gallinger, Roberto J Botelho, Leonardo Salmena.

Aggressive solid malignancies, including pancreatic ductal adenocarcinoma (PDAC), can exploit lysosomal exocytosis to modify the tumor microenvironment, enhance motility, and promote invasiveness. However, the molecular pathways through which lysosomal functions are co-opted in malignant cells remain poorly understood. In this study, we demonstrate that inositol polyphosphate 4-phosphatase, Type II (INPP4B) overexpression in PDAC is associated with PDAC progression. We show that INPP4B overexpression promotes peripheral dispersion and exocytosis of lysosomes resulting in increased migratory and invasive potential of PDAC cells. Mechanistically, INPP4B overexpression drives the generation of PtdIns(3,5)P2 on lysosomes in a PIKfyve-dependent manner, which directs TRPML-1 to trigger the release of calcium ions (Ca2+). Our findings offer a molecular understanding of the prognostic significance of INPP4B overexpression in PDAC through the discovery of a novel oncogenic signaling axis that orchestrates migratory and invasive properties of PDAC via the regulation of lysosomal phosphoinositide homeostasis.

DOI: https://doi.org/10.1083/jcb.202401012
Nat Chem Biol. 2024 Aug 05.

Single-cell sensor analyses reveal signaling programs enabling Ras-G12C drug resistance.

Jason Z Zhang, Shao-En Ong, David Baker, Dustin J Maly.

Clinical resistance to rat sarcoma virus (Ras)-G12C inhibitors is a challenge. A subpopulation of cancer cells has been shown to undergo genomic and transcriptional alterations to facilitate drug resistance but the immediate adaptive effects on Ras signaling in response to these drugs at the single-cell level is not well understood. Here, we used Ras biosensors to profile the activity and signaling environment of endogenous Ras at the single-cell level. We found that a subpopulation of KRas-G12C cells treated with Ras-G12C-guanosine-diphosphate inhibitors underwent adaptive signaling and metabolic changes driven by wild-type Ras at the Golgi and mutant KRas at the mitochondria, respectively. Our Ras biosensors identified major vault protein as a mediator of Ras activation through its scaffolding of Ras signaling pathway components and metabolite channels. Overall, methods including ours that facilitate direct analysis on the single-cell level can report the adaptations that subpopulations of cells adopt in response to cancer therapies, thus providing insight into drug resistance.

DOI: https://doi.org/10.1038/s41589-024-01684-4
Nat Cell Biol. 2024 Aug 05.

p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells.

Julia Majewska, Amit Agrawal, Avi Mayo, Lior Roitman, Rishita Chatterjee, Jarmila Sekeresova Kralova, Tomer Landsberger, Yonatan Katzenelenbogen, Tomer Meir-Salame, Efrat Hagai, Ilanit Sopher, Juan-Felipe Perez-Correa, Wolfgang Wagner, Avi Maimon, Ido Amit, Uri Alon, Valery Krizhanovsky.

The accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.

DOI: https://doi.org/10.1038/s41556-024-01465-0
Cell Syst. 2024 Aug 02. pii: S2405-4712(24)00183-2. [Epub ahead of print]

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

Alexander T F Bell, Jacob T Mitchell, Ashley L Kiemen, Melissa Lyman, Kohei Fujikura, Jae W Lee, Erin Coyne, Sarah M Shin, Sushma Nagaraj, Atul Deshpande, Pei-Hsun Wu, Dimitrios N Sidiropoulos, Rossin Erbe, Jacob Stern, Rena Chan, Stephen Williams, James M Chell, Lauren Ciotti, Jacquelyn W Zimmerman, Denis Wirtz, Won Jin Ho, Neeha Zaidi, Elizabeth Thompson, Elizabeth M Jaffee, Laura D Wood, Elana J Fertig, Luciane T Kagohara.

  This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

Keywords:  Visium; Xenium; imaging mass cytometry; machine learning; multi-omics; pancreatic adenocarcinoma; pancreatic intraepithelial neoplasia; spatial transcriptomics; transfer learning

DOI:  https://doi.org/10.1016/j.cels.2024.07.001
Nat Cell Biol. 2024 Aug 08.

Spermidine is essential for fasting-mediated autophagy and longevity.

Sebastian J Hofer, Ioanna Daskalaki, Martina Bergmann, Jasna Friščić, Andreas Zimmermann, Melanie I Mueller, Mahmoud Abdellatif, Raffaele Nicastro, Sarah Masser, Sylvère Durand, Alexander Nartey, Mara Waltenstorfer, Sarah Enzenhofer, Isabella Faimann, Verena Gschiel, Thomas Bajaj, Christine Niemeyer, Ilias Gkikas, Lukas Pein, Giulia Cerrato, Hui Pan, YongTian Liang, Jelena Tadic, Andrea Jerkovic, Fanny Aprahamian, Christine E Robbins, Nitharsshini Nirmalathasan, Hansjörg Habisch, Elisabeth Annerer, Frederik Dethloff, Michael Stumpe, Franziska Grundler, Françoise Wilhelmi de Toledo, Daniel E Heinz, Daniela A Koppold, Anika Rajput Khokhar, Andreas Michalsen, Norbert J Tripolt, Harald Sourij, Thomas R Pieber, Rafael de Cabo, Mark A McCormick, Christoph Magnes, Oliver Kepp, Joern Dengjel, Stephan J Sigrist, Nils C Gassen, Simon Sedej, Tobias Madl, Claudio De Virgilio, Ulrich Stelzl, Markus H Hoffmann, Tobias Eisenberg, Nektarios Tavernarakis, Guido Kroemer, Frank Madeo.

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.

DOI: https://doi.org/10.1038/s41556-024-01468-x
Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00281-X. [Epub ahead of print]

SLC25A48 controls mitochondrial choline import and metabolism.

Anthony R P Verkerke, Xu Shi, Mark Li, Yusuke Higuchi, Tadashi Yamamuro, Daisuke Katoh, Hiroshi Nishida, Christopher Auger, Ichitaro Abe, Robert E Gerszten, Shingo Kajimura.

  Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.

Keywords:  bioenergetics; brown adipose tissue; cancer metabolism; choline; mitochondria; purine nucleotides

DOI:  https://doi.org/10.1016/j.cmet.2024.07.010
Clin Cancer Res. 2024 Aug 06.

A Phase 1b/2 Randomized Clinical Trial of Oleclumab with or without Durvalumab plus Chemotherapy in Patients with Metastatic Pancreatic Ductal Adenocarcinoma.

Andrew L Coveler, Matthew J Reilley, Mark Zalupski, Teresa Macarulla, Christos Fountzilas, Mariano Ponz-Sarvise, Adnan Nagrial, Nataliya V Uboha, Sophia Frentzas, Michael Overman, Anne Noonan, Wells A Messersmith, Nick Pavlakis, Niharika B Mettu, Ina Bisha, Ying Wang, Paul Smith, Elina Murtomaki, Agata A Bielska, Veronique Bragulat, Zachary A Cooper, Rakesh Kumar, David R Spigel.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) upregulates CD73, potentially contributing to immune surveillance evasion. Combining oleclumab (CD73 inhibitor) and durvalumab with chemotherapy may identify an effective treatment option.
PATIENTS AND METHODS: Multicenter Phase 1b/2 randomized clinical trial in patients with metastatic PDAC, untreated (Cohort A) or previously received gemcitabine-based chemotherapy (Cohort B) (NCT03611556). During escalation, patients received oleclumab 1500 or 3000 mg, durvalumab 1500 mg, and gemcitabine plus nab-paclitaxel (GnP) (Cohort A; n=14) or modified FOLFOX (Cohort B; n=11). During expansion, Cohort A patients (n=170) were randomized to: GnP (Arm A1), oleclumab (recommended Phase 2 dose; RP2D) with GnP (Arm A2), or oleclumab (RP2D) with durvalumab plus GnP (Arm A3). Primary objectives were safety (escalation) and objective response rate (ORR) (expansion). Secondary objectives included progression-free survival (PFS) and overall survival (OS).
RESULTS: During escalation, 1/11 patients from Cohort B (oleclumab 3000 mg) experienced two dose-limiting toxicities. Oleclumab RP2D was 3000 mg. During expansion, Grade ≥3 treatment-related adverse events occurred in 67.7% (42/62) of patients in A1, 73.7% (28/38) in A2, and 77.1% (54/70) in A3. ORR was 29.0%, 21.1%, and 32.9% in A1, A2, and A3, respectively (A1 vs A3; p=0.650). PFS (hazard ratio [HR]=0.72; 95% confidence interval [CI]: 0.47, 1.11) and OS (HR=0.75; 95% CI: 0.50, 1.13) were similar for A3 versus A1. Patients with high CD73 expression had improved PFS and OS in A3 versus A1, although this should be interpreted with caution.
CONCLUSIONS: Although the safety profile was acceptable, this study did not meet its primary efficacy endpoint.

DOI: https://doi.org/10.1158/1078-0432.CCR-24-0499
STAR Protoc. 2024 Aug 02. pii: S2666-1667(24)00404-0. [Epub ahead of print]5(3): 103239

Protocol for establishing spontaneous metastasis in mice using a subcutaneous tumor model.

Shiqin Liu, Michelle Shen, Kewei Le, Alifiani B Hartono, Tanya Stoyanova.

  Recapitulating spontaneous metastasis in preclinical models is crucial for understanding mechanisms underlying cancer progression and testing effective therapeutic interventions. We present a protocol for establishing and characterizing the spontaneous metastasis model in mice. We describe steps for generating primary tumors, tumor resection, monitoring metastatic dissemination, and evaluating metastatic burden using histological and imaging techniques. This protocol provides a valuable tool for studying metastasis in vivo and testing therapeutic strategies aimed at preventing or targeting metastatic diseases. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Keywords:  Cancer; Cell Biology; Model Organisms; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2024.103239
Nature. 2024 Aug 07.

Membrane prewetting by condensates promotes tight-junction belt formation.

Karina Pombo-García, Omar Adame-Arana, Cecilie Martin-Lemaitre, Frank Jülicher, Alf Honigmann.

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.

DOI: https://doi.org/10.1038/s41586-024-07726-0
Sci Rep. 2024 08 04. 14(1): 18030

Distance-depending transcriptome changes of pancreatic stellate cells in paracrine pancreatic ductal adenocarcinoma co-culture models.

Anais Zourelidis, Bogusz Trojanowicz, Yoshiaki Sunami, Gerd Hause, David Vieweg, Jörg Kleeff.

Pancreatic stellate cells (PSC) are one source of cancer-associated fibroblasts (CAF) and play, therefore, an essential role in pancreatic ductal adenocarcinoma (PDA). Paracrine signalling between PDA cells and CAF has been widely studied, yet external influences on paracrine crosstalk are poorly understood. This study aimed to gain a deeper insight into the communication of PSC and cancer cells under different co-culture conditions via analysis of PSC gene expression profiles. Two contactless co-culture models with tumor cells from the p48-Cre; lox-stop-lox-KrasG12D/+; lox-stop-lox-Trp53R172H/+ mouse model (KPC) and murine PSC separated through a microporous membrane and grown in different compartments (standard co-culture) or on different sides of the same membrane (inverse co-culture), were established. RNA-Sequencing analysis of PSC mRNA was performed 24 h and 72 h after co-culture with KPC cells. For selected genes, results were confirmed by quantitative RT-PCR and immunocytochemistry. Standard co-culture displayed 19 differentially expressed genes (DEG) at 24 h and 52 DEG at 72 h. In inverse co-culture, 800 DEG at 24 h and 2213 DEG at 72 h were enriched. PSC showed great heterogeneity in their gene expression profiles; however, mutually regulated genes of both co-cultures, such as VCAN and CHST11, could be identified. VCAN-protein-protein interaction-network analysis revealed several shared genes between co-culture models, such as SDC4 and FN1. In conclusion, PSC show a varying susceptibility to cancer cell signals depending on the co-culture method, with intensified transcriptome changes with closer proximity.

DOI: https://doi.org/10.1038/s41598-024-68148-6
Mol Oncol. 2024 Aug 07.

Precision Cancer Medicine 2.0-Oncology in the postgenomic era.

Riccardo Masina, Carlos Caldas.

  Genomic medicine has transformed the lives of patients with cancer by enabling individualised and evidence-based clinical decision-making. Despite this progress, the implementation of precision cancer medicine is limited by its dependence on isolated biomarkers. The development of bulk and single-cell multiomic technologies has revealed the enormous complexity of the cancer ecosystem. Beyond the cancer cell, the tumour microenvironment, macroenvironment and host factors, including the microbiome, profoundly influence the cancer phenotype, and accounting for these enhances the resolution of precision medicine. The advent of robust multiomic profiling and interpretable machine learning algorithms mark the dawn of a new postgenomic era of personalised cancer medicine. In Precision Cancer Medicine 2.0, high-resolution personalised clinical decision-making is informed by the comprehensive multiomic profiling of tumour and host, integrated using artificial intelligence.

Keywords:  cancer; data integration; machine learning; precision cancer medicine; translational research; tumour biomarkers

DOI:  https://doi.org/10.1002/1878-0261.13707
Cell Rep. 2024 Aug 07. pii: S2211-1247(24)00916-1. [Epub ahead of print]43(8): 114587

A molecular pathway for cancer cachexia-induced muscle atrophy revealed at single-nucleus resolution.

Yichi Zhang, Matthieu Dos Santos, Huocong Huang, Kenian Chen, Puneeth Iyengar, Rodney Infante, Patricio M Polanco, Rolf A Brekken, Chunyu Cai, Ambar Caijgas, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Ning Liu, Eric N Olson.

  Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.

Keywords:  AAV; CP: Cancer; CP: Metabolism; atrophy; cachexia; denervation; myogenin; myostatin; single nucleus ATAC-seq; single nucleus RNA-seq; single nucleus multiome

DOI:  https://doi.org/10.1016/j.celrep.2024.114587
Methods Mol Biol. 2024 Aug 10.

The Chorioallantoic Membrane (CAM) Assay for the Analysis of Starvation-Induced Autophagy.

Nazlı Şevval Menemenli, Özün Özcan, H Hazal Hüsnügil, Aliye Ezgi Güleç Taşkıran, Göksu Oral, Aytekin Akyol, Sreeparna Banerjee.

  During avian development, the chorioallantoic membrane (CAM) is generated around 4 days after fertilization following the fusion of the allantois and the chorion. The CAM develops rapidly over the next several days and gets heavily vascularized and therefore has been explored widely as a tool for the study of angiogenesis. Additionally, being immunodeficient, the CAM can be used for tumor growth of human origin and its metastasis. Of note, the CAM assay is minimally invasive for the chicken embryo and lacks innervation, which gives this in vivo model a low ethical burden. Here, we describe the protocol for the generation of microtumors from human colorectal cancer cell lines on the CAM, incubated in a nutrient-deficient medium for the activation of autophagy. We show that pre-inoculation markers of autophagy induced through nutrient deficiency are retained in the microtumors generated on the CAM.

Keywords:  Autophagy; Chorioallantoic membrane; Colorectal cancer; LC3; Nutrient deficiency; p62

DOI:  https://doi.org/10.1007/7651_2024_562
Cell Metab. 2024 Jul 30. pii: S1550-4131(24)00274-2. [Epub ahead of print]

Hepatic BMAL1 and HIF1α regulate a time-dependent hypoxic response and prevent hepatopulmonary-like syndrome.

Vaishnavi Dandavate, Nityanand Bolshette, Rachel Van Drunen, Gal Manella, Hanna Bueno-Levy, Mirie Zerbib, Ippei Kawano, Marina Golik, Yaarit Adamovich, Gad Asher.

  The transcriptional response to hypoxia is temporally regulated, yet the molecular underpinnings and physiological implications are unknown. We examined the roles of hepatic Bmal1 and Hif1α in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on either Bmal1 or Hif1α, through shared and distinct roles that are daytime determined. We further show that hypoxia-inducible factor (HIF)1α accumulation upon hypoxia is temporally regulated and Bmal1 dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1α are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to extracellular signaling regulated kinase (ERK) activation, endothelial nitric oxide synthase, and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1α are key time-dependent regulators of the hypoxic response and can provide molecular insights into the pathophysiology of hepatopulmonary syndrome.

Keywords:  BMAL1; HIF1α; circadian clocks; contrast echocardiography; hepatopulmonary syndrome; hypoxia; lung single-cell RNA-seq; nitric oxide; pulmonary vasodilation

DOI:  https://doi.org/10.1016/j.cmet.2024.07.003
J Phys Chem B. 2024 Aug 09.

Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.

Jimmy Maillard, Ewa Grassin, Ekaterina Bestsennaia, Melinda Silaghi, Karolina Straková, José García-Calvo, Naomi Sakai, Stefan Matile, Alexandre Fürstenberg.

A milestone in optical imaging of mechanical forces in cells has been the development of the family of flipper fluorescent probes able to report membrane tension noninvasively in living cells through their fluorescence lifetime. The specifically designed Flipper-CF3 probe with an engineered inherent blinking mechanism was recently introduced for super-resolution fluorescence microscopy of lipid ordered membranes but was too dim to be detected in lipid disordered membranes at the single-molecule level (García-Calvo, J. J. Am. Chem. Soc. 2020, 142(28), 12034-12038). We show here that the original and commercially available probe Flipper-TR is compatible with single-molecule based super-resolution imaging and resolves both liquid ordered and liquid disordered membranes of giant unilamellar vesicles below the diffraction limit. Single probe molecules were additionally tracked in lipid bilayers, enabling to distinguish membranes of varying composition from the diffusion coefficient of the probe. Differences in brightness between Flipper-CF3 and Flipper-TR originate in their steady-state absorption and fluorescence properties. The general compatibility of the Flipper-TR scaffold with single-molecule detection is further shown in super-resolution experiments with targetable Flipper-TR derivatives.

DOI: https://doi.org/10.1021/acs.jpcb.4c02506
Methods Mol Biol. 2024 ;2845 219-235

Thermodynamic Characterization of LC3/GABARAP:Ligand Interactions by Isothermal Titration Calorimetry.

Johannes Dopfer, Martin P Schwalm, Stefan Knapp, Vladimir V Rogov.

  Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.

Keywords:  Autophagy; GABARAP; Isothermal titration calorimetry; Kd determination; LC3; LIR motifs

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_18
Cell Rep. 2024 Aug 02. pii: S2211-1247(24)00906-9. [Epub ahead of print]43(8): 114577

GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise.

Axel Labour, Marlène Lac, Lucas Frassin, Benjamin Lair, Enda Murphy, Claire Maslo, Laurent Monbrun, Marie-Lou Calmy, Marie Marquès, Nathalie Viguerie, Geneviève Tavernier, Pierre Gourdy, Donal O'Gorman, Emilie Montastier, Claire Laurens, Alexandra Montagner, Cedric Moro.

  Growth and differentiation factor 15 (GDF15) has recently emerged as a weight loss and insulin-sensitizing factor. Growing evidence also supports a role for GDF15 as a physiological, exercise-induced stress signal. Here, we tested whether GDF15 is required for the insulin-sensitizing effects of exercise in mice and humans. At baseline, both under a standard nutritional state and high-fat feeding, GDF15 knockout (KO) mice display normal glucose tolerance, systemic insulin sensitivity, maximal speed, and endurance running capacity when compared to wild-type littermates independent of sex. When submitted to a 4-week exercise training program, both lean and obese wild-type and GDF15 KO mice similarly improve their endurance running capacity, glucose tolerance, systemic insulin sensitivity, and peripheral glucose uptake. Insulin-sensitizing effects of exercise training were also unrelated to changes in plasma GDF15 in humans. In summary, we here show that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise.

Keywords:  CP: Metabolism; GDF15; exercise; exerkine; glucose tolerance; insulin sensitivity; mice

DOI:  https://doi.org/10.1016/j.celrep.2024.114577