bims-mepmim 2024-04-21 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2024‒04‒21
28 papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet.
LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.
Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism.
Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.
The emerging importance of the α-keto acid dehydrogenase complexes in serving as intracellular and intercellular signaling platforms for the regulation of metabolism.
Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.
Metabolic programming of organ-specific natural killer cell responses.
Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.
Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.
Regulated regeneration of adipose tissue in lipodystrophic Agpat2-null mice partially ameliorates hepatic steatosis.
iNOS regulates hematopoietic stem and progenitor cells via mitochondrial signaling and is critical for bone marrow regeneration.
ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress.
VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.
Rapid HPLC method reveals dynamic shifts in coenzyme Q redox state.
Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.
De novo purine metabolism is a metabolic vulnerability of cancers with low p16 expression.
Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.
Deuterium metabolic imaging differentiates glioblastoma metabolic subtypes and detects early response to chemoradiotherapy.
Context-dependent roles for ubiquitous mitochondrial creatine kinase CKMT1 in breast cancer progression.
Redox regulation of macrophages.
Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.
Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages.
GPR1 and CMKLR1 control lipid metabolism to support development of clear cell renal cell carcinoma.
ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates the Warburg effect in breast cancer.
A LTB4/CD11b self-amplifying loop drives pyogranuloma formation in chronic granulomatous disease.
25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.
Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets.
Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine-mediated regulation of c-Fos.

J Lipid Res. 2024 Apr 17. pii: S0022-2275(24)00047-6. [Epub ahead of print] 100542

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet.

Nicholas M Venetos, Colin T Stomberski, Zhaoxia Qian, Richard T Premont, Jonathan S Stamler.

  Nitric oxide (NO), produced primarily by nitric oxide synthase (NOS) enzymes, is known to influence energy metabolism by stimulating fat uptake and oxidation. The effects of NO on de novo lipogenesis, however, are less clear. Here we demonstrate that hepatic expression of eNOS is reduced following prolonged administration of a hypercaloric high-fat diet. This results in marked reduction in the amount of S-nitrosylation of liver proteins including notably Acetyl-CoA Carboxylase (ACC), the rate-limiting enzyme in de novo lipogenesis. We further show that ACC S-nitrosylation markedly increases enzymatic activity. Diminished eNOS expression and ACC S-nitrosylation may thus represent a physiological adaptation to caloric excess by constraining lipogenesis. Our findings demonstrate that S-nitrosylation of liver proteins is subject to dietary control and suggest that de novo lipogenesis is coupled to dietary and metabolic conditions through ACC S-nitrosylation.

Keywords:  Acetyl-CoA Carboxylase; Cell signaling; Dietary fat; Lipogenesis; Liver; S-nitrosylation; Triglycerides

DOI:  https://doi.org/10.1016/j.jlr.2024.100542
Cell Rep. 2024 Apr 15. pii: S2211-1247(24)00430-3. [Epub ahead of print] 114102

LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.

Ryo Terao, Tae Jun Lee, Jason Colasanti, Charles W Pfeifer, Joseph B Lin, Andrea Santeford, Keitaro Hase, Shinobu Yamaguchi, Daniel Du, Brian S Sohn, Yo Sasaki, Mitsukuni Yoshida, Rajendra S Apte.

  Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.

Keywords:  CD38; CP: Immunology; CP: Metabolism; NAD(+); NMN; age-related macular degeneration; cellular senescence; cholesterol efflux; neurodegeneration; nicotinamide adenine dinucleotide; nicotinamide mononucleotide

DOI:  https://doi.org/10.1016/j.celrep.2024.114102
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2318420121

Iron regulates the quiescence of naive CD4 T cells by controlling mitochondria and cellular metabolism.

Ajay Kumar, Chenxian Ye, Afia Nkansah, Thomas Decoville, Garrett M Fogo, Peter Sajjakulnukit, Mack B Reynolds, Li Zhang, Osbourne Quaye, Young-Ah Seo, Thomas H Sanderson, Costas A Lyssiotis, Cheong-Hee Chang.

  In response to an immune challenge, naive T cells undergo a transition from a quiescent to an activated state acquiring the effector function. Concurrently, these T cells reprogram cellular metabolism, which is regulated by iron. We and others have shown that iron homeostasis controls proliferation and mitochondrial function, but the underlying mechanisms are poorly understood. Given that iron derived from heme makes up a large portion of the cellular iron pool, we investigated iron homeostasis in T cells using mice with a T cell-specific deletion of the heme exporter, FLVCR1 [referred to as knockout (KO)]. Our finding revealed that maintaining heme and iron homeostasis is essential to keep naive T cells in a quiescent state. KO naive CD4 T cells exhibited an iron-overloaded phenotype, with increased spontaneous proliferation and hyperactive mitochondria. This was evidenced by reduced IL-7R and IL-15R levels but increased CD5 and Nur77 expression. Upon activation, however, KO CD4 T cells have defects in proliferation, IL-2 production, and mitochondrial functions. Iron-overloaded CD4 T cells failed to induce mitochondrial iron and exhibited more fragmented mitochondria after activation, making them susceptible to ferroptosis. Iron overload also led to inefficient glycolysis and glutaminolysis but heightened activity in the hexosamine biosynthetic pathway. Overall, these findings highlight the essential role of iron in controlling mitochondrial function and cellular metabolism in naive CD4 T cells, critical for maintaining their quiescent state.

Keywords:  heme; iron; mitochondria; tonic signaling

DOI:  https://doi.org/10.1073/pnas.2318420121
J Immunol. 2024 Apr 15. pii: ji2300649. [Epub ahead of print]

Iron Is Critical for Mucosal-Associated Invariant T Cell Metabolism and Effector Functions.

Eimear K Ryan, Christy Clutter, Conor De Barra, Benjamin J Jenkins, Simon O'Shaughnessy, Odhrán K Ryan, Chloe McKenna, Helen M Heneghan, Fiona Walsh, David K Finlay, Linda V Sinclair, Nicholas Jones, Daniel T Leung, Donal O'Shea, Andrew E Hogan.

Mucosal-Associated Invariant T (MAIT) cells are a population of innate T cells that play a critical role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells can rapidly respond via both TCR-dependent and -independent mechanisms, resulting in robust cytokine production. The metabolic and nutritional requirements for optimal MAIT cell effector responses are still emerging. Iron is an important micronutrient and is essential for cellular fitness, in particular cellular metabolism. Iron is also critical for many pathogenic microbes, including those that activate MAIT cells. However, iron has not been investigated with respect to MAIT cell metabolic or functional responses. In this study, we show that human MAIT cells require exogenous iron, transported via CD71 for optimal metabolic activity in MAIT cells, including their production of ATP. We demonstrate that restricting iron availability by either chelating environmental iron or blocking CD71 on MAIT cells results in impaired cytokine production and proliferation. These data collectively highlight the importance of a CD71-iron axis for human MAIT cell metabolism and functionality, an axis that may have implications in conditions where iron availability is limited.

DOI: https://doi.org/10.4049/jimmunol.2300649
Redox Biol. 2024 Apr 10. pii: S2213-2317(24)00131-9. [Epub ahead of print]72 103155

The emerging importance of the α-keto acid dehydrogenase complexes in serving as intracellular and intercellular signaling platforms for the regulation of metabolism.

Ryan J Mailloux.

The α-keto acid dehydrogenase complex (KDHc) class of mitochondrial enzymes is composed of four members: pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (KGDHc), branched-chain keto acid dehydrogenase (BCKDHc), and 2-oxoadipate dehydrogenase (OADHc). These enzyme complexes occupy critical metabolic intersections that connect monosaccharide, amino acid, and fatty acid metabolism to Krebs cycle flux and oxidative phosphorylation (OxPhos). This feature also imbues KDHc enzymes with the heightened capacity to serve as platforms for propagation of intracellular and intercellular signaling. KDHc enzymes serve as a source and sink for mitochondrial hydrogen peroxide (mtH2O2), a vital second messenger used to trigger oxidative eustress pathways. Notably, deactivation of KDHc enzymes through reversible oxidation by mtH2O2 and other electrophiles modulates the availability of several Krebs cycle intermediates and related metabolites which serve as powerful intracellular and intercellular messengers. The KDHc enzymes also play important roles in the modulation of mitochondrial metabolism and epigenetic programming in the nucleus through the provision of various acyl-CoAs, which are used to acylate proteinaceous lysine residues. Intriguingly, nucleosomal control by acylation is also achieved through PDHc and KGDHc localization to the nuclear lumen. In this review, I discuss emerging concepts in the signaling roles fulfilled by the KDHc complexes. I highlight their vital function in serving as mitochondrial redox sensors and how this function can be used by cells to regulate the availability of critical metabolites required in cell signaling. Coupled with this, I describe in detail how defects in KDHc function can cause disease states through the disruption of cell redox homeodynamics and the deregulation of metabolic signaling. Finally, I propose that the intracellular and intercellular signaling functions of the KDHc enzymes are controlled through the reversible redox modification of the vicinal lipoic acid thiols in the E2 subunit of the complexes.

DOI: https://doi.org/10.1016/j.redox.2024.103155
JCI Insight. 2024 Apr 16. pii: e168594. [Epub ahead of print]

Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.

Yijing Zhou, Vanessa B Sanchez, Peining Xu, Thomas Roule, Marco Flores-Mendez, Brianna Ciesielski, Donna Yoo, Hiab Teshome, Teresa Jimenez, Shibo Liu, Mike Henne, Tim O'Brien, Ye He, Clementina Mesaros, Naiara Akizu.

  Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an inter-organelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14 deficient mice. However, pre-degenerating SNX14 deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in pre-degenerating PCs, suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.

Keywords:  Lysosomes; Monogenic diseases; Neurodegeneration; Neuroscience

DOI:  https://doi.org/10.1172/jci.insight.168594
Immunol Rev. 2024 Apr 17.

Metabolic programming of organ-specific natural killer cell responses.

Rebecca B Delconte, Joseph C Sun.

  Cells of the mammalian innate immune system have evolved to protect the host from various environmental or internal insults and injuries which perturb the homeostatic state of the organism. Among the lymphocytes of the innate immune system are natural killer (NK) cells, which circulate and survey host tissues for signs of stress, including infection or transformation. NK cells rapidly eliminate damaged cells in the blood or within tissues through secretion of cytolytic machinery and production of proinflammatory cytokines. To perform these effector functions while traversing between the blood and tissues, patrolling NK cells require sufficient fuel to meet their energetic demands. Here, we highlight the ability of NK cells to metabolically adapt across tissues, during times of nutrient deprivation and within tumor microenvironments. Whether at steady state, or during viral infection and cancer, NK cells readily shift their nutrient uptake and usage in order to maintain metabolism, survival, and function.

Keywords:  NK cells; cancer; immunity; immunometabolism

DOI:  https://doi.org/10.1111/imr.13333
Nitric Oxide. 2024 Apr 16. pii: S1089-8603(24)00050-8. [Epub ahead of print]

Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.

Tarciso A Sellani, Samanta L Tomaz, Jéssica M Gonçalves, Adriana Lima, Carolina C de Amat Herbozo, Gabrielli N Silva, Mônica Gambero, Ieda M Longo-Maugéri, Karin A Simon, Hugo P Monteiro, Elaine G Rodrigues.

  Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

Keywords:  Hyperglycemic tumor microenvironment; IL-10; Murine melanoma; Nitric oxide; Streptozotocin-induced hyperglycemia; macrophages; nitric oxide synthases

DOI:  https://doi.org/10.1016/j.niox.2024.04.007
Nat Commun. 2024 Apr 17. 15(1): 3290

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.

Ying Zhu, Kerem Can Akkaya, Julia Ruta, Nanako Yokoyama, Cong Wang, Max Ruwolt, Diogo Borges Lima, Martin Lehmann, Fan Liu.

The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome. Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.

DOI: https://doi.org/10.1038/s41467-024-47569-x
iScience. 2024 Apr 19. 27(4): 109517

Regulated regeneration of adipose tissue in lipodystrophic Agpat2-null mice partially ameliorates hepatic steatosis.

Anil K Agarwal, Katie Tunison, Jay D Horton, Abhimanyu Garg.

  Both humans and mice with congenital generalized lipodystrophy due to AGPAT2 deficiency develop diabetes mellitus, insulin resistance, and hepatic steatosis, which have been attributed to the near total loss of adipose tissue (AT). Here, we show that regulated AT regeneration in doxycycline (dox)-fed Tg-AT-hAGPAT2;mAgpat2-/- mice partially ameliorates hepatic steatosis at 12 weeks of age and causes reduced expression of genes involved in hepatic de novo lipogenesis despite partial (∼30-50%) AT regeneration compared to that in wild-type mice. Compared to chow-fed Tg-AT-hAGPAT2;mAgpat2-/- mice, those fed dox diet had markedly reduced serum insulin levels, suggesting an improvement in insulin resistance. Interestingly, the fasting plasma glucose levels in dox-fed Tg-AT-hAGPAT2;mAgpat2-/- mice were no different than those in chow-fed wild-type mice. Indirect calorimetry revealed normalization in the energy balance of dox-fed Tg-AT-hAGPAT2;mAgpat2-/- mice compared to that in chow-fed mice. This study's findings suggest that partial AT regeneration in lipodystrophic mice can ameliorate metabolic derangements.

Keywords:  Lipid; Pathophysiology; Physiology

DOI:  https://doi.org/10.1016/j.isci.2024.109517
Free Radic Biol Med. 2024 Apr 16. pii: S0891-5849(24)00404-0. [Epub ahead of print]

iNOS regulates hematopoietic stem and progenitor cells via mitochondrial signaling and is critical for bone marrow regeneration.

Supriya Sinha, Priyanka Dhankani, Milind Nahiyera, Krishna Bhan Singh, Divya Singh, Madhav Nilakanth Mugale, Sharad Sharma, Jagavelu Kumaravelu, Madhu Dikshit, Sachin Kumar.

  Hematopoietic stem cells (HSCs) replenish blood cells under steady state and on demand, that exhibit therapeutic potential for Bone marrow failures and leukemia. Redox signaling plays key role in immune cells and hematopoiesis. However, the role of reactive nitrogen species in hematopoiesis remains unclear and requires further investigation. We investigated the significance of inducible nitric oxide synthase/ nitric oxide (iNOS/NO) signaling in hematopoietic stem and progenitor cells (HSPCs) and hematopoiesis under steady-state and stress conditions. HSCs contain low levels of NO and iNOS under normal conditions, but these increase upon bone marrow stress. iNOS-deficient mice showed subtle changes in peripheral blood cells but significant alterations in HSPCs, including increased HSCs and multipotent progenitors. Surprisingly, iNOS-deficient mice displayed heightened susceptibility and delayed recovery of blood progeny following 5-Fluorouracil (5-FU) induced hematopoietic stress. Loss of quiescence and increased mitochondrial stress, indicated by elevated MitoSOX and MMPhi HSCs, were observed in iNOS-deficient mice. Furthermore, pharmacological approaches to mitigate mitochondrial stress rescued 5-FU-induced HSC death. Conversely, iNOS-NO signaling was required for demand-driven mitochondrial activity and proliferation during hematopoietic recovery, as iNOS-deficient mice and NO signaling inhibitors exhibit reduced mitochondrial activity. In conclusion, our study challenges the conventional view of iNOS-derived NO as a cytotoxic molecule and highlights its intriguing role in HSPCs. Together, our findings provide insights into the crucial role of the iNOS-NO-mitochondrial axis in regulating HSPCs and hematopoiesis.

Keywords:  Bone marrow; Hematopoietic stem and progenitor cells (HSPCs); Inducible nitric oxide synthase (iNOS); Nitric oxide (NO); hematopoietic stress

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.04.225
J Biol Chem. 2024 Apr 17. pii: S0021-9258(24)01800-3. [Epub ahead of print] 107299

ABCG2 and SLC1A5 functionally interact to rewire metabolism and confer a survival advantage to cancer cells under oxidative stress.

Jia Shi, Kirk Pabon, Rui Ding, Kathleen W Scotto.

ABCG2, a member of the ABC transporter superfamily, is overexpressed in many human tumors and has long been studied for its ability to export a variety of chemotherapeutic agents, thereby conferring a multidrug resistance (MDR) phenotype. However, several studies have shown that ABCG2 can also confer an MDR-independent survival advantage to tumor cells exposed to stress. While investigating the mechanism by which ABCG2 enhances survival in stressful milieus, we have identified a physical and functional interaction between ABCG2 and SLC1A5, a member of the solute transporter superfamily and the primary transporter of glutamine in cancer cells. This interaction was accompanied by increased glutamine uptake, increased glutaminolysis and rewired cellular metabolism, as evidenced by an increase in key metabolic enzymes and alteration of glutamine-dependent metabolic pathways. Specifically, we observed an increase in glutamine metabolites shuttled to the TCA cycle, an increase in the synthesis of glutathione, accompanied by a decrease in basal levels of reactive oxygen species and a marked increase in cell survival in the face of oxidative stress. Notably, knockdown of SLC1A5 or depletion of exogenous glutamine diminished ABCG2-enhanced autophagy flux, further implicating this solute transporter in ABCG2-mediated cell survival. This is, to our knowledge, the first report of a functionally significant physical interaction between members of the two major transporter superfamilies. Moreover, these observations may underlie the protective role of ABCG2 in cancer cells under duress and suggest a novel role for ABCG2 in the regulation of metabolism in normal and diseased states.

DOI: https://doi.org/10.1016/j.jbc.2024.107299
J Clin Invest. 2024 Apr 15. pii: e173934. [Epub ahead of print]134(8):

VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Melissa M Wolf, Matthew Z Madden, Emily N Arner, Jackie E Bader, Xiang Ye, Logan Vlach, Megan L Tigue, Madelyn D Landis, Patrick B Jonker, Zaid Hatem, KayLee K Steiner, Dakim K Gaines, Bradley I Reinfeld, Emma S Hathaway, Fuxue Xin, M Noor Tantawy, Scott M Haake, Eric Jonasch, Alexander Muir, Vivian L Weiss, Kathryn E Beckermann, W Kimryn Rathmell, Jeffrey C Rathmell.

  Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Keywords:  Cancer; Macrophages; Metabolism; Oncology; T cells

DOI:  https://doi.org/10.1172/JCI173934
J Biol Chem. 2024 Apr 17. pii: S0021-9258(24)01802-7. [Epub ahead of print] 107301

Rapid HPLC method reveals dynamic shifts in coenzyme Q redox state.

Victor Vitvitsky, Roshan Kumar, Jutta Diessl, David A Hanna, Ruma Banerjee.

Ubiquinol or coenzyme Q (CoQ) is a lipid-soluble electron carrier in the respiratory chain and an electron acceptor for various enzymes in metabolic pathways that intersect at this cofactor hub in the mitochondrial inner membrane. The reduced form of CoQ is an antioxidant, which protects against lipid peroxidation. In this study, we have optimized a UV-detected HPLC method for CoQ analysis from biological materials, which involves a rapid single-step extraction into n-propanol followed by direct sample injection onto a column. Using this method, we have measured the oxidized, reduced and total CoQ pools, and monitored shifts in the CoQ redox status in response to cell culture conditions and bioenergetic perturbations. We find that hypoxia or sulfide exposure induces a reductive shift in the intracellular CoQ pool. The effect of hypoxia is however, rapidly reversed by exposure to ambient air. Interventions at different loci in the electron transport chain can induce sizeable redox shifts in the oxidative or reductive direction, depending on whether they are up- or downstream of complex III. We have also used this method to confirm that CoQ levels are higher and more reduced in murine heart versus brain. In summary, the availability of a convenient HPLC-based method described herein, will facilitate studies on CoQ redox dynamics in response to environmental, nutritional and endogenous alterations.

DOI: https://doi.org/10.1016/j.jbc.2024.107301
Nat Immunol. 2024 Apr 19.

Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

Meredith L Stone, Jesse Lee, Jae W Lee, Heather Coho, Mito Tariveranmoshabad, Max M Wattenberg, Hana Choi, Veronica M Herrera, Yuqing Xue, Shaanti Choi-Bose, Sofia K Zingone, Dhruv Patel, Kelly Markowitz, Devora Delman, Vinod P Balachandran, Gregory L Beatty.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

DOI: https://doi.org/10.1038/s41590-024-01820-1
Cancer Res Commun. 2024 Apr 16.

De novo purine metabolism is a metabolic vulnerability of cancers with low p16 expression.

Naveen Kumar Tangudu, Raquel Buj, Hui Wang, Jiefei Wang, Aidan R Cole, Apoorva Uboveja, Richard Fang, Amandine Amalric, Baixue Yang, Adam Chatoff, Claudia V Crispim, Peter Sajjakulnukit, Maureen A Lyons, Kristine Cooper, Nadine Hempel, Costas A Lyssiotis, Uma R Chandran, Nathaniel W Snyder, Katherine M Aird.

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in ~50% of all human cancers. In its canonical role, p16 inhibits the G1-S phase cell cycle progression through suppression of cyclin dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. However, the broader impact of p16/CDKN2A loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR KO libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/CDKN2A. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low CDKN2A expression. We determined that cells with low p16/CDKN2A expression are sensitive to multiple inhibitors of de novo purine synthesis, including anti-folates. Finally, tumors with p16 knockdown were more sensitive to the anti-folate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2Alow tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.

DOI: https://doi.org/10.1158/2767-9764.CRC-23-0450
Nat Commun. 2024 Apr 15. 15(1): 3226

Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.

iMAXT Consortium

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

DOI: https://doi.org/10.1038/s41467-024-47185-9
Cancer Res. 2024 Apr 18.

Deuterium metabolic imaging differentiates glioblastoma metabolic subtypes and detects early response to chemoradiotherapy.

Jacob Chen Ming Low, Jianbo Cao, Friederike Hesse, Alan J Wright, Anastasia Tsyben, Islam Alshamleh, Richard Mair, Kevin M Brindle.

Metabolic subtypes of glioblastoma have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. Here, we used 2H magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived glioblastoma xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial TCA cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H MRS and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for TCA cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2552
Cell Rep. 2024 Apr 12. pii: S2211-1247(24)00449-2. [Epub ahead of print]43(4): 114121

Context-dependent roles for ubiquitous mitochondrial creatine kinase CKMT1 in breast cancer progression.

Vinay Ayyappan, Nicole M Jenkinson, Caitlin M Tressler, Zheqiong Tan, Menglin Cheng, Xinyi Elaine Shen, Alejandro Guerrero, Kanchan Sonkar, Ruoqing Cai, Oluwatobi Adelaja, Sujayita Roy, Alan Meeker, Pedram Argani, Kristine Glunde.

  Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.

Keywords:  CKMT1; CP: Cancer; CP: Metabolism; breast cancer; creatine; metabolism; metastasis; reactive oxygen species

DOI:  https://doi.org/10.1016/j.celrep.2024.114121
Redox Biol. 2024 Mar 12. pii: S2213-2317(24)00099-5. [Epub ahead of print]72 103123

Redox regulation of macrophages.

Nhien Tran, Evanna L Mills.

Redox signaling, a mode of signal transduction that involves the transfer of electrons from a nucleophilic to electrophilic molecule, has emerged as an essential regulator of inflammatory macrophages. Redox reactions are driven by reactive oxygen/nitrogen species (ROS and RNS) and redox-sensitive metabolites such as fumarate and itaconate, which can post-translationally modify specific cysteine residues in target proteins. In the past decade our understanding of how ROS, RNS, and redox-sensitive metabolites control macrophage function has expanded dramatically. In this review, we discuss the latest evidence of how ROS, RNS, and metabolites regulate macrophage function and how this is dysregulated with disease. We highlight the key tools to assess redox signaling and important questions that remain.

DOI: https://doi.org/10.1016/j.redox.2024.103123
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2320934121

Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Lijie Gu, Yanhong Du, Jianglei Chen, Mohammad Nazmul Hasan, Yung Dai Clayton, David J Matye, Jacob E Friedman, Tiangang Li.

  Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.

Keywords:  cullin; fatty liver; insulin resistance; neddylation; reductive stress

DOI:  https://doi.org/10.1073/pnas.2320934121
Immunity. 2024 Apr 11. pii: S1074-7613(24)00141-9. [Epub ahead of print]

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages.

Jay K Mandula, Rosa A Sierra-Mondragon, Rachel V Jimenez, Darwin Chang, Eslam Mohamed, Shiun Chang, Julio A Vazquez-Martinez, Yu Cao, Carmen M Anadon, Sae Bom Lee, Satyajit Das, Léo Rocha-Munguba, Vincent M Pham, Roger Li, Ahmad A Tarhini, Muhammad Furqan, William Dalton, Michelle Churchman, Carlos M Moran-Segura, Jonathan Nguyen, Bradford Perez, Douglas J Kojetin, Alyssa Obermayer, Xiaoqing Yu, Ann Chen, Timothy I Shaw, Jose R Conejo-Garcia, Paulo C Rodriguez.

  Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

Keywords:  Jagged; Notch ligands; immunosuppression in cancer; immunosuppressive myelopoiesis; macrophage reprogramming; tumor-associated macrophages

DOI:  https://doi.org/10.1016/j.immuni.2024.03.020
Cancer Res. 2024 Apr 19.

GPR1 and CMKLR1 control lipid metabolism to support development of clear cell renal cell carcinoma.

Dazhi Wang, Iqbal Mahmud, Vijay S Thakur, Sze Kiat Tan, Daniel G Isom, David B Lombard, Mark L Gonzalgo, Oleksandr N Kryvenko, Philip L Lorenzi, Vanina T Tcheuyap, James Brugarolas, Scott M Welford.

Clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer, is largely incurable in the metastatic setting. ccRCC is characterized by excessive lipid accumulation that protects cells from stress and promotes tumor growth, suggesting that the underlying regulators of lipid storage could represent potential therapeutic targets. Here, we evaluated the regulatory roles of GPR1 and CMKLR1, two G-protein coupled receptors of the pro-tumorigenic adipokine chemerin that is involved in ccRCC lipid metabolism. Both genetic and pharmacological suppression of either receptor suppressed lipid formation and induced multiple forms of cell death, including apoptosis, ferroptosis and autophagy, significantly impeding ccRCC growth in cell lines and patient derived xenograft (PDX) models. Comprehensive lipidomic and transcriptomic profiling of receptor competent and depleted cells revealed overlapping and unique signaling of the receptors granting control over triglyceride synthesis, ceramide production, and fatty acid saturation and class production. Mechanistically, the receptors both enforced suppression of the triglyceride lipase ATGL but also demonstrated distinct functions, such as the unique ability of CMKLR1 to control lipid uptake through regulation of SREBP1c and the CD36 scavenger receptor. Treating PDX models with the CMKLR1-targeting small molecule α-NETA led to a dramatic reduction of tumor growth, lipid storage, and clear cell morphology. Together, these findings provide mechanistic insight into lipid regulation in ccRCC and identify a targetable axis at the core of the histological definition of this tumor that could be exploited therapeutically.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2926
Oncogene. 2024 Apr 17.

ULK1-dependent phosphorylation of PKM2 antagonizes O-GlcNAcylation and regulates the Warburg effect in breast cancer.

Zibin Zhou, Xiyuan Zheng, Jianxin Zhao, Aiyun Yuan, Zhuan Lv, Guangcan Shao, Bin Peng, Meng-Qiu Dong, Quan Xu, Xingzhi Xu, Jing Li.

Pyruvate kinase M2 (PKM2) is a central metabolic enzyme driving the Warburg effect in tumor growth. Previous investigations have demonstrated that PKM2 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification, which is a nutrient-sensitive post-translational modification. Here we found that unc-51 like autophagy activating kinase 1 (ULK1), a glucose-sensitive kinase, interacts with PKM2 and phosphorylates PKM2 at Ser333. Ser333 phosphorylation antagonizes PKM2 O-GlcNAcylation, promotes its tetramer formation and enzymatic activity, and decreases its nuclear localization. As PKM2 is known to have a nuclear role in regulating c-Myc, we also show that PKM2-S333 phosphorylation inhibits c-Myc expression. By downregulating glucose consumption and lactate production, PKM2 pS333 attenuates the Warburg effect. Through mouse xenograft assays, we demonstrate that the phospho-deficient PKM2-S333A mutant promotes tumor growth in vivo. In conclusion, we identified a ULK1-PKM2-c-Myc axis in inhibiting breast cancer, and a glucose-sensitive phosphorylation of PKM2 in modulating the Warburg effect.

DOI: https://doi.org/10.1038/s41388-024-03035-y
iScience. 2024 Apr 19. 27(4): 109589

A LTB4/CD11b self-amplifying loop drives pyogranuloma formation in chronic granulomatous disease.

Kelsey C Haist, Sophie L Gibbings, Jordan Jacobelli, Kara J Mould, Peter M Henson, Donna L Bratton.

  Sterile pyogranulomas and heightened cytokine production are hyperinflammatory hallmarks of Chronic Granulomatous Disease (CGD). Using peritoneal cells of zymosan-treated CGD (gp91phox-/-) versus wild-type (WT) mice, an ex vivo system of pyogranuloma formation was developed to determine factors involved in and consequences of recruitment of neutrophils and monocyte-derived macrophages (MoMacs). Whereas WT cells failed to aggregate, CGD cells formed aggregates containing neutrophils initially, and MoMacs recruited secondarily. LTB4 was key, as antagonizing BLT1 blocked neutrophil aggregation, but acted only indirectly on MoMac recruitment. LTB4 upregulated CD11b expression on CGD neutrophils, and the absence/blockade of CD11b inhibited LTB4 production and cell aggregation. Neutrophil-dependent MoMac recruitment was independent of MoMac Nox2 status, BLT1, CCR1, CCR2, CCR5, CXCR2, and CXCR6. As proof of concept, CD11b-deficient CGD mice developed disrupted pyogranulomas with poorly organized neutrophils and diminished recruitment of MoMacs. Importantly, the disruption of cell aggregation and pyogranuloma formation markedly reduced proinflammatory cytokine production.

Keywords:  Biological sciences; Immunology; Natural sciences; Pathophysiology; Physiology

DOI:  https://doi.org/10.1016/j.isci.2024.109589
Immunity. 2024 Apr 12. pii: S1074-7613(24)00142-0. [Epub ahead of print]

25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.

Jun Xiao, Shuang Wang, Longlong Chen, Xinyu Ding, Yuanhao Dang, Mingshun Han, Yuxiao Zheng, Huan Shen, Sifan Wu, Mingchang Wang, Dan Yang, Na Li, Chen Dong, Miao Hu, Chen Su, Weiyun Li, Lijian Hui, Youqiong Ye, Huiru Tang, Bin Wei, Hongyan Wang.

  Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response.

Keywords:  25-hydroxycholesterol; AMPK; CH25H; M2-like macrophage; STAT6; cholesterol matabolism; oxysterol; tumor immunotherapy; tumor-associated macrophage

DOI:  https://doi.org/10.1016/j.immuni.2024.03.021
STAR Protoc. 2024 Apr 12. pii: S2666-1667(24)00176-X. [Epub ahead of print]5(2): 103011

Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets.

Julian Swatler, Alicja Targońska, Laura Turos-Korgul, Grażyna Mosieniak, Katarzyna Piwocka.

  Extracellular vesicles (EVs) enable communication between cells and tissues and are implicated in modulation of tumor immunosuppression. Here, we present a protocol for isolating tumor-derived EVs and assessing their functional influence in cultures with different subsets of human T cells. We describe steps for differential ultracentrifugation, size exclusion chromatography, EVs quantification, and fluorescence-activated cell sorting of human T cells. We then detail procedures for culturing T cells with EVs and using high-resolution spectral flow cytometry phenotyping for the analysis thereof. For complete details on the use and execution of this protocol, please refer to Swatler et al.1 and Swatler et al.2.

Keywords:  Cancer; Cell Biology; Immunology

DOI:  https://doi.org/10.1016/j.xpro.2024.103011
Sci Immunol. 2024 Apr 19. 9(94): eadg8817

Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine-mediated regulation of c-Fos.

Sicong Ma, Roger Sandhoff, Xiu Luo, Fuwei Shang, Qiaozhen Shi, Zhaolong Li, Jingxia Wu, Yanan Ming, Frank Schwarz, Alaa Madi, Nina Weisshaar, Alessa Mieg, Marvin Hering, Ferdinand Zettl, Xin Yan, Kerstin Mohr, Nora Ten Bosch, Zhe Li, Gernot Poschet, Hans-Reimer Rodewald, Nina Papavasiliou, Xi Wang, Pu Gao, Guoliang Cui.

CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME influences Treg cell differentiation is not understood. Here, we measured 630 metabolites in the TME and found that serine and palmitic acid, substrates required for the synthesis of sphingolipids, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos. Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits antitumor immunity.

DOI: https://doi.org/10.1126/sciimmunol.adg8817