Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.

Restrictor synergizes with Symplekin and PNUTS to terminate extragenic transcription.

Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage, and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the SPT5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of noncoding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from, Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

WNT Oncogenic Transcription Requires MYC Suppression of Lysosomal Activity and EPCAM Stabilization in Gastric Tumors.

BACKGROUND & AIMS: WNT signaling is central to spatial tissue arrangement and regulating stem cell activity, and it represents the hallmark of gastrointestinal cancers. Although its role in driving intestinal tumors is well characterized, WNT's role in gastric tumorigenesis remains elusive. METHODS: We have developed mouse models to control the specific expression of an oncogenic form of ß-catenin in combination with MYC activation in Lgr5+ cells of the gastric antrum. We used multiomics approaches applied in vivo and in organoid models to characterize their cooperation in driving gastric tumorigenesis. RESULTS: We report that constitutive ß-catenin stabilization in the stomach has negligible oncogenic effects and requires MYC activation to induce gastric tumor formation. Although physiologically low MYC levels in gastric glands limit ß-catenin transcriptional activity, increased MYC expression unleashes the WNT oncogenic transcriptional program, promoting ß-catenin enhancer invasion without a direct transcriptional cooperation. MYC activation induces a metabolic rewiring that suppresses lysosomal biogenesis through mTOR and ERK activation and MiT/TFE inhibition. This prevents EPCAM degradation by macropinocytosis, promoting ß-catenin chromatin accumulation and activation of WNT oncogenic transcription. CONCLUSION: Our results uncovered a new signaling framework with important implications for the control of gastric epithelial architecture and WNT-dependent oncogenic transformation.

Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3) has a key role in the mitochondrial dysfunction induced by mutant huntingtin.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the IT15 gene that encodes the protein huntingtin (htt). Evidence shows that mutant htt causes mitochondrial depolarization and fragmentation, but the underlying molecular mechanism has yet to be clarified. Bax/Bak and BNip3 are pro-apoptotic members of the Bcl-2 family protein whose activation triggers mitochondrial depolarization and fragmentation inducing cell death. Evidence suggests that Bax/Bak and BNip3 undergo activation upon mutant htt expression but whether these proteins are required for mitochondrial depolarization and fragmentation induced by mutant htt is unclear. Our results show that BNip3 knock-out cells are protected from mitochondrial damage and cell death induced by mutant htt whereas Bax/Bak knock-out cells are not. Moreover, deletion of BNip3 C-terminal transmembrane domain, required for mitochondrial targeting, suppresses mitochondrial depolarization and fragmentation in a cell culture model of HD. Hence, our results suggest that changes in mitochondrial morphology and transmembrane potential, induced by mutant htt protein, are dependent and linked to BNip3 and not to Bax/Bak activation. These results provide new compelling evidence that underlies the molecular mechanisms by which mutant htt causes mitochondrial dysfunction and cell death, suggesting BNip3 as a potential target for HD therapy.

Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation.

BACKGROUND: Thanks to mechanotransductive components cells are competent to perceive nanoscale topographical features of their environment and to convert the immanent information into corresponding physiological responses. Due to its complex configuration, unraveling the role of the extracellular matrix is particularly challenging. Cell substrates with simplified topographical cues, fabricated by top-down micro- and nanofabrication approaches, have been useful in order to identify basic principles. However, the underlying molecular mechanisms of this conversion remain only partially understood. RESULTS: Here we present the results of a broad, systematic and quantitative approach aimed at understanding how the surface nanoscale information is converted into cell response providing a profound causal link between mechanotransductive events, proceeding from the cell/nanostructure interface to the nucleus. We produced nanostructured ZrO2 substrates with disordered yet controlled topographic features by the bottom-up technique supersonic cluster beam deposition, i.e. the assembling of zirconia nanoparticles from the gas phase on a flat substrate through a supersonic expansion. We used PC12 cells, a well-established model in the context of neuronal differentiation. We found that the cell/nanotopography interaction enforces a nanoscopic architecture of the adhesion regions that affects the focal adhesion dynamics and the cytoskeletal organization, which thereby modulates the general biomechanical properties by decreasing the rigidity of the cell. The mechanotransduction impacts furthermore on transcription factors relevant for neuronal differentiation (e.g. CREB), and eventually the protein expression profile. Detailed proteomic data validated the observed differentiation. In particular, the abundance of proteins that are involved in adhesome and/or cytoskeletal organization is striking, and their up- or downregulation is in line with their demonstrated functions in neuronal differentiation processes. CONCLUSION: Our work provides a deep insight into the molecular mechanotransductive mechanisms that realize the conversion of the nanoscale topographical information of SCBD-fabricated surfaces into cellular responses, in this case neuronal differentiation. The results lay a profound cell biological foundation indicating the strong potential of these surfaces in promoting neuronal differentiation events which could be exploited for the development of prospective research and/or biomedical applications. These applications could be e.g. tools to study mechanotransductive processes, improved neural interfaces and circuits, or cell culture devices supporting neurogenic processes.

Kainate induces mobilization of synaptic vesicles at the growth cone through the activation of protein kinase A.

Activation of protein kinase A (PKA) pathway at presynaptic terminals plays a crucial role in the supply of synaptic vesicles (SVs) from the reserve pool, affecting the steady-state level of activity and the reconstitution of the readily releasable pool after intense stimulation. However, the identity of the stimuli activating this pathway is undefined. Using fluorescence resonance energy transfer and molecular genetic, we show that kainate, through the activation of presynaptic kainate receptors, induces PKA activation and enhances synapsin I phosphorylation at PKA-specific residues. This leads to a dispersion of synapsin I immunoreactivity, which is accompanied by a PKA-dependent increase in the rate of SV recycling at the growth cone and by an enhanced miniature excitatory postsynaptic currents frequency in mature networks. Selective activation of this pathway is induced by the native neurotransmitter glutamate, when applied in the high nanomolar range. These data identify glutamate, specifically acting on KARs, as one of the stimuli able to induce phosphorylation of synapsin at PKA sites, both at the axonal growth cone and at the mature synapse, thus increasing SV availability and contributing to plasticity phenomena.

Mapping functional to morphological variation reveals the basis of regional extracellular matrix subversion and nerve invasion in pancreatic cancer.

Intratumor morphological heterogeneity of pancreatic ductal adenocarcinoma (PDAC) predicts clinical outcomes but is only partially understood at the molecular level. To elucidate the gene expression programs underpinning intratumor morphological variation in PDAC, we investigated and deconvoluted at single cell level the molecular profiles of histologically distinct clusters of PDAC cells. We identified three major morphological and functional variants that co-exist in varying proportions in all PDACs, display limited genetic diversity, and are associated with a distinct organization of the extracellular matrix: a glandular variant with classical ductal features; a transitional variant displaying abortive ductal structures and mixed endodermal and myofibroblast-like gene expression; and a poorly differentiated variant lacking ductal features and basement membrane, and showing neuronal lineage priming. Ex vivo and in vitro evidence supports the occurrence of dynamic transitions among these variants in part influenced by extracellular matrix composition and stiffness and associated with local, specifically neural, invasion.

Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer.

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.

Oxidative stress enhances the therapeutic action of a respiratory inhibitor in MYC-driven lymphoma.

MYC is a key oncogenic driver in multiple tumor types, but concomitantly endows cancer cells with a series of vulnerabilities that provide opportunities for targeted pharmacological intervention. For example, drugs that suppress mitochondrial respiration selectively kill MYC-overexpressing cells. Here, we unravel the mechanistic basis for this synthetic lethal interaction and exploit it to improve the anticancer effects of the respiratory complex I inhibitor IACS-010759. In a B-lymphoid cell line, ectopic MYC activity and treatment with IACS-010759 added up to induce oxidative stress, with consequent depletion of reduced glutathione and lethal disruption of redox homeostasis. This effect could be enhanced either with inhibitors of NADPH production through the pentose phosphate pathway, or with ascorbate (vitamin C), known to act as a pro-oxidant at high doses. In these conditions, ascorbate synergized with IACS-010759 to kill MYC-overexpressing cells in vitro and reinforced its therapeutic action against human B-cell lymphoma xenografts. Hence, complex I inhibition and high-dose ascorbate might improve the outcome of patients affected by high-grade lymphomas and potentially other MYC-driven cancers.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity.

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Targeting mitochondrial respiration and the BCL2 family in high-grade MYC-associated B-cell lymphoma.

Multiple molecular features, such as activation of specific oncogenes (e.g., MYC, BCL2) or a variety of gene expression signatures, have been associated with disease course in diffuse large B-cell lymphoma (DLBCL), although their relationships and implications for targeted therapy remain to be fully unraveled. We report that MYC activity is closely correlated with-and most likely a driver of-gene signatures related to oxidative phosphorylation (OxPhos) in DLBCL, pointing to OxPhos enzymes, in particular mitochondrial electron transport chain (ETC) complexes, as possible therapeutic targets in high-grade MYC-associated lymphomas. In our experiments, indeed, MYC sensitized B cells to the ETC complex I inhibitor IACS-010759. Mechanistically, IACS-010759 triggered the integrated stress response (ISR) pathway, driven by the transcription factors ATF4 and CHOP, which engaged the intrinsic apoptosis pathway and lowered the apoptotic threshold in MYC-overexpressing cells. In line with these findings, the BCL2-inhibitory compound venetoclax synergized with IACS-010759 against double-hit lymphoma (DHL), a high-grade malignancy with concurrent activation of MYC and BCL2. In BCL2-negative lymphoma cells, instead, killing by IACS-010759 was potentiated by the Mcl-1 inhibitor S63845. Thus, combining an OxPhos inhibitor with select BH3-mimetic drugs provides a novel therapeutic principle against aggressive, MYC-associated DLBCL variants.

Pendrin overexpression affects cell volume recovery, intracellular pH and chloride concentration after hypotonicity-induced cell swelling.

The pendrin (SLC26A4 or PDS) gene is responsible, when mutated, for the Pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid dysfunctions. Pendrin protein is an anion exchanger and we focused on a still unexplored function that it might play in view of its importance in the inner ear: Cl(-) fluxes regulation during cellular volume control. We challenged HEK-293 Phoenix cells over-expressing wild type pendrin (PDS HEK cells) together with the EYFP (Enhanced Yellow Fluorescent Protein) or over-expressing the EYFP alone (control HEK cells) with hypo-osmolar solutions. Taking advantage of the confocal optical sectioning we measured the cell volume. In addition, we determined the intracellular pH and chloride concentration with fluorescent probes (EYFP and seminaphthorhodafluor-5F, SNARF-5F). Consequently, we could estimate simultaneously Cl(-) fluxes, cellular volume and intracellular pH variations. Cl(-) movements markedly differed between PDS and control HEK cells upon hypotonic shock and are accompanied by an attenuation of the swelling induced pH drop in PDS HEK cells. The contemporary measurements of the three variables not yet reported in living cells, allowed to assess a possible influence of pendrin upregulation in volume homeostasis and evidenced its participation to Cl(-) fluxes.

Tonic modulation of GABA release by nicotinic acetylcholine receptors in layer V of the murine prefrontal cortex.

By regulating the neocortical excitability, nicotinic acetylcholine receptors (nAChRs) control vigilance and cognition and are implicated in epileptogenesis. Modulation of gamma-aminobutyric acid (GABA) release often accompanies these processes. We studied how nAChRs regulate GABAergic transmission in the murine neocortex with immunocytochemical and patch-clamp methods. The cholinergic fibers densely innervated the somatosensory, visual, motor, and prefrontal cortices (PFC). Laminar distribution was broadly homogeneous, especially in the PFC. The cholinergic terminals were often adjacent to the soma and dendrites of GABAergic interneurons, but well-differentiated synapses were rare. Tonically applied nicotine (1-100 microM) increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) on pyramidal neurons in PFC layer V. The contribution of nAChR types was assessed by using 1 microM dihydro-beta-erythroidine (DHbetaE), to block heteromeric nAChRs, and 10 nM methyllycaconitine (MLA), to block homomeric nAChRs. Both inhibitors antagonized the effect of nicotine on IPSCs, suggesting that mixed nAChR types control pyramidal neuron inhibition in layer V. To determine whether nAChRs are expressed on basket cells' terminals, we studied miniature IPSCs (mIPSCs). These were revealed using 0.5 microM tetrodotoxin and 50 microM Cd(2+) to isolate the GABAergic terminals from the action potential drive. The nicotinic stimulation of mIPSCs was antagonized by DHbetaE, but not MLA, indicating that heteromeric nAChRs prevail in GABAergic terminals. Immunocytochemistry confirmed the expression of nAChRs on basket cells' somata and terminals. Finally, when the ionotropic glutamatergic transmission was blocked, nicotine partially inhibited the IPSCs, an effect counteracted by both DHbetaE and MLA. Therefore, a fraction of nAChRs are capable of activating GABAergic interneurons that in turn inhibit other GABAergic interneurons, thereby reducing the IPSCs. We conclude that heteromeric nAChRs control GABA release presynaptically, whereas mixed nAChRs regulate both excitation and inhibition of interneurons, the balance depending on the overall glutamatergic drive.

Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA.

Pendred syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss, with malformations of the inner ear, ranging from enlarged vestibular aqueduct (EVA) to Mondini malformation, and deficient iodide organification in the thyroid gland. Nonsyndromic EVA (ns-EVA) is a separate type of sensorineural hearing loss showing normal thyroid function. Both Pendred syndrome and ns-EVA seem to be linked to the malfunction of pendrin (SLC26A4), a membrane transporter able to exchange anions between the cytosol and extracellular fluid. In the past, the pathogenicity of SLC26A4 missense mutations were assumed if the mutations fulfilled two criteria: low incidence of the mutation in the control population and substitution of evolutionary conserved amino acids. Here we show that these criteria are insufficient to make meaningful predictions about the effect of these SLC26A4 variants on the pendrin-induced ion transport. Furthermore, we functionally characterized 10 missense mutations within the SLC26A4 ORF, and consistently found that on the protein level, an addition or omission of a proline or a charged amino acid in the SLC26A4 sequence is detrimental to its function. These types of changes may be adequate for predicting SLC26A4 functionality in the absence of direct functional tests.

Radiation and adjuvant drug-loaded liposomes target glioblastoma stem cells and trigger in-situ immune response.

BACKGROUND: The radio- and chemo-resistance of glioblastoma stem-like cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the blood-brain barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma. METHODS: Liposomes (LIPs), surface-decorated with an Apolipoprotein E-modified peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXO-LIPs alone or concomitantly with radiation. RESULTS: Our results indicated that mApoE, through the engagement of the low-density lipoprotein receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused a significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response. CONCLUSIONS: Our results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of glioblastoma (GBM) recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.

Microtubule dysfunction precedes transport impairment and mitochondria damage in MPP+ -induced neurodegeneration.

Dysfunction of the microtubule (MT) system is an emerging theme in the pathogenesis of Parkinson's disease. This study was designed to investigate the putative role of MT dysfunction in dopaminergic neuron death induced by the neurotoxin 1-methyl-4-phenylpiridinium (MPP(+)). In nerve growth factor-differentiated PC12 cells, we have analyzed post-translational modifications of tubulin known to be associated with differently dynamic MTs and show that MPP(+) causes a selective loss of dynamic MTs and a concomitant enrichment of stable MTs. Through a direct live cell imaging approach, we show a significant reduction of MT dynamics following exposure to MPP(+) and a reorientation of MTs. Furthermore, these alterations precede the impairment of intracellular transport as revealed by changes in mitochondria movements along neurites and their accumulation into varicosities. We have also analyzed activation of caspase 3 and mitochondrial injury, well-known alterations induced by MPP(+), and found that they are noticeable only when MT dysfunction is already established. These data provide the first evidence that axonal transport impairment and mitochondrial damage might be a consequence of MT dysfunction in MPP(+) -induced neurodegeneration, lending support to the concept that alterations of MT organization and dynamics could play a pivotal role in neuronal death in Parkinson's disease.

Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

An Easy Path for Correlative Electron and Super-Resolution Light Microscopy.

A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.